(19)
(11)EP 2 181 330 B2

(12)NEW EUROPEAN PATENT SPECIFICATION
After opposition procedure

(45)Date of publication and mention of the opposition decision:
31.08.2016 Bulletin 2016/35

(45)Mention of the grant of the patent:
20.11.2013 Bulletin 2013/47

(21)Application number: 08784426.2

(22)Date of filing:  26.08.2008
(51)Int. Cl.: 
C12Q 1/04  (2006.01)
C12M 1/16  (2006.01)
C12Q 1/08  (2006.01)
C12Q 1/14  (2006.01)
C12M 1/20  (2006.01)
(86)International application number:
PCT/DK2008/000303
(87)International publication number:
WO 2009/026920 (05.03.2009 Gazette  2009/10)

(54)

COMPOSITIONS AND MEANS FOR DIAGNOSING MICROBIAL INFECTIONS

ZUSAMMENSETZUNGEN UND MITTEL ZUR DIAGNOSE MIKROBIELLER INFEKTIONEN

COMPOSITIONS ET MOYENS PERMETTANT DE DIAGNOSTIQUER DES INFECTIONS MICROBIENNES


(84)Designated Contracting States:
AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MT NL NO PL PT RO SE SI SK TR

(30)Priority: 31.08.2007 EP 07115480
31.08.2007 US 969260 P

(43)Date of publication of application:
05.05.2010 Bulletin 2010/18

(73)Proprietor: Statens Serum Institut
2300 Copenhagen S (DK)

(72)Inventor:
  • FRIMODT-MOLLER, Niels
    DK-2820 Gentofte (DK)

(74)Representative: Cornish, Kristina Victoria Joy et al
Kilburn & Strode LLP 20 Red Lion Street
London WC1R 4PJ
London WC1R 4PJ (GB)


(56)References cited: : 
WO-A-03/106696
US-A1- 2002 076 742
US-B1- 6 350 588
WO-A-2004/050675
US-B1- 6 251 624
US-B1- 6 750 038
  
  • RODRIGUEZ T ET AL: "Standardization of Neisseria meningitidis serogroup B colorimetric serum bactericidal assay" CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY, AMERICAN SOCIETY FOR MICROBIOLOGY, US, vol. 9, no. 1, January 2002 (2002-01), pages 109-114, XP003009515 ISSN: 1071-412X
  


Description

Technical field of the invention



[0001] The present invention relates to compositions, platforms, kits and methods for diagnosing, detecting and/or characterising a microbial infection or contamination. In particular the present invention relates to such compositions, platforms, kits and methods for diagnosing, detecting and/or characterising a urinary tract infection.

Background of the invention


Urinary Tract Infections (UTI):



[0002] Urinary tract infection is one of the most common infections in general practice as well as the most common nosocomial infection in the western world. The overall prevalence is 3-5% and the incidence 18/1,000 inhabitants per year1. Infections are most prevalent in females (six-fold higher risk than in men), which is usually explained by the fact that the opening of the urethra lies in direct contact with the vaginal flora and the the urethra is fairly short in women in contrast to the 4-5 times longer and therefore protected urethra in men. Regarding age groups, infections are most common early, i.e. below the age of one, and late in life, i.e. > 60 years of age.

[0003] Although most infections are uncomplicated lower UTI, i.e. bladder infections, which often cure themselves without antibiotic treatment, UTI can also be complicated with ascending renal infections which can spread to the blood leading to septicaemia with septic shock. Around 50% of Escherichia coli bacteraemias have the urinary tract as focus, and the mortality is around 20% for these infections in spite of effective antibiotics. Renal impairment with uraemia and dialysis treatment is caused by chronic urinary tract infections in more than a third of cases. Operative procedures and other iatrogenic manipulations of the urinary tract such as cystoscopy, catheterisation etc. results in a significant amount of infections, e.g. bladder catheterisation leads to bacteriuria in 30-50% of patients after a week and almost 100% after a month.

Pathogenesis and Etiology:



[0004] UTI can be caused by virus such as adenovirus and by flagellates such as Trichomonas vaginalis but the majority of infections (>98%) are caused by bacteria, which in most cases stem from the patients' own rectal flora. Due to chance or low hygiene bacteria can spread from the rectum and anus via the perineum to the vagina, where they can colonize the vagina and especially the area around meatus urethrae externae. From this point they gain access to the urethra helped by factors such as e.g. low temperatures inhibiting local immune function or by physical factors such as intercourse. If the bacteria contain specific virulence factors, which help them to adhere to the mucous membranes in the urethra and the bladder, they can penetrate into and through the epithelium and into the tissue underneath. An infective process ensues with intracellular micro-colony formation, apoptosis of the epithelial cells and release of cell material and bacteria into the bladder lumen with risk of renewed infection. In many cases the immune system will by and large remove the bacteria and restore normal function of the bladder wall tissue, but the bacteria in the bladder lumen can given time enough also ascend via the ureters to the renal pelvis, where they can spread further to the medulla and the cortex of the kidneys causing pyelonephritis, peri- or intrarenal abscess or other calamities.

[0005] The immune reaction can be seen as increasing numbers of leucocytes, mostly granulocytes, in the urine and in the tissues involved. Specific antibodies against the intruder can also be measured after 2-3 weeks in most persistent cases. E. coli is the main culprit causing 60- 90% of UTIs since this bacterium often holds the virulence factors necessary for causing UTI, i.e. adherence properties (fimbriae or pili with special predilection for receptors on the bladderepithelium), cell movement (flagellae) and a vast amount of other virulence factors, which enables the bacteria to circumvent the immune function and penetrate into human tissue. The rest of the infections are caused by other Enterobacteriaceae (Klebsiella and Enterobacter spp., Proteus spp.) and some Gram-positive bacteria (Enterococci, Staphylococcus saprophytics, Aerococcus spp.) and more rarely Candida spp. The possible role of Mycoplasma and Ureaplasma spp in UTI is still under debate.

[0006] Bacteria may be found in the urine of a patient who does not have symptoms or other signs (e.g. leucocyturia), i.e. so-called asymptomatic bacteriuria. This is particularly common in elderly patients and a prevalence of 10-15% has been found in several studies.

Clinical presentation:



[0007] The bladder infection leads to local pain, which can be felt behind the pubic bones or perhaps in the loins - but this is often a sign of renal involvement, as well as pain during voiding. Also, the irritation in the bladder will lead to frequent voiding. Fever can evolve although this is more common in case of pyelonephritis. The urine will change colour and turbidity to dark, cloudy and some times with hematuria, i.e. presence of erythrocytes due to minor bleeding from the scarred bladder epithelium. If bacteremia ensues the patient will develop signs of sepsis with general pain and malaise, high fever and shivering.

[0008] Even with uncomplicated UTI the patient is so invalidated by the condition that she (or he) will stay home from work and seek medical attention.

Diagnosis of UTI



[0009] Since the urinary tract is usually sterile with low numbers of leucocytes, the presence of bacteria and increased numbers of leucocytes is indicatory of infection. The diagnosis of UTI is based on the typical symptoms as well as presence of bacteria and increased numbers of leucocytes in a sterilely obtained urine sample. Due to the common colonisation of the external part of the urethra. it is difficult to obtain a sample during voiding without contamination of the sample. To avoid this contamination the best way to obtain a sterile urine sample is by suprapubic puncture, or via a bladder catheter or in case of renal pelvis infection via a percutaneous nephrostomy. But since these latter methods are rather cumbersome and often painful for the patient in the large majority of cases and also demands hospital admission, in most cases the urine is collected as a Mid Stream Urine (MSU), i.e. the meatus is cleaned with a cotton swab wetted with sterile saline, the patient then voids a small first part of the urine to cleanse the urethra and then voids - mid-stream - a sample collected in a sterile container - to end by voiding the rest of the urine volume in the toilet.

[0010] Other samples such as swabs from the urethra or blood cultures are also taken on indication of urethritis (e.g. gonorrhoea) or bacteraemia.

Quantitative criteria for diagnosis of UTI:



[0011] Due to the problems with contamination of the urine sample when taken as MSU and the subsequent evaluation of the results, and the fact that some patients may have asymptomatic bacteriuria it was in the 1950' ies found not the least in studies by Edward Kass2, that in order to discern between an asymptomatic patient and a patient with pyelonephritis, at least 105 bacteria/ml urine of one potential urinary pathogen (see above) must be present in two urine samples taken at least 24 h apart.

[0012] Later, it was found that counts down to 103 bacteria/ml of urine of a typical urinary pathogen (see above) is indicative of infection in patients, who have typical symptoms of UTI3, i.e. the Kass-criteria2 are used for patients with asymptomatic bacteriuria.

[0013] Currently used methods of diagnosing bacteria in the urine:

a) Direct methods



[0014] 
  1. 1. Microscopy: Bacteria can be visualized in the urine sample by either phase contrast microscopy of a wet smear, or by simple light microscopy of a Gram-coloured preparation. In the wet smear, the form and possible movement of the bacteria can be seen but naturally not the Gram-type of the bacteria. This can be discerned by the light microscopy of the Gram-stain, and in both cases leucocytes can be seen and roughly quantified. The problem with microscopy is the lack of specificity, i.e. only a presumptive bacterial diagnosis can be given, and the sensitivity, i.e. bacteria can only be visualized when present in numbers of at least 105 bacteria/ml of urine. Furthermore, microscopy will not reveal the antibiotic susceptibility of the bacteria. Phase contrast microscopy has a positive predictive value of 58% as related to quantitative culture with ≧ 105 bacteria/ml as criteria for UTI 4
  2. 2. Culture: The gold standard of diagnosis of UTI. In the laboratory this is performed by a quantitative culture, i.e. a standardized loop applying 1 or 10 µl urine on an agar plate. This allows quantification of bacteria by counting the number of colonies (colony forming units, CFU) on the incubated plate. If low numbers of bacteria are anticipated (e.g. suprapubic puncture or catheter sample) up to one ml of urine may be cultured allowing the counts of down to 1-2 CFU/ml urine. The culture can lead to further diagnosis of the bacteria by biochemical and other types of laboratory workup. Furthermore, a susceptibility test can be performed. Sensitivity of the method is by definition 100%, but in some cases bacteria may not grow if special media, atmosphere or temperature conditions are not used (some Aerococcus strains may only grow on blood agar and in CO2) or if the patient has started antibiotic treatment bacteria may not grow due to antibiotic suppression. The specificity is not 100%, since the positive culture may still contain contaminants - the result should be combined with the symptoms and signs as well as the presence of increased numbers of leucocytes in the urine. The quantitative loops have a variation of +/- ½ log CFU/ml, which means that only a rough estimate of the counts can be made5. The disadvantage of quantitative cultures in the laboratory is that the urine must be kept in a condition, where the bacteria do not multiply prior to culture; this can be obtained by cooling the urine during transportation (i.e. < 4° C) or by the use of transport media, which preserve the bacteria without promoting growth, e.g. by using boric acid. Boric acid containing tubes for transportation have therefore become popular in recent years, but this method carries the inherent problem of boric acid being toxic, e.g. carcinogenic to humans.
    Other culture methods: Dipslides with a plastic plate skeleton carrying agar media on one or both sides that are dipped into the urine have been used for 20-30 years (e.g. Uricult (Orion)). The advantage of these is the ease of inoculation and that the direct inoculation can be quantified by comparing the bacterial growth with pictures of dipslides used for cultures with known quantities of bacteria. The dipslide can also be used as a transport medium. A susceptibility test has been developed, where antibiotic containing discs are placed on the agar surfaces after inoculation (e.g. Sensicult (Orion)). The positive predictive value (PPV) of the susceptibility test has in some cases been as low as 0,6 , which can be explained by the lack of standardized inoculum and difficulty in interpreting a zone around the disc on a rounded surface, as the agar plate is not completely flat. Also, the small surface of the dipslide (i.e. approx. 2x5 cm) makes itdifficult to evaluate the growth characteristics of bacteria, and especially whether there are more than one species. Furthermore, evaluation of susceptibility is difficult without the knowledge of the bacterial species. A dipslide with chromogenic agar on one of the sides (DipStreak) has recently been marketed by Novamed in Israel.
  3. 3. Other methods: During the later years several methods have been tried for quantifying bacteria in urine such as turbidity measurements by spectrophotometry, cyto-centrifugation, ATP-measurement and others. So far, to our knowledge, none of these have been applied in routine laboratories and especially not so in primary care due to their cost and demand for technical skill.

b) Indirect methods:



[0015] 
  1. 1. Dipstick for nitrite and leucocyte-esterase: Determination of nitrite in the urine is used for diagnosis of UTI, since this substance can only be present if nitrate-reductase producing bacteria are present in the urine. Nitrate stems from protein-metabolism and is found in urine in varying concentrations depending on the intake of protein the day up to the sampling. Nitrite can be removed, however, if there are bacteria present, which produce nitrite-reductase (e.g. E. coli can contain both types of enzymes). The test is not very sensitive, since the reaction needs about 3 hours of incubation, some bacteria produce nitrite-reductase, and some urinary pathogens do not produce nitrate-reductase at all, e.g. staphylococci. The leucocyte-esterase test is more relevant, since it is the easiest way to prove the presence of leucocytes; the enzyme can only stem from leucocytes, and the amount of enzyme is correlated to the numbers of leucocytes. Whole leucocytes lyse easily and rapidly, which means that the urine microscopy for leucocytes must be performed within an hour after sampling in order to achieve a relevant quantitative microscopy, while the enzyme test can be performed several hours later due to the stability of the enzyme. The presence of leucocytes, however, is only predictive of infection in 50% of patients, since leucocyturia can be found in many patients without infection. Together, the test for the two enzymes combined (i.e. either one or both positive) has a rather low PPV (60-80%) due to the above mentioned factors.
  2. 2. Symptoms alone: In many cases in general practice, the general practitioner (GP) will initiate treatment based on symptoms alone. This can be due to reservations against diagnostic workup due to cost, geography or tradition, knowledge of susceptibility of the pathogens and/or use of broad spectrum antibiotics suspected to cover all possible pathogens.

Treatment of UTI:



[0016] In 30% of cases of uncomplicated UTI, the infection will be self-curable, i.e. disappear without antibiotic treatment6. But in most cases and especially in complicated cases i.e. all other patients than women in the age group 14-60 years of age, antibiotic treatment is the standard. Depending on the condition and the antibiotic used the uncomplicated infection will be cured in 3-7 days, while pyelonephritis needs 10-14 days of treatment, and the more chronic cases longer duration of treatment, which can in some cases be months to years. The effect depends upon the susceptibility of the bacterial pathogens6,7. The standard test performed in a laboratory takes time, especially when the disc susceptibility test is performed, since this test is based on several conditions being kept within certain limits (inoculum, incubation time, reading of the test, incubation atmosphere etc.). The most important factor is the inoculum, which must be within certain narrow limits to ensure quality of the test, why it is difficult to perform a meaningful disc diffusion susceptibility test directly on the primary urine sample, since the exact number of bacteria is unknown.

[0017] WO 99/18232 (by Chen et al) provides a multi-compartment assay device based on the combined use of medium capable of sustaining growth of total microbial organisms, a medium which is selective for the particular target organism and a medium which comprises an antimicrobial susceptibility interpretation medium. The application teaches the use of liquid medium and does not suggest a set-up which allows differentiation between the presence of multiple groups and/or strains of micro-organisms in the same sample and determination of the antimicrobial susceptibility of each group or strain of said micro-organisms.

[0018] Later Chen et al in WO03106696 discloses methods and devices for the detection of pathogenic microorganisms and their antimicrobial susceptibility. The use of a fluoresceent or chromogenic substrate can be included to get a visual signal of the presence of the microorganism, but the use of several different substances to determine different species is not mentioned.

[0019] WO0106000 discloses a test media for identification and differentiation of enterobacteriaceae. The medium comprises an antibiotic to prevent growth of other microorganisms than enterobacteriaceae.

[0020] US6750038 describes a rapid antibiotic susceptibility test. The use of a chromogene is not used to identify the microorganisms.

[0021] US6251624 discloses an apparatus and method for detecting, quantifying and characterizing microorganisms. Antibiotic susceptibility is tested by growth zone inhibiton.

[0022] WO2004050675 discloses a multichamber growth plate with selective broth for identification of particular microorganisms and with antibiotics in the media for testing susceptibility

[0023] Document WO 2004/050675 discloses a multi-chambered (with several compartments) plate (or platform) for diagnosing, detecting and/or characterising a microbial infection or contamination. Compartment can contain a semi-solid microbial growth medium. Some of the media used in WO 2004/050675 may contain a single chromogenic or fluorescent substance, for example pseudomonas agar P 64 and MUG MAC 65. Each chamber in the test plate contains a different media. WO 2004/050675 is detecting the same microorganisms as the present application, such as, e.g. Escherichia coli (ATCC 25922 strain), or Staphylococcus aureus, which are known as uropathogens.

[0024] Document US 2002/076742 describes the use of liquid medium and does not suggest any kind of set up which allows differentiation between the presence of multiple groups and/or strains of micro-organisms in the same sample.

[0025] Document US 6 251 624 relates to performing urine screening and instruments employed to do so, but there is no disclosure in document US 6 251 624 of the combination of ingredients in the semi-solid growth medium, in particular the three or more chromogenic substrates.

[0026] Hence, an improved technology for diagnosis, detection and characterisation of microbial infections or contamination, offering the possibility of differentiating between such multiple groups and/or strains of micro-organisms would be advantageous. In particular, such a technology would be advantageous if provided in the form of a platform which is efficient, reliable and possible to manufacture at a reasonable cost.

Summary of the invention



[0027] Thus, an object of the present invention relates to compositions, platforms, kits and methods for diagnosing, detecting and/or characterising a microbial infection or contamination.

Embodiments of the invention:



[0028] 
  1. 1. A platform comprising a solid support wherein said solid support comprises an indentation capable of acting as a receptacle for a sample with a possible microbial infection or contamination, said indentation being divided into three or more compartments, each compartment containing a test composition or a control composition and one or more integrated dividing members for dividing said indentation into said separate compartments, wherein
    1. i) said test composition comprises a semi solid microbial growth medium, an antimicrobial and three or more chromogenic substrates selected from the group consisting of 5-Bromo-6-chloro-3 -indolyl phosphate, 5 -Bromo-4-chloro-3-indolyl-3-D-galactopyranoside, 5-Bromo-6-chloro-3-indolyl-β-D-galactopyranoside, 6-Chloro-3-indolyl- β-D-galactopyranosidc, 5-Bromo-4-chloro-3-indolyl-β-D-glucopyranoside, 5-Bromo-4-chloro-3-indolylβ-D-glucuronide;
    2. ii) wherein each test composition comprises a different antimicrobial;
    3. iii) said control composition comprises the same semi solid microbial growth medium and the same chromogenic substrates as said test composition, but does not comprise an antimicrobial as defined in i);
    and wherein said semi solid microbial growth medium comprises tryptophan and one or more inducers; and wherein said platform comprises a control composition.
  2. 2. The platform according to embodiment 1, wherein the antimicrobial in the test composition is selected from the group consisting of aminoglycosides, ansamycins, beta-lactam antibiotics, glycopeptides, macrolides, lincosamides, polypeptides, quinolones, sulphonamides, tetracyclines, cyclic lipopeptides, glycylcyclines, oxazolidinones, diaminopyrimidines, nitrofurans, rifamycins, antibiotic peptides, amphenicols, nitroimidazoles, streptogramins and phosphomycins.
  3. 3. The platform according to embodiment 1 or embodiment 2, wherein said composition is present in each of said compartments in an amount corresponding to from 25-45% of the volume of the compartment.
  4. 4. The platform according to any one of embodiment s 1 to 3, said platform having 7 or more compartments.
  5. 5. The platform according to any one of embodiments 1 to 4, wherein said compartments are separated by dividing members that have been treated to prevent diffusion of an antimicrobial between the compartments.
  6. 6. The platform according to any one of embodiments 1 to 5, wherein said solid support is manufactured from a plastic/polymer substrate, from a glass substrate or from a metal substrate.
  7. 7. The platform according to any one of embodiments 1 to 6, wherein said solid support is a Petri dish, optionally having a diameter of from 80 to 100 mm.
  8. 8. The platform according to any one of embodiments 1 to 7, wherein each of said compartments comprising a test composition has an area of from 4-9 cm2 and/or 25 wherein said compartment comprising a control composition has an area of from 15-25 cm2.
  9. 9. The platform according to any one of embodiments 1 to 8, wherein one or more inducers are present in said test composition and in said control composition in 30 amounts of 0.001 to 1.0 g/l.
  10. 10. The platform according to any one of embodiments 1 to 9, wherein said one or more inducers are selected from the group consisting of 4-minophenyl-3-D-galactopyranoside, isopropyl-J3-D-thiogalactopyranoside,1 -O-Methylf3 -Dgalactopyranoside, Methyl-f3-D-thiogalactopyranosidc, 1 -o-Methyl-cL-D5 galactopyranoside, Isopropyl-f3-D-thioglucopyranoside, 1 -O-methyl-3-D-glucopyranoside, Isopropyl-f3-D-thioglucouronic acid, and 1 -O-Methyl-3-D-glucouronic acid, sodium salt.
  11. 11. The platform according to any one of embodiments 1 to 10, wherein
    1. i) colonies of E. coli, if present, will appear Salmon-red/pink;
    2. ii) colonies of Citrobacter sp. , if present, will appear greenish-blue;
    3. iii) colonies of Kiebsiella, Enterobacter or Citrobacter spp. will appear dark blue;
    4. iv) colonies of Proteus inirabilis/Morganella morganii, if present, will appear light brown;
    5. v) colonies of Proteus vulgaris, if present, will appear dark green;
    6. vi) colonies of Enterococcusfaecalis or Enterococcusfaeciutn, if present, will appear greenish or blue;
    7. vii) colonies of Staphylococcus saprophyticus, if present, will appear red or salmon-red;
    8. viii) colonies of Staphylococcus or Pseudoinonas aeruginosa, if present, will appear white or yellow; and
    9. ix) colonies of candida spp. , if present, will appear white.
  12. 12. The platform according to any one of embodiments 1 to 11, wherein said antimicrobial is selected from the group consisting of Amikacin, Amoxicillin, 5 Amoxicillin-clavulanic acid, Amphothericin-B, Ampicillin, Ampicllin-sulbactam, Apramycin, Azithromycin, Aztreonam, Bacitracin, Benzylpenicillin, Caspofungin, Cefaclor, Cefadroxil, Cefalexin, Cefalothin, Cefazolin, Cefdinir, Cefepimc, Cefixime, Cefmenoxime, Cefoperazone, Cefoperazone-sulbactam, Cefotaxime, Cefoxitin, Cefpirome, Cefpodoxime, Cefpodoxime-clavulanic acid, Cefpodoxime-sulbactam, 10 Cefprozil, Cefquinome, Ceftazidime, Ceftibutin, Ceftiofur, Ceftobiprole, Ceftriaxon, Cefuroxime, Chloramphenicole, Florfenicole, Ciprofloxacin (or other fluoroquinolone), Clarithromycin, Clinafloxacin, Clindamycin, Cloxacillin, Colistin, Cotrimoxazol (Trimthoprimlsulphamethoxazole). Dalbavancin, DalfopristinlQuinopristin, Daptomycin, Dibekacin, Dicloxacillin, Doripenem. Doxycycline, Enrofloxacin, Ertapenem, Erythromycin, Flucloxacillin, Fluconazol, Flucytosin, Fosfomycin, Fusidic acid, Garenoxacin, Gatifloxacin, Gemifloxacin, Gentamicin, Imipenem, Itraconazole, Kanamycin, Ketoconazole, Levofloxacin, Lincomycin, Linezolid, Loracarbef, Mecilinam (amdinocillin), Meropenem, Metronidazole, Meziocillin, Mezlocillin-sulbactam, Minocycline, Moxifloxacin, Mupirocin, Nalidixic acid, Neomycin, Netilmicin, Nitrofurantoin, Norfioxacin, Ofloxacin, Oxacillin, Pefloxacin, Penicillin V, Piperacillin, Piperacillin-sulbactam, Piperacillin-tazobactam, Rifampicin, Roxythromycin, Sparfioxacin, Spectinomycin, Spiramycin, Streptomycin, Sulbactam, Sulfamethoxazole, Teicoplanin, Telavancin, Telithromycin, Temocillin, Tetracyklin, Ticarcillin, Ticarcillinclavulanic acid, Tigecycline, Tobramycin, Trimethoprim, Trovafloxacin, Tylosin, Vancomycin, Virginiamycin, Voriconazole, preferably selected from the group consisting of Amoxicillin, cluvulanic acid/ampicillin, sulbactam, a fluoroquinolone, Sulphamethoxazole, trimethoprim, an oral cephalosporin, Nitrofurantoin and Fosfomycin (fosfomycintrometerole) or is selected from the group consisting of 30 trimethoprim, sulfamethoxazole, ampicillin, nitrofurantoin and mecillinam (amdinocillin) and combinations of these.
  13. 13. The platform according to embodiments 16, wherein said fluoroquinolone is Ciprofloxacin and/or wherein said oral cephalosporin is selected from the group consisting of Cefalexin, cefuroxime, cefadroxil and cefaclor.
  14. 14. The platform according to any one of embodiments 1 to 13, wherein said semi solid microbial growth medium is selected from the group consisting of:
    1. i) A medium composed of: li g/l Hydrolysed casein, 3 gll Peptones, 2 gll Glucose, 3 g/l Sodium Chloride, 1 gll soluble starch, 2 g/l Disodium hydrogen Phosphate, 1 g/l Sodium Acetate, 0.2 g/l Magnesium glycerophosphate, 0.1 g/l Calcium gluconate, 0.001 g/l cobaltous sulphate, 0.001 g/l Cupric sulphate, 0.001 g/l Zinc sulphate, 0.001 gll Ferrous Sulphate, 0.002 gll Magnesium Chloride, 0.001 gil Menadione, 0.001 g/l Cyanobalamin, 0.02 g/l L-Cysteine hydrochloride, 0.02 g/l Tryptophan, 0.003 g/l pyridoxine, 0.003 g/l 15 pantothenate, 0.003 gll nicotinamide, 0.0003 g/l Biotin, 0.00004 g/l Thiamine, 0.01 g/l Adenine, 0.01 g/l Guanine, 0.01 g/l Xanthine, 0.01 g/l Uracil, 8 g/l agar, in distilled water;
    2. ii) A medium composed of: 2 g Na2HPO4 12 H20, 625 g tryptone, 250 20 g starch, 833.6 g Potassium Chloride, 2.5 g detergent, 74.8 g meat broth (Oxoid CM975K), 800g D(+)Glucose-monohydrate, 1.75 g Xanthin, 1.75 g Guanin, 17.5 g Magnesium Sulphate 7 H20, 19.2 g CaC12 2 H20, 2,720 g Agar, 5 N HCl to pH 7.4, solution of vitamins, and 12.5 1 horse blood per 250 liter distilled water;
    3. iii) A medium comprising: 14.5 g/l Peptone, 2 gll glucose, 5.5 g/l salt mix, 1 g/l Soluble starch, 1.5 g/l chromogenic mix, and 8 g/l Agar; and
    4. iv) A medium comprising: 2 g/l Beef extract powder/beef extract, 17.5 g/l 30 Acid Digest of Casein, 1.5 g/l starch and 17 g/l Agar;
  15. 15. The platform according to any one of embodiments 1 to 14, wherein said 3 or more chromogenic substrates comprise 5-Bromo-4-chloro-3-indolyl-3-D-galactopyranoside, 6-Chloro-3-indo1yl-3-D-galactopyranoside, 5-Bromo-4-chloro-3- indolyl -f3-D-glucopyranoside.
  16. 16. The platform according to any one of embodiments 1 to 14, wherein said three or more chromogenic substances is selected from the group consisting of 6-Chloro-3- indolyl-3-D-galactopyranoside, 5 -Bromo-4-chloro-3 -indolyl-3-D-glucopyranoside, 5- Bromo-6-chloro-3-indolyl-phosphate, 5-Bromo-4-chloro-3-indolyl-f3-D-glucuronide and 5-Bromo-4-chloro-3-indolyl-f3-D-galactopyranoside.
  17. 17. The platform according to any one of claims 1 to 14, wherein said test and said control composition comprises 6-Chloro-3-indolyl-3-D-galactopyranoside in combination with 5 -Bromo-4-chloro-3 -indolyl-f3-D-glucopyranoside, 5-Bromo-4-15 chloro-3-indolyl-j3-D-glucuronide, 5-Bromo-4-chloro-3-indolyl-f3-D-galactopyranoside and 5-Bromo-6-chloro-3-indolyl phosphate.
  18. 18. The platform according to any one of embodiments 1 to 14, wherein said three or more chromogenic substances includes the group of 5-bromo-6-chloro-3-indolyl-beta- D-galactopyranoside, 5 -bromo-4-chloro-3 -indolyl-beta-D-glucopyranoside and 6- chloro-3 -indolyl-beta-D-galactopyranoside.
  19. 19. The platform according to any one of embodiments 1 to 14, wherein said three or more chromogenic substances includes the group of 5-Bromo-4-chloro-3-indolyl- beta-D-galactopyranoside, 6-chloro-3-indolyl-beta-D-galactopyra-noside, 5-Bromo-4- chloro-3 -indolyl-beta-D-glucopyranoside and 5 -Bromo-4-chloro-3 -indolyl-beta-Dglucuronide.
  20. 20. The platform according to any one of embodiments 1 to 19, wherein said test composition and said control composition comprise four chromogenic substrates.
  21. 21. The platform according to any one of embodiments 1 to 20, wherein said semi solid microbial growth medium comprises 0.25-3.0 g/l tryptophan is present.
  22. 22. The platform according to any one of embodiments 1 to 21, wherein said solid support is a Petri dish divided into 5 test compartments and 1 control compartment.
  23. 23. A kit comprising a platform according to any one of embodiments 1 to 22.
  24. 24. The kit according to embodiment 23, said kit further comprising a standard illustrating the amount of growth on the platform, which results from contacting said platform with a suspension having a predetermined titre of a microbial reference strain.
  25. 25. The kit according to embodiment 24 wherein said standard is a photographic or printed reproduction of the platform.
  26. 26. The kit according to embodiment 24 wherein said standard has been generated by contacting the platform with a reference strain of E. coli bacteria (optionally E. coli ATCC 29522), and/or a reference strain of Staphylococcus aureus (optionally Staphylococcus aureus ATCC 25913).
  27. 27. The kitaccording to embodiment 25, said kit further comprising one or more separately packaged antimicrobials.
  28. 28. Use of a platform of according to any one of embodiments 1 to 22 in detection and/or diagnosis of infections selected from the group consisting of urinary tract infections, skin and soft tissue infections, infections with Staphylococcus aureus (including methicilin resistant Staphylococcus aureus), infections with meningococcy infection with gonococci, infections with streptococci including infections with pneumococci.

Brief description of the figures



[0029] 

Figure 1 shows two presently preferred embodiments relating to the platform according to the present invention: according to figure 1A the platform comprises an indentation (10) being capable of acting as a receptacle for a sample with a possible a microbial infection or contamination. The indentation is divided into separate compartments (11, 12, 13, 14, 15, and 16), by means of one or more integrated dividing members (17). Each compartment contains a composition comprising a semi-solid microbial growth medium, two or more chromogenicorfluorogenic substances or substrates and an antibiotic; or a composition that comprises a semi-solid microbial growth medium, two or more chromogenic or fluorogenic substances or substrates, but does not comprise an antimicrobial. According to figure 1B the platform comprises multiple indentations (18), each indentation being capable of acting as a receptacle for a sample with a possible microbial infection or contamination. Each indentation contains a composition comprising a semi-solid microbial growth medium, two or more chromogenic or fluorogenic substances or substrates and an antibiotic; or a composition that comprises a semi-solid microbial growth medium, two or more chromogenic or fluorogenic substances or substrates, but does not comprise an antimicrobial.

Figure 2 illustrates one of the presently preferred embodiments of the invention according to which the platform comprises a composition according to the inventions comprising Trimethoprim (19), a composition according to the inventions comprising Sulphamethizole (20) a composition according to the inventions comprising Ampicillin (21), a composition according to the inventions comprising Mecillinam (22) and a composition according to the inventions comprising Nitrofurantoin (23). The platform further comprises a composition according to the invention in which there is no antimicrobial (24). Finally, the figure illustrates yet a preferred embodiment according to which the indentation or compartment containing said composition not comprising an antimicrobial has a size which is twice the size of any of the other indentations or compartments.

Figure 3 shows a standard for determining the quantity or titre of E. coli in a sample analysed using the platform according a preferred embodiment of the invention (illustrated in figure 2). The photos show growth of E. coli at different quantities of bacteria/ml urine. The illustrated E. coli is resistant to sulfamethizole and ampicillin since there is growth in these two antibiotic compartments but it is susceptible to trimethoprim, nitrofurantoin and mecillinam.

Figure 4 shows a standard for determining the different colony types of urinary pathogens in a sample analysed using the platform according a preferred embodiment of the invention. Growth conditions and colours for ordinary urinary tract pathogenic bacteria and their susceptibility/resistance to the five antibiotics in the Flexicult plate.

BacteriumColony sizeColony colourAgar colourSusceptibility = S, or Resistance = R
TrimethoprimSulfamethizoleAmpicillinMecillinamNitrofurantoin
E.coli Large Red/ brownish - S R R S S
Klebsiella spp. Large, Fat Dark blue - S S R S R
Enterobacter spp Large Dark blue - S R R R S
Proteus mirabilis Large (swarm) Light brown/ Brown Brown S S S R S
P.vulgaris Large (swarm) Greenish brown Brown S S R/S R R/S
Morganella spp. Large (swarm) Light brown Brown S S R R R/S
Citrobacter spp. Large Green/greenish blue - S S R S S
P.aureginosa Small Greyish white/ greenish Greenish R R R R R
E.faecalis Small Green/greenish blue Dark ring around colony S R S S R
E.faecium Small Greenish Dark ring around colony S R S S R
S.saprophytic us Small White/Reddish - S S S S S
Candida spp. Large/small White - R R R R R


[0030] The present invention will now be described in more detail in the following.

Detailed description of the invention


Definitions



[0031] Prior to discussing the present invention in further details, the following terms and conventions will first be defined:

Microbial



[0032] In the present context the term "microbial" is to be interpreted broadly as meaning "pertaining to a microbe". The term "microbe" refers collectively to bacteria, fungi, archaea and protists.

Semi-solid growth medium



[0033] The expression "semi-solid growth medium" as used herein refers to a growth medium which allows micro-organisms to form colonies on its surface, such as a medium which has a gel-like appearance or is in the form of a gel, a gel being a colloidal system in which a porous network of interconnected particles spans the volume of a liquid medium. It is further understood that a gel is mostly liquid in composition and thus exhibit densities similar to that of the particular liquid, however have the structural coherence of a solid. Preferably, the semi-solid growth medium as used herein is prepared by adding to a liquid growth medium a sufficient amount of a substance which melts when heated and solidifies when cooled again, such as gelatin or agar. It will be understood that the porous network of interconnected particles in the medium will allow nutrients and antimicrobial to diffuse through the medium to become available to the micro-organisms.

Growth medium



[0034] In the context of the present invention the term "growth medium" refers to a substance in or on which microbes can grow. The term in particular comprises nutrient broth (liquid nutrient medium) or Lysogeny Broth (L-B medium) and agar, which are the most common growth media for microbes.

[0035] Likewise, the term covers liquid medium in which microbes may grow in suspension, as well as semi-solid medium as defined above, allowing microbes to form colonies on its surface. The term "growth medium" also comprises specialized media which are sometimes required for growth of certain microorganism including fastidious organisms, requiring specialized environments due to complex nutritional requirements.

[0036] In general, a growth medium will comprise a carbon source such as glucose or succinate for bacterial growth, water, various salts provide essential elements needed for microbial growth, such as magnesium, nitrogen, phosphorous, and sulfur to allow the bacteria to synthesize protein and nucleic acid, and a source of amino acids and nitrogen (e.g., beef, yeast extract).

[0037] It is also to be understood that the term "growth medium" includes defined media, also known as chemical defined media, as well as undefined media, also known as basal or complex media. A defined medium for microbes will have known quantities of all ingredients, including trace elements and vitamins required by the microbe and especially a defined carbon source such as glucose or glycerol, and a defined nitrogen source such as an ammonium salt or a nitrate. An undefined medium on the contrary, has some complex ingredients, such asyeast extractor casein hydrolysate, which consist of a mixture of many chemical species in unknown proportions.

Chromogenic substrate



[0038] In the context of the present invention the terms "chromogenic substrate", "chromogenic substance" and "chromogen" are used interchangeably referring to a precursor of a biochemical pigment. It is to be understood that, in particular, the chromogen may be a substrate, compound or substance, which when metabolized by a microbe produces a characteristic colour or pigment that is useful as a means of detection and/or identification of said microbe.

Fluorogenic substrate



[0039] The term "fluorogenic substrate" is used interchangeably with "fluorogenic substance", referring to a precursor of a fluorescent compound. A fluorescent compound is a compound in which the molecular absorption of a photon triggers the emission of another photon with a longer wavelength and wherein the energy difference between the absorbed and emitted photons ends up as molecular vibrations or heat. In particular, the absorbed photon is in the ultraviolet range, and the emitted light is in the visible range. Hence detection of fluorescent compounds may typically be performed by exposing them to ultraviolet light (UV light) and then subsequently registering the light (often visible light) which is emitted by the fluorescent compound. It is to be understood that, in particular, the fluorescent substrate may be a substrate, compound or substance, which when metabolized by a microbe emits light that is useful as a means of detection and/or identification of said microbe.

Antimicrobial



[0040] In the present application the terms "antibiotic" and "antimicrobial" are used interchangeably to define a chemical compound that inhibits or abolishes the growth of microorganisms, such as bacteria, fungi or protozoans, that is, a chemical compound with anti-bacterial, anti-fungal, and/or anti-parasitical activity. The term includes antibiotic or antimicrobial compounds produced and isolated from living organisms, for example, the penicillin-class produced by fungi in the genus Penicillium or streptomycin from bacteria of the genus Streptomyces. The terms also include antibiotic or antimicrobial compounds obtained by chemical synthesis, such as sulfonomide drugs.

[0041] The terms in particular include anti-bacterial antibiotics, which are antibiotics that do not have activity against viruses, fungi and other non-bacterial microbes. The anti-bacterial antibiotics include bactericidal antibiotics, which destroy bacteria, and bacteriostatic antibiotics which prevent bacteria from multiplying. The anti-bacterial antibiotics further include "narrow-spectrum" antibiotics which target particular types of bacteria, such as Gram negative or Gram-positive bacteria, and broad spectrum antibiotics which affect a wide range of bacteria. Likewise, the anti-bacterial antibiotics include antibiotics for ingestion as well as antibiotics for intravenous administration which are often used to treat serious infections such as deep-seeded systemic infections, and antibiotics for topical administration. The anti-bacterial antibiotics comprise antibiotics within the following presently recognised classes: Aminoglycosides, Ansamycins, Beta-lactam antibiotics, (including the carbacephem, carbapenems, cephalosporins (first, second, third and fourth generations), monobactams and penicillins) Glycopeptides, Macrolides, lincosamides, Polypeptides, Quinolones, Sulphonamides, Tetracyclines, Cyclic lipopeptides, Glycylcyclines, Oxazolidinones, diaminopyrimidines, Nitrofurans, Rifamycins, antibiotic peptides, amphenicols, nitroimidazoles, streptogramins and phosphomycins.

Aspects and embodiments of the invention



[0042] In a first and broadest aspect, the invention provides a composition comprising a semi-solid microbial growth medium, three or more chromogenic or fluorescent substances or substrates, and an antimicrobial. The fact that a semi-solid microbial growth medium is employed according to the present invention has the advantage that microbial growth can be observed as single colonies on the surface of said medium. The combined use of chromogenic or fluorescent substances or substrates offers the possibility of distinguishing multiple types or species of microorganisms growing on the medium by the colour of the pigment or fluorescence produced in the colonies. Simultaneously, it is possible to determine the sensitivity of each microbial type or species against the antimicrobial present in the composition according to the invention.

[0043] Another advantage of the present invention is that when the platform is used to test urine for infections the number of colonies formed on the semi-solid microbial growth medium directly correlates with the number of bacteria per volume of urine added to the platform. Thereby making it easy to determine the concentration of bacteria in the urine.

[0044] The semi-solid medium also comprises tryptophan. The advantage of including tryptophan is that it enables easy detection of Enterobacteria of the Proteus-Morganella-Providencia and at the same time tryptophan does not interfere with determination of antibiotic susceptibility.

[0045] The semi-solid medium in the composition according to the disclosure comprises a galactose polymer (Agar-agar) or Gelatin.

[0046] Preferably the composition according to the invention comprises a microbial growth medium selected from the group consisting of: Iso-sensitest agar, Danish Blood Agar, Discovery medium and Mueller-Hinton medium. For those media which do not inherently comprise tryptophan it may be a further advantage to include tryptophan in these media. The concentration of tryptophan in the medium may be between 0.25 - 3.0 g/liter.

[0047] The Iso-sensitest agar is currently available from Oxoid and its composition is specified in The Oxoid Manual 1998. The Iso-sensitest agar is composed by 11 g/l Hydrolysed casein, 3 g/l Peptones, 2 g/l Glucose, 3 g/l Sodium Chloride, 1 g/l soluble starch, 2 g/l Disodium hydrogen Phosphate, 1 g/l Sodium Acetate, 0.2 g/l Magnesium glycerophosphate, 0.1 g/l Calcium gluconate, 0.001 g/l cobaltous sulphate, 0.001 g/l Cupric sulphate, 0.001 g/l Zinc sulphate, 0.001 g/l Ferrous Sulphate, 0.002 g/l Magnesium Chloride, 0.001 g/l Menadione, 0.001 g/l Cyanobalamin, 0.02 g/l L-Cysteine hydrochloride, 0.02 g/l Tryptophan, 0.003 g/l pyridoxine, 0.003 g/l pantothenate, 0.003 g/l nicotinamide, 0.0003 g/l Biotin, 0.00004 g/l Thiamine, 0.01 g/l Adenine, 0.01 g/l Guanine, 0.01 g/l Xanthine, 0.01 g/l Uracil, 8 g/l agar, in distilled water.

[0048] As for the Danish Blood Agar this is known by the skilled person to have the following composition: 2 g Na2HPO4 12 H2O, 625 g tryptone, 250 g starch, 833.6 g Potassium Chloride, 2.5 g detergent, 74.8 g meat broth (Oxoid CM975K), 800g D(+)Glucose-monohydrate, 1.75 g Xanthin, 1.75 g Guanin, 17.5 g Magnesium Sulphate 7 H2O, 19.2 g CaCl2 · 2 H2O, 2,720 g Agar, 5 N HCl to pH 7.4, solution of vitamins, and 12.5 I horse blood per 250 liter distilled water.

[0049] The Discovery medium is manufactured by Oxoid (product code CM 1087). According to the manufacturer the medium has the following composition: 14.5 g/l Peptone, 2 g/l glucose, 5.5 g/l salt mix, 1 g/l Soluble starch, 1.5 g/l chromogenic mix, and 8 g/l Agar.

[0050] The Mueller-Hinton medium comprises 2 g/l Beef extract powder/beef extract, 17.5 g/l Acid Digest of Casein, 1.5 g/l starch and 17 g/l Agar.

[0051] It is to be understood that some variation in the amount of each component in said medium will be tolerated; in general a variation of ± 20% will be tolerated. However, as the skilled person will know certain components may be varied to an even larger extent: the amount of agar may be varied substantially such as from 4-25 g/l without significantly altering the performance of the medium. It is thus generally preferred that the medium in the composition according to the disclosure comprises from 4-25g/l of a galactose polymer.

[0052] These media, together with other suitable media are characteristic in offering great reliability when used for determining sensitivity towards antimicrobials. Such reliability is not seen for all media as some media comprise compounds which interfere with the antibiotics and thereby affects the ability to use these media for testing for antibiotic susceptibility. The great reliability is witnessed for instance by fact that on these media:
  1. i) the addition of 16 mg/l ampicillin causes a reduction in the growth of/numbers of colonies formed by reference strain Escherichia coli ATCC 25922 of at least 5 logCFU/ml, as determined after contacting the composition with a suspension containing 105 CFU/ml and incubating the composition for 18-24 hours at 37°C and at ambient atmosphere;
  2. ii) the addition of 32 mg/l nitrofurantoin causes a reduction in the growth of/numbers of colonies formed by reference strain Staphylococcus saprophyticus ATCC 49907 of at least 4 log CFU/ml, as determined after contacting the composition with a suspension containing 105 CFU/ml and incubating the composition for 18-24 hours at 37°C and at ambient atmosphere;
  3. iii) the addition of 700 mg/l sulphamethizole causes a reduction in the growth of/numbers of colonies formed by reference strain Escherichia coli ATCC 25922 of at least 3 log CFU/ml, as determined after contacting the composition with a suspension containing 105 CFU/ml and incubating the composition for 18-24 hours at 37°C and at ambient atmosphere
  4. iv) the addition of 16 mg/l trimethoprim causes a reduction in the growth of/numbers of colonies formed by reference strain Escherichia coli ATCC 25922 of at least 3 log CFU/ml, as determined after contacting the composition with a suspension containing 105 CFU/ml and incubating the composition for 18-24 hours at 37°C and at ambient atmosphere
  5. v) the addition of 16 mg/l mecilinam causes a reduction in the growth of/numbers of colonies formed by reference strain Escherichia coli ATCC 25922 of at least 5 log CFU/ml, as determined after contacting the composition with a suspension containing 105 CFU/ml and incubating the composition for 18-24 hours at 37°C and at ambient atmosphere.


[0053] In further embodiments the microbial growth medium is a selective medium capable of applying a selective pressure to organisms growing on it, such as a medium which is selective for Gram-negative bacteria or for Gram-positive bacteria.

[0054] The semi-solid media may further comprise sulpha inhibitors and/or metal ions as they may affect the antibiotic susceptibility. For example variations in the concentrations Mg2+ or Ca2+, may affect results of aminoglycoside and tetracycline tests with Ps. aeruginosa. Furthermore, excess zinc ions may reduce zone sizes of carbapenems. Excessive cation content will reduce antibiotic activity, whereas low cation content may result in enhanced activity. The Ca2+ and Mg2+ ions may in particular be present in the semi-solid medium in the form of soluble salts. Sulfonamide is inhibited by thymidine, which bacteria can use and therefore grow in spite of sulfonamide. The presence of thymidin-phosphorylase will inhibit thymidine and thus restore the function of sulfonamide. The concentration needed of thymidine-phosphorylase will depend on the concentration of thymidine in the medium.

[0055] Another parameter of the semi-solid media is the concentration of thymidine as this may affect testing of trimethoprim and methicillin-resistant staphylococci. Most agar media contain small amounts of sulphonamide and trimethoprim antagonists that may affect the results of susceptibility testing (especially if blood is not added) with low antibiotic content in the medium. Hence in a particular embodiment Susceptibility test media should contain less than 0.03 mg/l thymidine, otherwise small colonies are seen on the trimethoprim agar. If the medium contains slightly more thymidine than recommended, it is possible to reduce the concentration by adding thymidine-phosphorylase: 0.025 to 0.1 IU enzyme/ml medium or 5% haemolysed horse blood, which contains the same enzyme.

[0056] Whereas the presence of only one antibiotic in the composition according to the invention may be desirable in respect of some antibiotics and for some purposes, the composition according to the invention may also comprise 2 or more antimicrobials, such as 3 or more antimicrobials, such as 4 or more antimicrobials, or such as 5 or more antimicrobials.

[0057] The composition according to the invention may be characterised in that the antimicrobial or, if more than one antimicrobial is present, that at least one of the antimicrobials, such as 2, 3, 4, 5 or more of the antimicrobials or all of the antimicrobials are selected from the group consisting of: Amikacin, Amoxicillin, Amoxicillin-clavulanic acid, Amphothericin-B, Ampicillin, Ampicllin-sulbactam, Apramycin, Azithromycin, Aztreonam, Bacitracin, Benzylpenicillin, Caspofungin, Cefaclor, Cefadroxil, Cefalexin, Cefalothin, Cefazolin, Cefdinir, Cefepime, Cefixime, Cefmenoxime, Cefoperazone, Cefoperazone-sulbactam, Cefotaxime, Cefoxitin, Cefpirome, Cefpodoxime, Cefpodoxime-clavulanic acid, Cefpodoxime-sulbactam, Cefprozil, Cefquinome, Ceftazidime, Ceftibutin, Ceftiofur, Ceftobiprole, Ceftriaxon, Cefuroxime, Chloramphenicole, Florfenicole, Ciprofloxacin, Clarithromycin, Clinafloxacin, Clindamycin, Cloxacillin, Colistin, Cotrimoxazol (Trimthoprim/sulphamethoxazole), Dalbavancin, Dalfopristin/Quinopristin, Daptomycin, Dibekacin, Dicloxacillin, Doripenem, Doxycycline, Enrofloxacin, Ertapenem, Erythromycin, Flucloxacillin, Fluconazol, Flucytosin, Fosfomycin, Fusidic acid, Garenoxacin, Gatifloxacin, Gemifloxacin, Gentamicin, Imipenem, Itraconazole, Kanamycin, Ketoconazole, Levofloxacin, Lincomycin, Linezolid, Loracarbef, Mecillnam (amdinocillin), Meropenem, Metronidazole, Mezlocillin, Mezlocillin-sulbactam, Minocycline, Moxifloxacin, Mupirocin, Nalidixic acid, Neomycin. Netilmicin, Nitrofurantoin, Norfbxacin, Ofloxacin, Oxacillin, Pefloxacin, Penicillin V, Piperacillin, Piperacillin-sulbactam, Piperacillin-tazobactam, Rifampicin, Roxythromycin, Sparfloxacin, Spectinomycin, Spiramycin, Streptomycin, Sulbactam, Sulfamethoxazole, Teicoplanin, Telavancin, Telithromycin, Temocillin, Tetracyklin, Ticarcillin, Ticarcillin-clavulanic acid, Tigecycline, Tobramycin, Trimethoprim, Trovafloxacin, Tylosin, Vancomycin, Virginiamycin and Voriconazole.

[0058] A complete list of antimicrobials which could be incorporated into the composition according to the invention either alone or in combinations is shown in Table 1. The concentrations used in the agar is preferably related to the S/R breakpoint for the particular drug, however, it will be within the capacity of a skilled person to determine the exact concentration needed for a particular purpose.
Table 1. List of antibiotics, IUPAC codes and concentration range for possible concentrations used in agar.
AntibioticChemical name (IUPAC)Range of concentrations covered Mg/l
Amikacin   2S)-4-amino-N-[(2S,3S,4R,5S)-5-amino-2-[(2S,3R,4S,5S,6R)-4-amino-3,5-dihydroxy-6-(hydroxymethyl)oxan-2-yl] oxy-4-[(2R,3R,4S,5R,6R)-6-(aminomethyl)-3,4,5-trihydroxy-oxan-2-yl]oxy-3-hydroxy-cyclohexyl]-2-hydroxy-butanamide 2-128
Amoxicillin   7-[2-amino-2-(4-hydroxyphenyl)-acetyl] amino-3,3-dimethyl-6-oxo-2-thia-5-azabicyclo[3.2.0]heptane -4-carboxylic acid 0,1 - 32
Amoxicillin-clavulanic acid   Amoxicillin:7-[2-amino-2-(4-hydroxyphenyl)-acetyl]amino-3,3-dimethyl-6-oxo-2-thia-5-azabicyclo[3.2.0] heptane -4-carboxylic acid - Clavulanic acid: (2R,5R,Z)-3-(2-hydroxyethylidene)-7-oxo-4-oxa-1-aza-bicyclo[3.2.0] heptane-2-carboxylic acid 0.1 - 32
Amphothericin-B   (1R-1R*,3S*,5R*,6R*,9R*,11R*, 15S*, 16R*,17R*, 18S*,19E, 21E, 23E, 25E, 27E, 29E, 31E, 33R*, 35S*,36R*,37S*))-33-((3-Amino-3,6-dideoxy-beta-D-mannopyranosyl)oxy)-1,3,5,6,9,11,17,37-octahydroxy-15,16,18-trimethyl -13-oxo-14,39-dioxabicyclo(33.3.1)nonatriaconta-19,21,23,25,27,29,31-heptaene-36-carboxylic acid 0.1 - 64
Ampicillin   7-(2-amino-2-phenyl-acetyl)amino-3,3 -dimeihyl-6-oxo-2-thia-5-azabicyclo [3.2.0]heptane-4-carboxylic acid 0.1 - 32
Ampicllin sulbactam   7-(2-amino-2-phenyl-acetyl)amino-3,3 -dimethyl-6-oxo-2-lhia-5-azabicyclo [3.2.0]heptane-4-carboxylic acid (2R,5R)-3,3-dimethyl-4,4,7-trioxo-4λ-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid 0.1 - 32
Apramycin   (2R,3R,4R,5S,6R)-5-amino-2-[((1R,2R, 3R,4R,6R,8R)-8-amino-9[(1R,2S,3R,4R, 6R)-4,6-diamino-2,3-dihydroxy-cyclohexyl]oxy-2-hydroxy-3-methylamino-5,10dioxabicyclo[4.4.0]dec-4-yl)oxy]-6-(hydroxymethyl)oxane-3,4-diol 0.5 - 128
Azithromycin   9-deoxy-9a-aza-9a-methyl-9a-homoerythromycin A 0.03 - 64
Aztreonam   3-[2-(2-azaniumyl-1,3-thiazol-4-yl)-2-(1-hydroxy-2-methyl-1-oxo-propan-2-yl) oxyimino- acetyl]amino-2-methyl-4-oxo-azetidine-1-sulfonate 0.25 - 16
Bacitracin     1-128
Benzylpenicillin   4-Thia-1-azabicyclo(3.2.0)heptane-2-carboxylic acid, 3,3-dimethyl-7-oxo-6-((phenylacetyl)amino)- (2S-(2alpha, 5alpha,6beta))- 0.03-256
Caspofungin   1-[(4R,5S)-5-[(2-aminoethyl)amino] -N2-(10,12-dimethyl-1-oxotetradecyl)-4-hydroxy-L-ornithine]-5-[(3R)-3-hydroxy-L-ornithine] pneumocandin B0 0.1 - 64
Cefaclor   7-[(2-amino-2-phenyl-acetyl)amino]- 3-chloro-8-oxo-5-thia-1-azabicyclo[4.2.0] oct-2-ene-2-carboxylic acid 0.05-256
Cefadroxil   8-[2-amino-2-(4-hydroxyphenyl)-acetyl]a mino-4-methyl-7-oxo-2-thia-6-azabicyclo [4.2.0] oct-4-ene-5-carboxylic acid 1 - 256
Cefalexin   8-(2-amino-2-phenyl-acetyl)amino-4-methyl-7-oxo-2-thia-6-azabicyclo[4.2.0] oct-4-ene-5-carboxylic acid 4-256
Cefalothin   (6R,7R)-3-(acetoxymethyl)-8-oxo-7-(2-(thiophen-2-yl)acetamido)-5-thia-1-aza-bicyclo[4.2.0]oct-2-ene-2-carboxylic acid 1 - 256
Cefazolin   3-[(5-methyl-1,3,4-thiadiazol-2-yl) sulfanylmethyl]-8-oxo-7-([2-(tetrazol-1-yl) acetyl]amino)-5-thia-1-azabicyclo[4.2.0] oct-2-ene-2-carboxylate 2-32
Cefdinir   8-[2-(2-amino-1,3-thiazol-4-yl)-1-hydroxy-2-nitroso-ethenyl]amino-4-ethenyl-7-oxo-2-thia-6-azabicyclo[4,2.0] oct-4-ene-5-carboxylic acid 0.1 - 128
Cefepime   (6R,7R,Z)-7-(2-(2-aminothiazol-4-yl)-2-(methoxyimino)acetamido)-3-((1-methylpyrrolidinium-1-yl)methyl)-8-oxo-5-thia-1-aza-bicyclo[4.2.0]oct-2-ene-2-carboxylate 0.1 - 16
Cefixime   (6R,7R)-7-{[2-(2-amino-1,3-thiazol-4-yl)-2-(carboxy methoxyimino)acetyl]amino}-3-ethenyl-8-oxo-5-thia-1-azabicyclo [4.2.0]oct-2-ene-2-carboxylic acid 0.01 - 256
Cefmenoxime   (6R,7R)-7-{[(2E)-2-(2-amino-1,3-thiazol-4-yl)-2-methoxyimino-acetyl]amino}-3-[(1-methyltetrazol-5-yl)sulfanylmethyl]-8-oxo-5-thia-1-azabicyclo[4.2.0] oct-2-ene-2-carboxylic acid 0.1 - 256
Cefoperazone   (6R,7S)-7-{[2-[(4-ethyl-2,3-dioxo-iperazine-1-carbonyl)amino]-2-(4-hydroxyphenyl)acetyl}amino]-3-[(1-methyltetrazol-5-yl)sulfanylmethyl]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 0.1 - 520
Cefoperazone sulbactam   (6R,7S)-7-{[2-[(4-ethyl-2,3-dioxo-piperazine-1-carbonyl)amino]-2-(4-hydroxyphenyl)acetyl}amino]-3-[(1-methyltetrazol-5-yl)sulfanylmethyl]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (2R,5R)-3,3-dimethyl-4,4,7-trioxo-4λ6-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid 0.1-520
Cefotaxime   (6R,7R,Z)-3-(acetoxymethyl)-7-(2-(2-aminothiazol-4-yl)-2-(methoxyimino) acetamido)-8-oxo-5-thia-1-azabicyclo [4.2.0] oct-2-ene-2-carboxylic acid 0.03 - 32
Cefoxitin   (6S,7R)-4-(carbamoyloxymethyl)-7-methoxy-8-oxo-7-[(2-thiophen-2-ylacetyl) amino]-5-thia-1-zabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 4-64
Cefpirome   1-[[(6R,7R)-7-[[(2Z)-2-(2-amino-4-thiazolyl)-2-(methoxyimino)acetyl] amino]-2-carboxy-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-en-3-yl]methyl]-6,7-dihydro-5H-Cyclopenta[b]pyridinium 0.01 - 256
Cefpodoxime   (6R,7R)-7-{[(2Z)-2-(2-amino-1,3-thiazol-4-yl)-2-methoxyimino-acetyl]amino}-3-(methoxymethyl)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 0,05 - 16
Cefpodoxime clavulanic acid   2R,5R,Z)-3-(2-hydroxyethylidene)-7-oxo-4-oxa-1-aza-bicyclo[3.2.0] heptane-2-carboxylic acid (2R,5R,Z)-3-(2-hydroxyethylidene)-7-oxo-4-oxa-1-aza-bicyclo[3.2.0] heptane-2-carboxylic acid 0.05 - 16
Cefpodoxime sulbactam   (6R,7R)-7-{[(2Z)-2-(2-amino-1,3-thiazol-4-yl)-2-methoxyimino-acetyl]amino}-3-(methoxymethyl)-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (2R,5R)-3,3-dimethyl-4,4,7-trioxo-4λ6-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid 0.05- 16
Cefprozil   8-[2-amino-2-(4-hydroxyphenyl)-acetyl] amino-7-oxo-4-prop-1-enyl-2-thia-6-azabicyclo[4.2.0]oct-4-ene-5-carboxylic acid 1 - 512
Cefquinome   Pharmacotherapeutic group: Cephalosporins and related substancesATCvet code: QJ51DA92 1 - 512
Ceftazidime   (6R, 7R,Z)-7-(2-(2-aminothiazol-4-yl)-2-(2-carboxypropan-2-yloxyimino) acetamido)-8-oxo-3-pyridinium-1-ylmethyl)-5-thia-1-aza-bicyclo[4.2.0] oct-2-ene-2-carboxylate 0.1 - 32
Ceftibutin   (+)-(6R,7R)-7-[(Z)-2-(2-Amino-4-thiazolyl)-4-carboxycroton-amido]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid 0.5-32
Ceftiofur   6r-[6a,7b(z)]]-7-[[(2-amino-4-thiazolyl) (methoxyimino)acetyl]amino]-3-[[2-furanylcarbonyl)thio]methyl]-8-oxo-5-thia-1-azabicyclo [4.2.0] oct-2-ene-2-carboxylic acid; (6R-7R)-7-[[2-amino-4-thiazolyl)-z-methoxyimino)acetyl]amino]-3-[[(2-furanylcarbonyl)thio]methyl]-8-oxo-5-thia-1-azabicyclo [4.2.0]oct-2-ene-carboxylic acid 0.1-128
Ceftobiprole   Ceftobiprole medocaril 0.03-512
Ceftriaxon   (6R,7R,Z)-7-(2-(2-aminothiazol-4-yl)-2-(methoxyimino)acetamido)-3-((6-hydroxy-2-methyl-5-oxo-2,5-dihydro-1,2,4-triazin-3-ylthio)methyl)-8-oxo-5-thia-1-aza-bicyclo[4.2.0]oct-2-ene-2-carboxylic acid 0.01 - 16
Cefuroxime   4-(carbamoyloxymethyl)-8-[2-(2-furyl)-2-methoxyimino-acetyl]amino-7-oxo-2-thia-6-aza bicyclo[4.2.0]oct-4-en e-5-carboxylic acid 0.1 - 64
Chloramphenicole   2,2-dichlor-N-[(aR,bR)-b-hydroxy-a-hydroxymethyl-4-nitrophenethyl] acetamide 0.5 - 128
Florfenicole     2-128
Ciprofloxacin   1-cyclopropyl-6-fluoro-4-oxo-7-piperazin-1-yl-quinoline-3-carboxylic acid 0.01 - 32
Clarithromycin   6-(4-dimethylamino-3-hydroxy-6-methyl-etrahydropyran-2-yl)oxy-14-ethyl-12,13-dihydroxy-4-(5-hydroxy-4-methoxy-4,6-dimethyl-tetrahydropyran-2-yl)oxy-7-methoxy-3,5,7,9,11,13-hexamethyl-1-oxacyclotetradecane-2,10-dione 0.03 - 64
Clinafloxacin   7-(3-amino-1-pyrrolidinyl)-8-chloro-1-cyclopro pyl-6-fluoro-1,4-dihydro-4-oxo-3-Quinolinecarboxylic acid, 0.01 - 64
Clindamycin   (2S,4R)-N-((1R)-2-chloro-1-((3R,4R,5S, 6R)-3,4,5-trihydroxy-6-(methylthio)-tetrahydro-2H-pyran-2-yl) propyl)-1-methyl-4-propylpyrrolidine-2-carboxamide 0.05 - 32
Cloxacillin   (2S,5R,6R)-6-{[3-(2-chlorophenyl)-5-methyl-oxazole-4-carbonyl]amino}-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0] heptane-2-carboxylic acid 0.5 - 128
Colistin   colistin sulfate and colistimethate sodium (colistin methanesulphonate sodium, colistin sulfomethate sodium 0.5- 16
Cotrimoxazol (Trimthoprim/sul phamethoxazole)   Trimethoprim:5-(3,4,5-trimethoxybenzyl)pyrimidine-2,4-diamine/Sulphamethoxazole:4-amino-N-(5-methylisoxazol-3-yl)-benzenesulfonamide 1-128
Dalbavancin   5,31-dichloro-38-de(methoxycarbonyl)-7-demethyl-19-deoxy-56-O-[2-deoxy-2-[(10-methyl-1-oxoundecyl)amino]-b-D-glucopyranuronosyl]-38-[[[3-(dimethylamino)propyl]amino] carbonyl]-42-O-a-D-mannopyranosyl-1-N15-methyl-Ristomycin A aglycone 0.05-16
Dalfopristin/Quin opristin quinupristine N-[(6R,9S,10R,13S,15aS,18R,22S,24aS)-22-[p-(dimethylamino)benzyl]-6-ethyldocosahydro- 0.25-256
  10,23-dimethyl-5,8,12,15,17,21,24-heptaoxo-13-phenyl-18-[["(3S)-3-quinuclidinylthio]methyl]-12H-pyrido[2,1-f]pyrrolo-[2,1-/] [1,4,7,10,13,16] oxapentaazacyclononadecin-9-yl]-3-hydroxypicolinamide  
  dalfopristin (3R,4R,5E,10E,12E,14S,26R,26aS)-26-[[2-(diethylamino)ethyl]sulfonyl]-8,9,14,15,24,25,26,26a-octahydro-14-hydroxy-3-isopropyl-4,12-dimethyl-3H-21,18-nitrilo-1H,22H-pyrrolo[2,1-c][1,8,4,19]-dioxadiazacyclotetracosine-1,7,16,22(4H,17H)-tetrone  
Daptomycin N-decanoyl-L-tryptophyl-L-asparaginyl-L-aspartyl-L-threonylglycyl-L-ornithyl-L-aspartyl-D-alanyl-L-aspartylglycyl-D-seryl-threo-3-methyl-L-glutamyl-3-anthraniloyl-L-alanine[egr]1-lactone 0.1 - 32
Dibekacin D-Streptamine, O-3-amino-3-deoxy-alpha-D-glucopyranosyl-(1-6)-O-(2,6-diamino-2,3,4,6-tetradeoxy-alpha-D-erythro-hexopyranosyl-(1-4))-2-deoxy 1-128
Dicloxacillin (2S,5R,6R)-6-{[3-(2,6-dichlorophenyl)-5-methyl-oxazole-4-carbonyl]amino}-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0] heptane-2-carboxylic acid 0.2 - 128
Doripenem carbapenem

0.01 - 256
Doxycycline (2-(amino-hydroxy-methylidene)-4-dimethylamino-5,10,11,12a-tetrahydroxy-6-methyl-4a,5,5a,6-tetrahydro-4H-tetracene-1,3,12-trione 0.1-16
Enrofloxacin 1-Cyclopropyl-7-(4-ethylpiperazin-1-yl)-6-fluor-4-oxo- 1,4-dihydrochinolin- 3-carboxylic acid 0.01 - 32
Ertapenem   3-[5-[(3-carboxyphenyl) carbamoyl] pyrrolidin-3-yl] sulfanyl-7-(1-hydroxyethyl)- 2-methyl-6-oxo-5-azabicyclo[3.2.0]hept-3-ene-4-carboxylic acid 0.01 - 128
Erythromycin   6-(4-dimethylamino-3-hydroxy-6-methyl-oxan-2-yl)oxy-14-ethyl-7,12,13-trihydroxy-4-(5-hydroxy-4-methoxy-4,6-dimethyl-oxan-2-yl)oxy-3,5,7,9,11,13-hexamethyl-1-oxacyclotetradecane-2,10-dione 0.03 - 64
Flucloxacillin   6-((S)-3-(2-chloro-6-fluorophenyl)-5-methylisoxazole-4-carboxamido)-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo [3.2.0] heptane-2-carboxylic acid 0.5 - 128
Fluconazol   2-(2,4-difluorophenyl)-1,3-bis(1H-1,2,4-triazol-1-yl)propan-2-ol 0.25-512
Flucytosin   4-amino-5-fluoropyrimidin-2(1H)-one 1 - 512
Fosfomycin   [(2R,3S)-3-methyloxiran-2-yl]phosphonic acid 1 - 64
Fusidic acid   2-(16-acetyloxy-3,11-dihydroxy-4,8,10,14-tetramethyl- 2,3,4, 5,6,7, 9,11,12,13,15,16-dodecahydro-1H-cyclopenta [a]phenanthren-17-ylidene)-6-methyl-hept-5-enoic acid 0.05 - 16
Garenoxacin   fluoroquinolone 0.01 - 32
Gatifloxacin   1-cyclopropyl-6-fluoro-8-methoxy-7-(3-methylpiperazin-1-yl)-4-oxo-quinoline-3-carboxylic aci 0.01 - 32
Gemifloxacin   7-[(4Z)-3-(aminomethyl)-4-methoxyimino-pyrrolidin-1-yl]-1-cyclopropyl-6-fluoro-4-oxo-1,8-naphthyridine-3-carboxylic acid 0.01 - 32
Gentamicin   2-[4,6-diamino-3-[3-amino-6-(1-methylaminoethyl)tetrahydropyran-2-yl] oxy-2-hydroxy-cyclohexoxy]-5-methyl-4-methylamino-tetrahydropyran-3,5-diol 0.5 - 128
Imipenem   (5R,6S)-3-[2-(aminomethylideneamino) ethylsulfanyl]-6-(1-hydroxyethyl)-7-oxo-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid 0.01-128
Itraconazole   4-[4-[4-[4-[[2-(2,4-dichlorophenyl)-2-(1H-1,2,4-triazol-1-ylmethyl)-1,3-dioxolan-4-yl]methoxy]phenyl]piperazin-1-yl]phenyl]-2-(1-methylpropyl)-2,4-dihydro-1,2,4-triazol- 3-one 0.1 - 256
Kanamycin   2-(aminomethyl)-6-[4,6-diamino-3-[4-amino-3,5-dihydroxy-6-(hydroxymethyl) tetrahydropyran-2-yl]oxy-2-hydroxy-cyclohexoxy]-tetrahydropyran-3,4,5-triol 2-256
Ketoconazole   1-[4-[4-[[(2S,4R)-2-(2,4-dichlorophenyl)-2-(imidazol-1-ylmethyl)-1,3-dioxolan-4-yl] methoxy]phenyl]piperazin-1-yl]ethanone 0.03-512
Levofloxacin   (-)-(S)-9-fluoro-2,3-dihydro-3-methyl-10-(4-methyl-1-piperazinyl)-7-oxo-7H-pyrido [1,2,3-de]-1,4-benzoxazine-6-carboxylic acid 0.01 - 32
Lincomycin  

1 - 32
Linezolid   N-[[3-(3-fluoro-4-morpholinophenyl)-2-oxooxazolidin-5-yl]methyl]acetamide 1 - 32
Loracarbef   8-(2-amino-2-phenyl-acetyl)amino-4-chloro-7-oxo-6-azabicyclo[4.2.0] oct-4-ene-5-carboxylic acid 0.1 - 128
Mecillnam (amdinocillin)   6-Amidinopenicillanic acid derivatives 6-[[(hexahydro-1H-azepin-1-yl) methylene]amino]-3,3-dimethyl-7-oxo-, (2S,5R,6R)- 4-Thia-1-azabicyclo[3.2.0] heptane-2-carboxylic acid, 0.25 - 254
Meropenem   3-[5-(dimethylcarbamoyl) pyrrolidin-2-yl] sulfanyl-6-(1-hydroxyethyl)-4-methyl-7-oxo-1-azabicyclo[3.2.0] hept-2-ene-2-carboxylic acid 0.03 - 256
Metronidazole   2-(2-methyl-5-nitro-1H-imidazol-1-yl) ethanol 1 - 512
Mezlocillin   (2S,5R,6R)-3,3-dimethyl-6-[[(2R)-2-[(3-methylsulfonyl-2-oxo-imidazolidine-1-carbonyl)amino]-2-phenyl-acetyl]amino]-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid 0.5-512
Mezlocillin-sulbactam   Mezlocillin: (2S,5R,6R)-3,3-dimethyl-6-[[(2R)-2-[(3-methylsulfonyl-2-oxo-imidazolidine-1-carbonyl)amino]-2-phenyl-acetyl]amino]-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid Sulbactam:(2R,5R)-3,3-dimethyl-4,4,7-trioxo-4λ6-thia-1-azabicyclo[3.2.0] heptane-2-carboxylic acid 0.5-512
Minocycline   2-(amino-hydroxy-methylidene)-4,7-bis (dimethylamino)-10,11,12a-trihydroxy-4a,5,5a,6-tetrahydro-4H-tetracene-1,3,12-trione (synonym 7-Dimethylamino-6-demethyl-6-deoxytetracycline) 0.1 - 256
Moxifloxacin,,   1-cyclopropyl-7-[(1S,6S)-2,8-diazabicyclo [4.3.0]non-8-yl]-6-fluoro-8-methoxy-4-oxo-quinoline-3-carboxylic acid 0.01 - 32
Mupirocin   9-[[(2Z)-3-methyl-1-oxo-4-[(2S,3R,4R, 5S)-tetrahydro-3,4-dihydroxy-5-[[(2S, 3S)-3-[(1S,2S)-2-hydroxy-1-methylpropyl]-2-oxiranyl]methyl]-2H-pyran-2-yl]-2-buten-1-yl]oxy]-Nonanoic acid 0.1 - 256
Nalidixic acid   1-ethyl-7-methyl-4-oxo-[1,8] naphthyridine-3-carboxylic acid 4 - 1024
Neomycin   C23H46N6O13 (MW 614.644 q/mol) 0.5-256
Netilmicin.   O-3-deoxy-4-C-methyl-3-(methylamino)-b-L-arabinopyranosyl-(1->6)-O-[2,6-diamino-2,3,4,6-tetradeoxy-a-D-glycero-hex-4-enopyranosyl-(1->4)-2-deoxy-N1-methyl-D-Streptamine, C21H41N5O7 (MW 475.58 g/mol) 0.5-512
Nitrofurantoin   1-[(5-nitro-2-furyl)methylideneamino] imidazolidine-2,4-dione 1 - 1028
Norfloxacin   1-ethyl-6-fluoro-4-oxo-7-piperazin-1-yl-1 H-quinoline- 3-carboxylic acid 0.5 - 128
Ofloxacin   (+/-)-9-fluoro-2, 3-dihydro-3-methyl-10-(4-methyl-1-piperazinyl)-7-oxo-7H-pyrido [1,2,3-de]-1,4-benzoxazine-6-carboxylic acid 0.01 - 32
Oxacillin   (2R,5R,6S)-3,3-dimethyl-6-[(5-methyl-3-phenyl-1,2-oxazole-4-carbonyl)amino]-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid 0.25-128
Pefloxacin   1-ethyl-6-fluoro-7-(4-methylpiperazin-1-yl)-4-oxo-quinoline-3-carboxylic acid 0.01 - 32
Penicillin V   Phenoxymethylpenicillin 0.1 - 256
Piperacillin   (2S,5R,6R)-6-{[(2R)-2-[(4-ethyl-2,3-dioxo-piperazine-1-carbonyl)amino]-2-phenyl-acetyl]amino}-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid 2 - 1024
Piperacillin-sulbactam   Piperacillin: (2S,5R,6R)-6-{[(2R)-2-[(4-ethyl-2,3-dioxo-piperazine-1-carbonyl) amino]-2-phenyl-acetyl]amino}-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0] heptane-2-carboxylic acid Sulbactam: (2R,5R)-3,3-dimethyl-4,4,7-trioxo-4λ6-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid 2 - 1024
Piperacillin-tazobactam   Piperacillin: as above; Tazobactam: (2S, 3S,5R)-3-methyl-4,4,7-trioxo-3-(triazol-1-ylmethyl)-4$|^{6}-thia-1-azabicyclo[3.2.0] heptane-2-carboxylic acid 2 - 1024
Rifampicin   5,6,9,17,19,21-Hexahydroxy-23-methoxy-2,4,12,16,18,20,22-heptamethyl-8-[N-(4-methyl-1-piperazinyl)formimidoyl]-2,7-(epoxypentadeca[1,11,13] trienimino)-naphtho[2,1-b]furan-1,11 (2H)-dione 21-acetate 0.005 - 128
Roxythromycin   Erythromycin-[O-[(2-methoxyethoxy)-methyl]oxime] 0.03 - 64
Sparfloxacin   5-amino-1-cyclopropyl-7-[(3R,5S)3,5-dimethylpiperazin-1-yl]-6,8-difluoro-4-oxo-quinoline-3-carboxylic acid 0.01 - 128
Spectinomycin   (2R,4aR,5aR,6S,7S,8R,9S,9aR,10aS)-4a,7,9-trihydroxy-2-methyl-6,8-bis (methylamino) decahydro-4H-pyrano[2,3-b][1,4]benzodioxin-4-one 32-512
Spiramycin   C43H74N2O14, (MW 843.053 g/mol) 0.05 - 8
Streptomycin   5-(2,4-diguanidino-3,5,6-trihydroxy-cyclohexoxy)- 4-[4,5-dihydroxy-6-(hydroxymethyl)-3-methylamino-tetrahydropyran-2-yl] oxy-3-hydroxy-2-methyl-tetrahydrofuran-3-carbaldehyde 2-1024
Sulbactam   (2R,5R)-3,3-dimethyl-4,4,7-trioxo-4λ6-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid 2 - 1024
Sulfamethoxazol e   4-amino-N-(5-methylisoxazol-3-yl)-benzenesulfonamide 16-2050
Teicoplanin   Glycopeptide - no |UPAC-code 0.05 - 128
Telavancin   N3"-[2-(decylamino)ethyl]-29-[[(phosphonomethyl)amino] methyl]-Vancomycin 0.25- 128
Telithromycin   (1S,2R,5R,7R,8R,9S,11R,13R,14R)-8-[(2S,3R,4S,6R)-4-dimethylamino-3-hydroxy-6-methyl-oxan-2-yl]oxy-2-ethyl-9-methoxy-1,5,7,9,11,13-hexamethyl-15-[4-(4-pyridin-3-ylimidazol-1-yl)butyl]-3,17-dioxa-15-azabicyclo[12.3.0] heptadecane-4,6,12,16-tetrone 0.01 - 64
Temocillin   (2S,SR,6S)-6-[(Carboxy-3-thienylacetyl) amino]- 6-methoxy-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0] heptane-2-carboxylic acid, 0.1 - 1024
Tetracyklin   2-(amino-hydroxy-methylidene)-4-dimethylamino-6,10,11,12a-tetrahydroxy-6-methyl-4,4a,5,5a-tetrahydroletracene-1,3,12-trione OR 4-(dimethylamino)-1,4,4a,5,5a,6,11,12a-octahydro-3,6,10,12,12a-pentahydroxy-1,11dioxo-naphihacene-2carboxamide 0.1 - 32
Ticarcillin   2S,5R,6R)-6-[[(2R)-2-carboxy-2-thiophen-3-yl-acetyl]amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid 2-512
Ticarcillin-clavulanic acid   Ticarcillin:2S,5R,6R)-6-[[(2R)-2-carboxy-2-thiophen-3-yl-acetyl]amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo [3.2.0]heptane-2-carboxylic acid-Clavulanic acid: (2R,5R,Z)-3-(2-hydroxyethylidene)-7-oxo-4-oxa-1-azabicyclo[3.2.0]heptane-2-carboxylic acid 2-512
Tigecycline   N-[(5aR,6aS,7S,9Z,10aS)-9-(amino-hydroxy-methylidene)-4,7-bis (dimethylamino)-1,10a,12-trihydroxy-8,10,11-trioxo-5a,6,6a,7-tetrahydro-5H-tetracen-2-yl]-2-(tert-butylamino) acetamide 0.1 - 256
Tobramycin   4-amino-2-[4,6-diamino-3- [3-amino-6-(aminomethyl) -5-hydroxy-etrahydropyran-2-yl]oxy- 2-hydroxy-cyclohexoxy]-6-(hydroxymethyl) tetrahydropyran-3,5-diol 0.5 - 128
Trimethoprim   5-(3,4,5-trimethoxybenzyl)pyrimidine-2,4-diamine 0.25 - 64
Trovafloxacin   7-(6-amino-3-azabicyclo[3.1.0]hex-3-yl)-1-(2,4-difluorophenyl)- 6-fluoro-4-oxo-[1,8] naphthyridine-3-carboxylic acid 0.1 - 32
Tylosin   macrolide 0.1 - 32
Vancomycin   C66H75Cl2N9O24 0.5 - 1024
Virginiamycin   C71H84N10O17 0.25 - 256
Voriconazole   2-(2,4-difluorophenyl)-3- (5-fluoropyrimidin-4-yl)-1-(1H-1,2,4-triazol-1-yl) butan-2-ol 0.004 - 128


[0059] The prevalence of antibiotic resistance has been and is continuing to increase in all bacteria including E. coli, and it is now becoming increasingly necessary to combine urine culture with a susceptibility test even in primary care.

[0060] In a recent study surveying resistance rates in E. coli from North America (USA and Canada)8, of the 1142 E. coli collected, 75.5% (862) were collected from the USA and 280 (24.5%) were from Canada. Overall, resistance to ampicillin was 37.7%, followed by SMX/TMP (21.3%), nitrofurantoin (1.1%), ciprofloxacin (5.5%) and levofloxacin (5.1 %). This study reported higher rates of antibiotic resistance in US versus Canadian outpatient urinary isolates of E. coli and demonstrated the continuing evolution of resistance to antimicrobial agents. In a European survey from 2002-3, the Eco-Sens study9, antibiotic resistance rates were determined in E. coli from a range of European countries. The results are shown in table 2.
Table 2. Antimicrobial resistance of E. coli in European countries in the Eco-Sens study9.
  Antimicrobial agenta (resistance in per cent)      
CountrynAMPAMCMECCFRTMPSULSXTNALCIPNITFOFGEN
Austria 126 17.5 2.4 1.6 0.8 9.5 25.4 9.5 2.4 0 0.8 0 0.8
Belgium 137 30.7 2.9 1.5 0.7 13.9 32.8 14.6 6.6 2.9 0.7 0.7 0.7
Canada 166 29.5 3.6 1.2 1.8 10.8 25.3 12.0 0.6 0 1.2 0.6 0.6
Denmark 85 22.4 1.2 1.2 1.2 10.6 21.2 8.2 3.5 0 1.2 1.2 0
Finland 182 19.8 4.9 0.5 1.6 5.5 15.4 4.9 1.6 0.5 0.5 1.1 0.5
France 199 27.6 1.5 1.5 1.0 15.6 31.7 15.1 3.5 2.0 1.0 1.0 0
Germany 138 29.0 2.2 2.2 1.4 22.5 34.8 21.0 3.6 2.2 0.7 0 0.7
Greece 132 22.0 0.8 0.8 3.0 13.6 19.7 11.4 6.8 1.5 3.0 1.5 0.8
Ireland 154 44.8 5.8 0.6 0.6 22.1 40.3 20.8 1.9 0 0 1.3 0.6
Luxembourg 24 41.7 0 0 0 16.7 25.0 16.7 8.3 4.2 4.2 0 0
The                          
Netherlands 195 28.7 2.6 1.5 4.6 12.3 25.6 10.3 5.1 2.1 1.0 0.5 0.5
Norway 168 23.8 3.6 0 2.4 13.1 25.0 11.3   0 0 1.2 0
Portugal 86 45.3 9.3 2.3 2.3 26.7 44.2 26.7 11.6 5.8 5.8 0 3.5
Spain 191 53.9 4.2 1.0 3.1 25.1 48.7 25.7 26.7 14.7 4.2 0.5 4.7
Sweden 193 15.5 5.7 1.6 5.2 8.8 16.6 8.3 2.6 0 0 0.5 0
Switzerland 122 27.0 2.5 0 0.8 18.9 31.1 18.9 6.6 2.5 0.8 0.8 3.3
United                          
Kingdom 180 37.2 2.8 1.7 1.7 13.3 31.7 12.2 2.2 0.6 0 0 0
Total 2478 29.8 3.4 1.2 2.1 14.8 29.1 14.1 5.4 2.3 1.2 0.7 1.0
aAMP, ampicillin; AMC, co-amoxiclav; MEC, mecillinam; CFR, cefadroxil; TMP, trimethoprim; SUL, sulfamethoxazole; SXT, trimethoprim/sulfamethoxazole; NAL, nalidixic acid; CIP, ciprofloxacin; NIT, nitrofurantoin; FOF, fosfomycin; GEN, gentamicin.


[0061] As can be deducted from these data, the resistance rates for commonly used antibiotics such as ampicillin, sulfonamides and trimethoprim are now so high (i.e. 15 - 45%), that these drugs cannot be used empirically without a susceptibility test. The use of fluoroquinolones, which are effective broad-spectrum drugs in some countries still covering most urinary pathogens, is directly related to development of resistance, which on even short-term scale is problematic due to the importance of these drugs for treating serious infections in hospitals. Since the resistance rates vary between geographic areas different antimicrobials may be preferred depending on the purpose for which the composition according to the invention is used.

[0062] In a presently preferred embodiment compositions according to the invention comprises one or more antimicrobials, wherein the antimicrobial/at least one of the antimicrobials is selected from the group consisting of: aminoglycosides, piperacillin/tazobactam, carbapenems, cephalosporins, glycopeptides, lipopeptides and antimicrobial peptides (e.g. polymycin or colistin) and combinations of these.

[0063] In an equally preferred embodiment, compositions according to the invention are developed for use in diagnosing urinary tract infections in Denmark and the Scandinavian countries. In these compositions the antimicrobial or antimicrobials are preferably selected from the group consisting of: Ampicillin/amoxicillin, Sulfonamide, Trimethoprim, Nitro-furantoin, Mecillinam, and Ciprofloxacin (or other fluoroquinolone), and combinations of these.

[0064] For the same purpose it may be preferred that the antimicrobial is selected from the group consisting of: trimethoprim, sulfamethizole, ampicillin, nitrofurantoin and mecillinam (amdinocillin) and combinations of these.

[0065] For similar reason compositions have been developed for use in UTI diagnostics in Europe outside Scandinavia. In these compositions the antimicrobial or antimicrobials is/are preferably selected from the group consisting of: Amoxicillin, cluvulanic acid/ampicillin, sulbactam, Ciprofloxacin (or other fluoroquinolone), Sulphamethoxazole, trimethoprim, Cefalexin/cefuroxime/cefadroxil (or other oral cephalosporin), Nitrofurantoin and Fosfomycin (fosfomycin-trometerole) and combinations of these.

[0066] For the purpose of diagnosing UTI in the United Stated it may be preferred that antimicrobial or antimicrobials is/are selected from the group consisting of: Amoxicillin, cluvulanic acid/ampicillin, sulbactam, Ciprofloxacin (or other fluoroquinolone), Sulphamethoxazole, trimethoprim, Cefalexinicefuroxime/cefadroxil/cefaclor (or other oral cephalosporin), Nitrofurantoin and Fosfomycin (fosfomycin-trometerole) and combinations of these.

[0067] It is to be understood in particular that amoxicillin may be included either alone or in combination with cluvulanic acid. Similarly, ampicillin may be included alone or in combination with sulbactam, and Sulphamethoxazole amy be used alone or in combination with trimethoprim.

[0068] For purposes other than diagnosing UTIs the antimicrobial or antimicrobials may also be selected from the group consisting of: Amoxicillin/clavulanic acid (or sulbactam), Phenoxymethyl-penicillin, Cephalosporin (e.g. cefuroxime axetil, cefalexin), Ciprofloxacin, levofloxacin, ofloxacin, fleroxacin, Sulphametoxazole and trimethoprim (optionally in combination as used for oral treatment), Tetracyclin (doxycycline or any other tetracyclin group), Chloramphenicol, Fosfomycin, Macrolide/clindamycin and Rifampicin, and combinations of these.

[0069] If the test would be used in a hospital laboratory, antibiotics for intravenous use might be considered also, including antimicrobials selected from the group consisting of: Aminoglycosides, Cephalosporins (cefotaxime, ceftazidime, cefepime, cefpodoxime, ceftriaxone and others), Piperacillin/tazobactam, Carbapenems, cephalosporins, Glycopeptides, Lipopeptides, Linezolid and antimicrobial peptides (e.g. polymycin or colistin) and combination of these.

Important additives to the selective medium



[0070] Sulfa inhibitors and Metal ions: Variation in Mg and Ca, will affect results of aminoglycoside and tetracycline tests with Ps. aeruginosa. Excess zinc ions may reduce zone sizes of carbapenems. Excessive cation content will reduce antibiotic acitivty, whereas low cation content may result in enhanced activity Ca and Mg should be available in the medium in the form of soluble salts.

[0071] The thymidine content of the medium affects testing of trimethoprim and methicillin-resistant staphylococci. Most agar media contain small amounts of sulphonamide and trimethoprim antagonists that may affect the results of susceptibility testing (especially if blood is not added) with low antibiotic content in the medium. Susceptibility test media should contain less than 0.03 mg/l thymidine, otherwise small colonies are seen on the trimethoprim agar. If the medium contains slightly more thymidine than recommended, it is possible to reduce the concentration by adding thymidinephosphorylase: 0.025 to 0.1 IU enzyme/ml medium or 5% haemolysed horse blood, which contains the same enzyme.

[0072] Prior to adding the antibiotic to the semi-solid media it may be an advantage to dissolve the antibiotic.

[0073] In particular solvents used to dissolve the antimicrobial agent(s) may include one or more of the following:

Water

Physiological saline 0.85%

Phosphate buffer, pH 6.0, 0.1 mol/L

Phosphate buffer, pH 8.0, 0.1 mol/L

Phosphate buffer, pH 7.2, 0.01 mol/L

Saturated solution sodium bicarbonate

Aqueous sodium bicarbonate 0.1%

Ethanol 95%

Methanol

Glacial acetic acid

Dimethylformamide (DMF)

Dimethyl sulfoxide (DMSO)

DMSO 1/10 vol

DMSO plus glacial acetic acid

½ volume of water plus drops of 1 mol/L Sodium hydroxide

½ volume of water plus drops of 0.1 mol/L Sodium hydroxide

½ volume of water plus drops of 2.5 mol/L Sodium hydroxide

Hydrogen chloride acid 0.04 mol/L

Polysorbate-80 (0.002%) in water



[0074] Stability of the antimicrobial in the semi-solid media may depend on the pH-value. The pH-value must not exceed certain limits as this will influence the antimicrobial effect. The stability of Ampicillin and Mecillinam has been improved by adjusting the pH-value of the semi-solid media to 6.0.

[0075] According to the invention the three or more chromogenic substances present in the composition is preferably selected from the group consisting of: 5-Bromo-4-chloro-3-indolyl phosphate, 5-Bromo-6-chloro-3-indolyl phosphate p-toluidine, 3,3'-(3,3'-dimethoxy-4,4'-biphenylylene)-bis-2-(p-nitrophenyl)-5-phenyl-2H-tetrazolium chloride, 5-Bromo-4-chloro-3-indolyl-β-D-galactopyranoside, 5-Bromo-6-chloro-3-indolyl-β-D-galactopyranoside, 5-Bromo-3-indolyl-β-D-galactopyranoside, 6-Bromo-2-naphthyl-β-D-galactopyranoside, 6-Chloro-3-indolyl- β -D-galactopyranoside, 6-Bromo-3-indolyl- β-D-galactopyranoside, 1-Methyl-3-indolyl-β-D-galactopyranoside, o-Nitrophenyl- β-D-galactopyranoside, p-Nitro-phenyl-β-D-galactopyranoside, 3,4-cyclohexenoesculetin-β-D-galactoside, 8-hydroxychinoline-β-D-galactoside, 5-Bromo-4-chloro-3-indolyl-α-D-galactopyranoside, 5-Bromo-4-chloro-3-indolyl-β-D-glucopyranoside, 5-Bromo-4-chloro-3- indolyl-β-D-glucuronide, 5- Bromo-6-chloro-3-indolyl-β-D-glucuronide, 8-hydroxyquinoline-β-D-glucuronide, 2,2'-Azinobis(3-ethylbenzothiazoline-6-sulfonic acid), 4-Chloro-1-naphthol, 3,3'-Diaminobenzidine tetrahydrochloride, o-Phenylenediamine, 3,3',5,5'-Tetramethylbenzidine, 4-[2-(4-octanoyloxy-3,5-dimethoxyphenyl)-vinyl]-quinolinium-1-(pro-pan-3-yl-carboxylic-acid)-bromide, 5-Bromo-6-chloro-3-indolyl-caprylate and 5-bromo-4-chloro-3-indoxyl-myoinositol1-phosphate and combinations of these.

[0076] As the skilled person will know, the components listed above are normally used in an amount of 0.001-1.0 g/l.

[0077] In table 3 below, these chromogenes are listed according to the enzyme for which they serve as substrate:
Table 3. Chromogenic enzyme substrates
EnzymeSubstrateConcentrationColor
Alkaline Phosphatase 5-Bromo-4-chloro-3-indolyl phosphate 0.001 to 1.0 g/l Blue
5-Bromo-6-chloro-3-indolyl phosphate p- toluidine 0.001 to 1.0 g/l Red
3,3'-(3,3'-dimethoxy-4,4'-biphenylylene)-bis-2-(p-nitrophenyl)-5-phenyl-2H-tetrazolium chloride 0.001 to 1.0 g/l Red
β-galactosidase 5-Bromo-4-chloro-3-indolyl-β-D-galactopyranoside 0.001 to 1.0 g/l Blue
5-Bromo-6-chloro-3-indolyl-β-D-galactopyranoside 0.001 to 1.0 g/l Magenta
5-Bromo-3-indolyl-β-D- galactopyranoside 0.001 to 1.0 g/l Blue
6-Bromo-2-naphthyl-β-D-galactopyranoside 0.001 to 1.0 g/l -
6-Chloro-3-indolyl-β-D- galactopyranoside 0.001 to 1.0 g/l Salmon
6-Bromo-3-indolyl-β-D- galactopyranoside 0.001 to 1.0 g/l -
1-Methyl-3-indolyl-β-D- galactopyranoside 0.001 to 1.0 g/l Green
o-Nitrophenyl-β-D-galactopyranoside 0.001 to 1.0 g/l Yellow
p-Nitrophenyl-β-D-galactopyranoside 0.001 to 1.0 g/l Yellow
3,4-cyclohexenoesculetin-β-D-galactoside 0.001 to 1.0 g/l Brown/Black
8-hydroxychinoline-β-D-galactoside 0.001 to 1.0 g/l -
α-galactosidase 5-Bromo-4-chloro-3-indolyl-α-D-galactopyranoside 0.001 to 1.0 g/l Blue
β-glucosidase 5-Bromo-4-chloro-3-indolyl-β-D-glucopyranoside 0.001 to 1.0 g/l Blue
β-glucuronidase 5-Bromo-4-chloro-3-indolyl-β-D-glucuronide 0.001 to 1.0 g/l Blue
5-Bromo-6-chloro-3-indolyl-β-D-glucuronide 0.001 to 1.0 g/l Magenta
8-hydroxyquinoline-β-D-glucuronide 0.001 to 1.0 g/l Black
Peroxidase 2,2'-Azino-bis(3-ethylbenzothiazoline-6- sulfonic acid) 0.001 to 1.0 g/l Green
4-Chloro-1-naphthol 0.001 to 1.0 g/l Blue
3,3'-Diaminobenzidine tetrahydrochloride 0.001 to 1.0 g/l Brown
o-Phenylenediamine 0.001 to 1.0 g/l -
3,3',5,5'-Tetramethylbenzidine 0.001 to 1.0 g/l Blue
Esterase 4-[2-(4-octanoyloxy-3,5-dimethoxyphenyl)-vinyl]-quinolinium1-(propan-3-yl-carboxylic-acid)-bromide 0.001 to 1.0 g/l Burgundy
5-Bromo-6-chloro-3-indolyl-caprylate 0.001 to 1.0 g/l Magenta
Phosphatidylinositol phospholipase C 5-bromo-4-chloro-3-indoxyl-myoinositol-1-phosphate 0.001 to 1.0 g/l Blue


[0078] For the purpose of detecting or diagnosing urinary tract infections at least one of said three or more chromogenic substance is preferably selected from the group consisting of: 5-Bromo-4-chloro-3-indolyl-β-D-galactopyranoside, 6-Chloro-3-indolyl-β-D-galactopyranoside, 5-Bromo-4-chloro-3-indolyl-β-D-glucopyranoside, 5-Bromo-4-chloro-3-indolylβ-D-glucuronide, and 5-Bromo-6-chloro-3-indolyl-phosphate or combinations of these. It may be preferred that the composition comprises 3, 4, or 5 chromogenic substances, all selected from this group.

[0079] The fluorogenic substance of the composition according to the invention may be selected from the group consisting of: 4-Methylumbelliferyl phosphate, 4-Methylumbelliferyl-β-D-galactopyranoside, 4-Methylumbelliferyl β-D-lactopyranoside and 4-Methylumbelliferyl b-D-glucuronide or combinations of these.

[0080] As described previously the semi-solid media comprises tryptophan, such as L-tryptophan. L-tryptophan present in the semi-solid media will be degraded by bacteria expressing tryptophanase to indole, pyruvate and ammonia. Enterobacteria of the Proteus-Morganella-Providencia group forms a pigment from L-tryptophan colouring the agar surrounding the colony reddish brown to brown. Bacteria belonging to the Proteus group: Proteus mirabilis, P. vulgaris, P. penneri and P. myxofaciens. Bacteria belonging to the Morganella group: Morganella morganii. Bacteria belonging to the Providencia group: Providencia rettgeri, P. stuartii, P. alcalifaciens, P. rustigianii and P. heimbachae. Proteus spp., Morganella spp. and Providencia spp. producing tryptophan deaminase will deaminate the amino acid L-Tryptophan to indolepyruvic acid and ammonia. Indolepyruvic acid, ferric ions and hydrazine compounds in the culture media produces a reddish to brown colour in the media. Bacteria degrading L-tryptophan to indole can further be detected by addition of a paper disc to the lid of the petri dish. The disc prepared with Ehrlich-Böhme's reagent (Paradimethylaminobenzaldehyd dissolved in ethylalkohol /HCl) will give a red colour with indol vapour formed during incubation.

[0081] The semi-solid media of the present invention also comprises on or more inducers which are substances that positively regulate the expression of one or more genes. Induction is common in metabolic pathways that result in the catabolism of a substance and the inducer is normally the substrate for the pathway. The effects of inducers can be critical to the function and sensitivity of many routine assays. Adding inducers to the medium can increase the coloration. Common inducers are shown in table 4 below:
Table 4 Inducers
EnzymeInducersConcentration
B-galactosidase 4-Aminophenyl-β-D-galactopyranoside 0.001 to 1.0 g/l
Isopropyl-β-D-thiogalactopyranoside
1-O-Methyl-β-D-galactopyranoside
Methyl-β-D-thiogalactopyranoside
A-galactosidase 1-O-Methyl-α-D-galactopyranoside 0.001 to 1.0 g/l
B-glucosidase Isopropyl-β-D-thioglucopyranoside 1-O-Methyl-β-D-glucopyranoside 0.001 to 1.0 g/l
B-glucuronidase Isopropyl-β-D-thioglucuronic acid, sodium salt 1-O-Methyl-β-D-glucuronic acid, sodium 0.001 to 1.0 g/l


[0082] As the skilled person will know, the components listed above are normally used in an amount of 0.001-1.0 g/l.

[0083] The composition according to the invention may comprise 3 or more chromogenic or fluorescent substrates, such as 4 or more chromogenic or fluorescent substrates, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more or 10 or more chromogenic or fluorescent substrates. In particular the composition according to the invention may comprise 3, 4, 5, 6, 7, 8, 9, 10 or 11 chromogenic or fluorescent substrates.

[0084] A particular advantage of the composition according to the present invention is its great stability. In preferred embodiments the composition is stable for a period of up to 12 months, such as for a period of up to 11 months, a period of up to 11 months, up to 9 months, up to 8 months, up to 7 months, up to 6 months, up to 5 months, up to 4 months, up to 3 months, up to 2 months or such as for a period of up to 1 month when stored at a temperature of 4°C or less.

[0085] A further aspect of the invention provides a platform for diagnosing, detecting and/or characterising a microbial infection or contamination comprising a composition as defined above.

[0086] In particular, the platform according to the invention may comprise a multiple of compositions as defined above, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 50, or 90 compositions, wherein at least 2 of the compositions comprises different antimicrobials, such as wherein at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, 14, 15, 20, 50 of the compositions comprises different antimicrobials or wherein each composition comprises a different antimicrobial.

[0087] In further embodiments the platform according to the invention comprises
  1. i) a test composition that comprises a semi-solid microbial growth medium as defined above, and three or more chromogenic or fluorogenic substances (or substrates), such as a chromogenic or fluorogenic substance (or sub-strate) as defined above, and one or more antibiotics, such as one or more antibiotics as defined above;
  2. ii) a control composition that comprises a semi-solid microbial growth medium as defined above, and three or more chromogenic or fluorogenic substances (or substrates), as defined above, but does not comprise an antimicrobial, such as an antimicrobial as defined above.


[0088] The control composition which does not comprise any antimicrobial, or which at least does not comprise any of the antimicrobials defined above (i.e. an antibiotic which is different from the antibiotic(s) present in the test composition), is useful for determining the number of micro-organisms as well as the species and/or groups of micro-organisms in a given sample. The test composition that comprises one or more antibiotics is useful for determining the susceptibility of these species and/or groups of micro-organisms to any antibiotic present in the test composition.

[0089] For the sake of convenience the platform according to the invention comprises a solid support, rendering the compositions according to the invention easy to handle.

[0090] In particular, the platform according to the invention may be based on a solid support, wherein said solid support comprises:
  1. i) an indentation (10) being capable of acting as a receptacle for a sample with a possible microbial infection or contamination, said indentation being divided into three or more separate compartments (11 - 16), each compartment containing a test composition as defined above or a control composition as defined above, that is a medium which does not comprise any of the antimicrobials mentioned above or which does not comprise any antimicrobials at all; and one or more integrated dividing members (17) for dividing said indentation into said separate compartments; or
  2. ii) multiple indentations (18), each indentation being capable of acting as a receptacle for a sample with a possible microbial infection or contamination, each containing a test composition as defined above or a control composition as defined above, wherein said platform comprises a control composition.


[0091] The numbers refer to the illustrations in figure 1, which are representative examples of platforms according to the present invention.

[0092] According to a preferred embodiment of the invention, the platform comprises test compositions containing antimicrobials selected from the group consisting of: Ampicillin/amoxicillin, Sulfonamide, Trimethoprim, Nitrofurantoin, Mecillinam, Ciprofloxacin (or other fluoroquinolone). According to this embodiment the platform may comprise one test composition comprising Ampicillin/amoxicillin, one comprising Sulfonamide, one comprising Trimethoprim, one comprising Nitrofurantoin, one comprising Mecillinam, and/or one comprising Ciprofloxacin (or other fluoroquinolone). Such a platform may be preferred for the purpose of diagnosing UTIs in Scandinavia.

[0093] Likewise the platform may comprise one test composition comprising rimethoprim, one comprising sulfamethizole, one comprising ampicillin, one comprising nitrofurantoin and one comprising mecillinam (amdinocillin).

[0094] For the purpose of diagnosing UTIs in the non-Scandinavian European countries a platform may be preferred comprising test compositions containing Amoxicillin, cluvulanic acid/ampicillin, sulbactam, Ciprofloxacin (or other fluor-oquinolone), Sulphamethoxazole, trimethoprim, Cefalexin/cefuroxime/cefadroxil (or other oral cephalosporin), Nitrofurantoin and Fosfomycin (fosfomycin-trometerole).

[0095] Finally, for the purpose of diagnosing UTIs in North America/the United States a platform may be preferred comprising test compositions containing Amoxicillin, cluvulanic acid/ampicillin, sulbactam, Ciprofloxacin (or other fluoroquinolone), Sulphamethoxazole, trimethoprim, Cefalexin/cefuroxime/cefadroxil/cefaclor (or other oral cephalosporin), Nitrofurantoin and Fosfomycin (fosfomycin-trometerole)

[0096] In order to serve its purpose, the indentation or the said multiple indentations of the platform must have a volume sufficiently large to accommodate a suitable volume of sample. In particular embodiments indentation or the said multiple indentations has/have a depth of from 1-40 mm, such as from 1-35 mm, from 1-30 mm, from 1-25 mm, from 5-40 mm, from 5-35 mm, from 5-30 mm, from 5-25 mm, from 10-40 mm, from 10-35 mm, from 10-30 mm, from 10-25 mm, from 15-40 mm, from 15-35 mm, from 15-30 mm or such as from 25-25 mm.

[0097] For urinary tract infections as for many other infections particular policies apply concerning the use of diagnostics in general practice. General Practitioner treat the majority of patients experiencing UTI's and the antibiotic consumption for treatment of UTI amounts to about 25% of the total antibiotic use outside hospitals - and since 90% of the total amount of antibiotics used in a western society is used in the community, the antibiotic use for UTI is substantial. For the patient there is ample evidence that it is of benefit to receive the right diagnosis including bacterial susceptibility as early as possible. There are therefore many good reasons for the GP to perform the diagnosis of UTI at the clinic: It shortens the time to treatment by decreasing the transport time to a distant, central laboratory, and avoiding the transport improves the quality of the culture. Since the GP has little experience in microbiology, the method of culture should be easy to inoculate and read, and the susceptibility test should be independent of the inoculum and also easy to read, i.e. the GP or his assistant has little time to measure an inhibition zone and translate it into a susceptibility group. At the same time, the culture system should be easy to transport to the laboratory, if more sophisticated bacterial diagnostic workup is needed. Other important factors: The test should be able to measure and read counts down to 103 CFU/ml, and it should be able to discern between pathogens and contaminants. All these factors are incorporated into platform according to the invention.

[0098] In order to meet these requirements the platform according to of the invention preferably has compartments with test compositions each of which has an area of from 4-9 cm2, such as from 5-8 cm2, from 6.5-7.5 cm2. Likewise it is preferred that the compartment or compartments with control composition has an area of from 15-25 cm2, such as from 17-23 cm2, or from 19.5-21.5 cm2.

[0099] The currently most preferred version of the platform has indentations or compartments with test compositions having an area of 6.93 cm2 and a volume of 6.24 cm3, and a compartment or an indentation with a control composition having an area of 20.78 cm2 and a volume of 18.7 cm3. This particular design is adapted to the use in detection, diagnosing and/characterising urinary tract infections based on the experience that these areas and volumes are sufficient if the platform is to be used for detection of microbial infections, including urinary tract infections, in which the micro-organisms/bacteria are present in amounts of 103 cfu/ml of sample/urine.

[0100] The said test and control compositions may be present in the respective indentations or compartments in an amount corresponding to from 5-75% of the volume of the indentation or compartment, such as from 10-75%, from 10-65%, from 10-55% from 10-45%, from 10-35%, from 20-75%, from 20-65%, from 20-55% from 20-45%, from 20-35% such as from 25-75%, from 25-65%, from 25-55% from 25-45%, from 25-35%, or such as in an amount corresponding to from 25-35% of the volume of the indentation or compartment.

[0101] In the platform according to the invention said indentation may, according to certain embodiments be divided into 3 or more compartments, such as 4 or more, 5 or more, 6 or more, 7 or more 8 or more, 9 or more, 10 or more, 15 or more, 20, or more, 40 or more, 60 or more or 90 or more compartments.

[0102] Further the platform according to the invention may have 3 or more indentations, such as 4 or more, 5 or more, 6 or more, 7 or more 8 or more, 9 or more, 10 or more, 15 or more, 20, or more, 40 or more, 60 or more or 90 or more indentations.

[0103] According to certain promising embodiments, the platform according to the invention comprises dividing members for dividing said indentation into separate compartments wherein said dividing members have been treated to prevent diffusion of an antimicrobial between the compartments. It is contemplated that surface tension present in the compositions according to the invention when they are poured into the indentations or wells of the platform is causing diffusion of antimicrobials between the compartments if great care is not taken. In order to avoid this said one or more dividing members may be polished so as to have a smooth surface. Other means for reducing the surface tension and diffusion af antimicrobials involve the addition ofadetergentsuch as Tween to the compositions according to the invention.

[0104] Whereas many useful designs may be contemplated the said solid support is preferably in the form of a closed or open container, such as a tray, a test tube, a bottle, a multi-well plate, a microtiter plate, a stick (dipstick) or a slide.

[0105] The solid support may in particular be manufactured from a plastic/polymer substrate, such as a polyvinyl chloride substrate, a polyethylene substrate, a polypropylene substrate, a polycarbonate substrate, an acrylonitrile butadiene styrene substrate, a polymethyl metacrylate substrate or a polystyrene substrate, from a glass substrate or from a metal substrate.

[0106] According to presently preferred embodiments the platform according to the invention comprises a solid support, which is in the form of a Petri dish, such as a Petri dish having a diameter of from 50 to 150 mm, from 60-130 mm, from 70-110 mm or from 80-100 mm. According to one particular embodiment, the support is in the form of a 90 mm Petri dish.

[0107] The platform of the invention may be adapted to use in particular in the detection and/or diagnosis of infections selected from the group consisting of urinary tract infections, skin and soft tissue infections, infections with S. aureus (including methicillin resistant S. aureus), infections with meningococci, infections with gonococci and infections with streptococci.

[0108] Other tests may be incorporated into the platform, for instance glued to the internal surface of a lid covering said support, to the internal side of the support or onto a central part of the support, for instance a part where said dividing members coalesce. For use in such tests the platform may comprise an enzyme, such as a lecocyte esterase. When incorporated into the platform the lecocyte esterase enables the concomitant diagnosis of leucocyturia (could also be done for nitrite test). In accordance herewith the enzyme may be contained within a separate or an integrated member of said support. For example as previously described bacteria degrading L-tryptophan to indole can further be detected by addition of a paper disc to the lid of the petri dish. The disc prepared with Ehrlich-Böhme's reagent (Paradimethyl-aminobenzaldehyd dissolved in ethylalkohol /HCl) will give a red colour with indol vapour formed during incubation.

[0109] Another aspect of the invention provides kit comprising a composition as described above.

[0110] A related aspect provides a kit comprising a platform as described above.

[0111] Such kits may in a particular be for diagnosing, detecting and/or characterising a microbial infection or contamination.

[0112] According to either aspect the kit may further comprise a standard illustrating the amount of growth on a platform as defined above, which results from contacting said platform with a suspension having a predetermined titre of a microbial reference strain. A representative standard is illustrated in figure 3 of the present application.

[0113] The kit may also comprise a standard illustrating the development of biochemical pigment and/or fluorescence on a platform as defined above, which results from growth on the platform of one or more microbial reference strains. A representative standard is illustrated in figure 4 of the present application.

[0114] For the sake of convenience the standard is preferably a photographic or printed reproduction of a platform as defined above.

[0115] According to a particular embodiment the said standard illustrating the amount of growth on the platform has been generated by contacting a platform according to the invention with a reference strain of E. coli bacteria, such as E. coli (ATCC 29522) bacteria, and/or a reference strain of Staphylococcus aureus, such as Staphylococcus aureus, ATCC 25913.

[0116] According to a further particular embodiment the said standard illustrating the development of biochemical pigment and/or fluorescence on a platform has been generated by contacting a platform according to the invention with one or more reference strains selected from the group consisting of: E. coli, such as E. coli strain ATCC 25922, K. pneumoniae, such as K. pneumoniae strain ATCC 10031, E. cloacae such as E. cloacae strain ATCC 13047, P. mirabilis such as P. mirabilis strain ATCC 12453, P. vulgaris such as P. vulgaris strain ATCC 13315, M. morganii such as ATCC 25830, C. freundii such as C. freundii strain 8090, P. aeruginosa such as P. aeruginosa strain ATCC 27853, E. faecalis such as E. faecalis strain ATCC 29212, E. faecium such as E. faecium strain ATCC 35667, S. saprophyticus such as S. saprophyticus strain 49907, C. albicans, such as C. albicans strain ATCC 200955.

[0117] A standard which is useful in the context of the present invention is shown in figure 4.

[0118] The standards illustrating the amount of growth may be generated by a process comprising:
  1. i) providing a suspension at a density of 0.5 McFarland/1+E08 CFU/mL of said reference strain or reference strains in 0.9% brine;
  2. ii) providing a series of 10-fold dilutions of said suspension in i), down to a density of +E03 CFU/mL;
  3. iii) contacting each of said dilutions in ii) with a platform according to the invention for 2-3 seconds and subsequently decanting the solutions;
  4. iv) incubating each platform over night at 35°C at ambient atmosphere.


[0119] According to further embodiments, the kit comprises one or more separately packaged antimicrobials. Such one or more antibiotics may be included for on-site addition by the user to one or more compartments of the platform.

[0120] According to a currently preferred embodiment, the kit according to the invention is a kit for point-of-care diagnosis and susceptibility testing of urinary tract pathogens. The kit was developed for urine culture in the primary health care setting. It is designed preferably as a nine cm Petri dish divided into six compartments: One larger compartment for quantitative analysis and five smaller compartments for susceptibility testing. The agar in each small compartment contains one of five antimicrobials (for Denmark and Scandinavia preferably: trimethoprim, sulfamethizole, ampicillin, nitrofurantoin and mecillinam (amdinocillin)) at a concentration adjusted to the breakpoint, such that growth in these compartments indicates resistance, and no growth indicates susceptibility. The Petri dish preferably has higher sides, approximately 2.48 cm, than the usual dish, i.e. ca. 1.48 cm. This allows a larger amount, maximum 100 ml, of urine to be poured onto the dish for inoculation.

[0121] The agar in the plate is preferably composed of:

Chromogenic substances,

Isosensitest agar,

Antibiotics mixed in agar in five compartments:

Sulfamethizole

Trimethoprim

Ampicillin

Nitrofurantoin

Mecillinam



[0122] An illustration of this embodiment is found in figure 2.

[0123] Inoculation occurs by pouring urine (5-10 ml is sufficient) over the plate and rotating the plate with one hand such that the fluid covers all six field. Hereafter the urine is poured off; presence of urine for 2-3 seconds is sufficient for inoculation. The plate is now incubated, preferably at 35-37° C for 18-24 h (room temperature can be sufficient for most urinary pathogens, but growth will be slower). Reading of the plate falls in three phases:
1) evaluation of quantity of growth: If any growth is present it is compared with a picture scheme showing the different quantities of CFU's for E. coli at 103, 104, 105, 106, and 107 CFU/ml.
2) The type of growth may now be evaluated, by comparing again with pictures/colour schemes showing the different types of urinary pathogens. Also the number of different colony types is noted, and the quantities of different colony types according to the above scheme. The bacterial families/species can be discerned by the colour and the colony type as illustrated in Table 5:
Table 5: Colour codes for colonies and agar of the different urinary pathogens on the Flexicult plate:
Species/groupColourAgar Colony (diameter in mm)
E. coli   Salmon-red/pink
Citrobacter sp   Greenish-blue
Klebsiella/Enterobacter/Citrobacter spp   Dark-blue
Proteus mirabilis/Morganella morganii Brown Light brown
Proteus vulgaris Brown Dark green
Enterococcus faecalis Green-blue Greenish/blue (1-2 mm)
Enterococcus faecium Green-blue Greenish/blue (½-1 mm)
Staphylococcus saprophyticus   Red/salmon-red
Staphylococcus, other   White/yellow
Pseudomonas aeruginosa Colour-less (white/yellow - greenish after 24-36 h incubation
Candida spp.   White (hyphae after 48 h)

3) Reading the susceptibility of the bacterial growth: Each bacterium (if more than one) is read individually: The growth in each of the antibiotic fields is compared with the growth on the control agar. If there is any growth on an antibiotic agar, and the amount of growth is similar to the control agar, then the bacterium is considered to be resistant to the antibiotic in the agar. If there is no growth (or substantially less than the control agar), then the bacterium is considered to be susceptible to the antibiotic. If there is more than no growth and less than substantially less growth, it is necessary to consider which antibiotic and which bacteria: For sulfamethizole and trimethoprim, which are bacteriostatic drugs, scanty minute colonies may cover the entire agar, but the growth is substantially reduced as compared to the control agar: this is recorded as susceptible to the antibiotic in question. When nitrofurantoin and mecillinam are used as antibiotics, a few single colonies may be seen of the same type as the bacterium on the control agar: These are not considered to be resistant mutants but "persisters", i.e. if retested they will appear susceptible. This is commonly seen also by disc diffusion especially for mecillinam.
4) The susceptibility/resistance of the different bacteria can also be used to diagnose the different pathogenic species, since some bacteria are "born" resistant to some antibiotics: i.e. they are inherently resistant to one or more antibiotics in contrast to resistance bacteria acquire because they are exposed to antibiotics. These resistance-"pattern" can be seen in Table 6.
Table 6: Normal susceptibility/resistance of common urinary pathogens. Most of the bacteria can become resistant to the antibiotics in question by mutation or transfer of genes harbouring resistance-mechanisms
BakterieSusceptible = S, or Resistant = R
Trime ToprimSulfonamideAmpicillinNitrofurantoinMecillinam
E. coli S S S S S
Citrobacter S S R S S
Klebsiella S S R S/R S
Entero-bacter S S R S/R S
P. mirabilis S S S S/R S/R
P. vulgaris S S R R R
 (Bakterie Susceptible = 5, or Resistant = R
Trime ToprimSulfonamideAmpicillinNitrofurantoinMecillinam
Morganella S S R R R
S.saprophyticus S S S S S/R
E.faecalis S R S S R
E.faecium S R S S R
S. agalactiae S S S S R
Pseud. Aeruginosa R R R R R
Candida sp. R R R R R


[0124] Yet another aspect of the invention provides the use of a composition according to the invention and as defined hereinbefore in the detection and/or diagnosis of infections selected from the group consisting of urinary tract infections, skin and soft tissue infections, infections with S. aureus (including methicillin resistant S. aureus), infections with meningococci, Infections with gonococci, infections with streptococci including infections with pneumococci. In particular the invention provides the use of a said composition in the detection and/or identification of an uropathogenic microorganism.

[0125] Still another aspect provides a composition according to the invention and as defined hereinbefore, for use in detection and/or diagnosis of infections selected from the group consisting of urinary tract infections, skin and soft tissue infections, infections with S. aureus (including methicillin resistant S. aureus), infections with meningococci, infections with gonococci, infections with streptococci including infections with pneumococci.

[0126] The invention further provides a method of diagnosing, detecting and/or characterising a microbial infection or contamination comprising the steps of:
  1. i) providing a sample with a possible microbial infection or contamination; and
  2. ii) contacting said sample with a platform as defined above.


[0127] According to the invention is also provided a method of diagnosing, detecting and/or characterising a microbial infection or contamination comprising the steps of:
  1. i) providing a sample with a possible a microbial infection or contamination; and
  2. ii) contacting said sample with a test composition (such as two or more, 3 or more, 4 or more, 5 or more, 6 or more, or 7 or more test compositions) as defined herein before and with a control composition as also defined hereinbefore.


[0128] As it will be understood, the test composition, comprises a semi-solid microbial growth medium, three or more chromogenic or fluorogenic substances or substrates, and an antimicrobial. The control composition as explained above, comprises said semi-solid growth medium and said three or more chromogenic or fluorogenic substances (or substrates) but does not comprise any antimicrobial or comprises an antimicrobial different from the antimicrobial(s) present in the test composition.

[0129] The sample which is analysed in the method and by use of the composition, platform or kit according to the invention may be selected from the group consisting of: a sample of body fluid, a faecal sample, a mucous sample, a skin sample, a soft tissue sample, a sample of a food or food ingredient, a sample of an animal feed and a microbial (e.g. bacterial) pure culture.

[0130] In preferred embodiments in particular embodiments relating to diagnosing, detecting and/or characterising urinary tract infections, the sample is a urine sample.

[0131] The method so provided may further comprise a step of incubating said platform for a period of 10 hours or more, such as of 11 hours or more, 12 hours or more, 13 hours or more, 14 hours or more, 15 hours or more, 16 hours or more, 17 hours or more, or 18 hours or more preferably at a temperature of 15-39°C, and preferably at ambient atmosphere.

[0132] The method may further be characterised by comprising a step of visually inspecting the platform for microbial growth. This step of visually inspecting the compositions for microbial growth may in particular comprise:
  1. i) evaluating a quantity of any microbial growth on said control composition, optionally by reference to a standard showing different quantities of colony forming units; and
  2. ii) evaluating a type of any microbial growth on said control composition comprising said semi-solid growth medium and said three or more chromogenic or fluorogenic substances (or substrates) but not comprising any antimicrobial/ antimicrobial different from the antimicrobial(s) present in the test composition, optionally by reference to a standard (such as a picture and/or colour scheme) illustrating growth of different groups or strains of microorganisms; and
  3. iii) determining antimicrobial susceptibility of said microbial growth by comparing the amount of growth on said test composition (such as two or more, 3 or more, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 50 or 90 or more test compositions) with the amount of growth on said control composition.


[0133] Suitable standards showing different quantities of colony forming units and standards illustrating growth of different groups or strains of micro-organisms are described above.

[0134] The step of visually inspecting the compositions for microbial growth may further comprise determining the number of different colony types and optionally the quantity of colonies of each type on said test composition (such as on two or more, 3 or more, 4 or more, 5 or more, 6 or more or 7 or more compositions) as defined hereinbefore or on said control composition.

[0135] The method may further comprise determining whether a micro-organism in said sample is susceptible to an antimicrobial in said test composition, susceptibility being indicated by:
  1. i) microbial growth being absent on said test composition or on one or more of said test compositions, while being present on said control composition; or
  2. ii) microbial growth being present on said test composition or on one or more of said test compositions as well as on said control composition, the number of colonies/area on said test or on one or more of said test composition being at least 100 fold less than the number/area on said control composition.


[0136] In a further aspect the invention provides a method of manufacturing the composition according to the invention, comprising the step of combining a semi-solid microbial growth medium, three or more chromogenic or fluorogenic substances (or substrates), and an antimicrobial.

[0137] Likewise the invention disclosed a method of manufacturing a platform according to the invention, comprising the step of combining a semi-solid microbial growth medium, three or more chromogenic or fluorogenic substances (or substrates), and an antimicrobial.

[0138] Finally the invention provide a method of manufacturing the diagnostic kit according to the invention, comprising the step of combining a semi-solid microbial growth medium, three or more chromogenic or fluorogenic substances (or substrates), and an antimicrobial.

[0139] It should be noted that embodiments and features described in the context of one of the aspects of the present invention also apply to the other aspects of the invention.

[0140] All patent and non-patent references cited in the present application, are hereby incorporated by reference in their entirety.

[0141] The invention will now be described in further details in the following non-limiting examples.

Examples


Example 1: Preparation of a platform based on a 6-compartment Petri dish (Flexicult)



[0142] Iso-sensitest agar was produced and combined with 1.5 g/l of a mixture of chromogenic substrate comprising 5-Bromo-4-chloro-3-indolyl-β-D-galactopyranoside, 6-Chloro-3-indolyl-β-D-galactopyranoside, 5-Bromo-4-chloro-3-indolyl-β-D-glucopyranoside, 5-Bromo-4-chloro-3-indolyl-β-D-glucuronide, 5-Bromo-6-chloro-3-indolyl-phosphate.

[0143] Alternatively Discovery agar (Oxoid, CM 1087) was used.

[0144] Antibiotic solutions were produced: trimethoprime, sulphamethizole, ampillicine, nitrofurantoine, and mecilliname.

[0145] Preparation of hot plates and temperature regulation via PT-100 sensor:
  1. 1. The bottles with Iso-sensitest/Discovery agar were melted at 105°C for 60 min. After melting, the bottles were placed in a water bath at 56°C for approx. 60 min.
  2. 2. The equipment for the filling of Flexicult was prepared: Variomag (Multitherm Stirring Block Thermostat and Telemodul 40 CT were interconnected and then connected to a socket. Telemodul 40 CT is the unit that controls both the temperature and the stirring speed of the heat block. The temperature was adjusted by means of a PT-100 sensor, which was installed at the back of the Telemodul 40 CT.
  3. 3. The bottles with Iso-sensitest/Discovery agar were taken from the water bath and placed on the Multitherm Stirring Block; each bottle was provided with a sterile magnet.
  4. 4. The cable for the PT-100 sensor was installed at the back of the Telemodul 40 CT, the contact point was singed and is then thoroughly wiped with a sterile cloth wetted with surgical spirits. The contact point was then placed in the flask with mecilliname. Telemodul 40 CT now adjusted the temperature in the agar to the required set point - approx. 50.0°C ± 2.0°C. The speed of the magnetic stirrerwas adjusted in relation to the contents of the flask (start): 235/min.

Preparation of dispensers:



[0146] The dispensers were prepared (Fill-Master 251 and Fill-Master 311). To the Fill-Master 251 was attached one 4 mm sterile silicone tube and to the Fill-Master 311 were attached five 2 mm sterile silicone tubes. The respective dispensers were set at the right tube size (2 mm and 4 mm, respectively).

[0147] The filling volume was set so that the filled volume + the weight of the dish are in the range of 31 - 33 g. This was checked regularly.

Addition of the antibiotic solutions:



[0148] Note: The 5 antibiotic solutions should not be added to the agar until the temperature is below 54.0°C. The temperature of the agar was checked in the display of the Telemodul CT 40.

Trimethoprime (16 µg/mL):



[0149] The trimethoprime stock solution was added with a graduated pipette to the Iso-sensitest/Discovery agar. The solution was thoroughly mixed on the Multitherm hot plate. The suction hose was placed in the bottle and was connected to the correct filling needle.

Sulphamethizole (700 µg/mL):



[0150] The sulphamethizole stock solution was added with a graduated pipette to the Iso-sensitest/Discovery agar. The solution was thoroughly mixed on the Multitherm hot plate. The suction hose was placed in the bottle and is connected to the correct filling needle.

Ampicilline (32 µg/mL):



[0151] The ampicilline stock solution was then added with a graduated pipette to the Iso-sensitest/Discovery agar. The solution was thoroughly mixed on the Multitherm hot plate. The suction hose was placed in the bottle and was connected to the correct filling needle.

Nitrofurantoine (32 µg/mL):



[0152] The nitrofurantoine stock solution was asses with a graduated pipette to the Iso-sensitest/Discovery agar. The solution was thoroughly mixed on the Multitherm hot plate. The suction hose was placed in the bottle and is connected to the correct filling needle.

Mecilliname (16 µg/mL):



[0153] The mecilliname stock solution was then added with a graduated pipette to the Iso-sensitest/Discovery agar. The solution was thoroughly mixed on the Multitherm hot plate. The suction hose was placed in the bottle and was connected to the correct filling needle.

Iso-sensitest/Discovery agar, without antibiotics:



[0154] The suction hose is placed in the bottle and was connected to the correct filling needle.

Filling of the Petri dish in 6 parts:



[0155] The 6 filling needles were placed in the dispenser head ensuring that the dosage corresponds to figure 2.

Example 2: Stability test



[0156] The stability of the chromogenic agar in Flexicult has been determined by testing Flexicult agars kept up to 4 months in refrigerator (< 4° C) and tested for growth conditions (quantity, colony size, colour of colonies and agar) at 1, 2, 3 and 4 months after production for clinical strains of all the bacteria mentioned in Table 2, and the same results were seen for up to 4 months after production (as present at day 1 after production) regarding:

Agar stability (water content/dryness, form)

Colourisation of bacterial colonies

Susceptibility/resistance towards the antibiotics (sulfamethizole, trimethoprim, ampicillin, nitrofurantoin and mecillinam.


Example 3: Clinical validation



[0157] The Flexicult agar kit has been tested in two GP clinics. Both clinics have 9 - 10 GP's working in the clinic and perform routine urine culture (either by dipslide or agar plate with subsequent antibiotic disc diffusion susceptibility in certain cases), performed by clinical biochemistry technician staff.

[0158] For the study, the Flexicult plate was inoculated with urine from patients with suspected UTI as based on history and symptoms in parallel with the routine culture systems for each clinic. The plates were incubated at 35-37°C in ambient atmosphere for 18 - 24 h (tests performed Friday, which could not be read Saturday, were not included). Plates were read by the technicians and the results recorded (by the use of material provided together with the plates from the Statens Serum Institut: A picture scheme showing the quantities of bacteria on the Flexicult plate, see Figure 3, and a picture scheme showing the different colony types of urinary pathogens, see Figure 4). Hereafter the plates were transported to the National Center for Antimicrobials and Infections Control, Statens Serum Institut, where the plates were read again, and from plates with relevant growth of one or two potential urinary pathogens colonies were processed for diagnosis using API-20E, Vitec or biochemical tests according to laboratory routine. Further, for all relevant bacteria the MIC towards sulfamethizole, trimethoprim, ampicillin, nitrofurantoin and mecilinam were determined by agar dilution in Mueller-Hinton agar according to CSLI (former NCCLS).

[0159] Results: The diagnoses of the bacteria are recorded in Table 7. showing the tentative and the actual diagnosis, and the percentage correct diagnoses (to the group or species level)
Table 7. Results of clinical validation trial, tentative diagnosis at reading of the test as compared with the subsequent final diagnosis of the bacteria.
 TotalWrong diagnosis No. isolatesCorrect diagnosis No. isolatesCorrect diagnosis % of total
E. coli 205 16 189 92,2
Klebsiellal Enterobacter sp 23 0 23 100,0
Prot/Morg 19 4 15 78,9
Enterobacteriaceae, other 4 4   0
Ps. aeruginosa 6 0 6 100,0
E. faecalis 67 1 66 98,5
Gr.B strep 12 9 3 25,0
S.saprophyticus. 6 0 6* 100,0
A. viridans 2 1 1 50,0
other 22 12 10 45,4
Candida 23 0 23 100,0
Total 389 47 342 88


[0160] Regarding the determination of susceptibility, i.e. comparing the measured MIC's with reading of the test, of the bacteria, Tables 7 and 8 show the results for the Gram-negative bacteria in Table 8 and the Gram-positive bacteria in Table 9. The results have been analysed according to whether the reading and reporting of the result could be classified as an error (i.e. the result being S for I, I for R, R for I or I for S), a major error (i.e. recorded as R but was actually S, which means that the patient could be treated with another drug) and very major error (i.e. reported as S but was R, which could carry the risk that the patient was treated with an antibiotic that did not have effect).
Table 8: Results of reading the susceptibility/resistance of Gram-negative bacteria growing on the Flexicult agar as compared to the subsequently determined MIC by agar-dilution. Numbers indicate number of strains with recorded result (% of total).
 Trimethoprim N= 248Sulfametizol N= 231Ampicillin N= 247Nitrofurantoin N= 183Mecillinam N= 246
Error S/R >< I 0 1 (0.4%) 2 (0.8%) 3 (1.6%) 11 (4.5%)
Major error 2 7 5 4 17
R><S (0.8%) (3.0%) (2.0%) (2.2%) (6.9%)
Very major error 2 1 1 8* 1
S><R (0.8 %) (0.5 %) (0.4 %) (4.4%) (0.4 %)
Table 9: Results of reading the susceptibility/resistance of Gram-Positive bacteria growing on the Flexicult agar as compared to the subsequently determined MIC by agar-dilution. Numbers indicate number of strains with recorded result (% of total).
 TrimethoprimSulfametizolAmpicilin N= 26Nitrofurantoin N= 26Mecillinam
Error - - 0 0 -
S/R >< I (0%) (0%)
Major error - - 1 0 -
R><S (3.8%) (0%)
Very major error - - 0 0 -
S><R (0%) (0%)


[0161] Conclusion of clinical validation: The results show a high degree of precision of the Flexicult regarding diagnosis to the group or species level of urinary pathogens. Also, the result of the susceptibility test incorporated in the Flexicult test is comparable to a disc-diffusion test by having < 5% very major errors. Certainly, the test can be used to diagnose UTI in a patient by finding the quantity of the bacteria at all relevant counts, to diagnose the urinary pathogen (up to almost 90% of the pathogens were correctly diagnosed to the group or species level). A retest of a patient with former UTI would help showing whether the same or a new type of pathogen was present i.e. it would within certain limits be possible to discern between a relapse or a new infection. When including the susceptibility of the isolate in question, this could help in the diagnosis of a possible relapse/reinfection by viewing the "resisto-type" of the isolates found.

[0162] Example 4: Flexicult vs. other diagnostic methods in primary care. Comparing the Flexicult with other available methods for diagnosis in general practice and susceptibility testing of urinary pathogens:
Table 10: Comparison of different diagnostic tests for UTI in primary care.
TestLevel of CFU/mlSpeed of result for Possible diagnosis and susceptibilityDiagnosis of pathogenSusceptibility test directlyComment
Microscopy 105 NA* NA NA  
Dipstick (nitrite/ 105 NA NA NA  
Leucocyte)          
Dipslide 103 3 days Yes,Some dipslides use Chromo-Genic agar NA Poor PPV of susc. test
Agar Plate with Depend on loop 3 days NA NA  
Subs. Disc          
diffusion          
Flexicult 103 2 days Yes Yes  
* NA, not applicable

References



[0163] 
  1. (1) Laupland KB et al. Infection, 2007, 35: 150-3.
  2. (2) Kass EH Ann Intern Med. 1962, 56:46-53.
  3. (3) Stamm WE et.al. N Engl J Med 1982, 307(8):463-468.
  4. (4) Frimodt-Møller N et al. Ugeskr Læger 1989, 151: 3062-4
  5. (5) Frimodt-Møller N et al APMIS 2000, 108: 525-30.
  6. (6) Mabeck CE. Postgraduate Medical Journal 1972, 48:69-75.
  7. (7) Ferry S et al Scand J Prim Health Care 1989, 7: 123-128
  8. (8) Zhanel GG et al. Int JAntimicrob Agents. 2006, 27: 468-75.
  9. (9) Kahlmeter G et al. J Antimicrob Chemother. 2003, 51: 69-76.



Claims

1. A platform comprising a solid support wherein said solid support comprises an indentation capable of acting as a receptacle for a sample with a possible microbial infection or contamination, said indentation being divided into three or more compartments, each compartment containing a test composition or a control composition and one or more integrated dividing members for dividing said indentation into said separate compartments, wherein

i) said test composition comprises a semi solid microbial growth medium, an antimicrobial
and
three or more chromogenic substrates selected from the group consisting of 5-Bromo-6-chloro-3-indolyl phosphate, 5-Bromo-4-chloro-3-indolyl-β-D-galactopyranoside, 5-Bromo-6-chloro-3-indolyl-β-D-galactopyranoside, 6-Chloro-3-indolyl-β-D-galactopyranoside, 5-Bromo-4-chloro-3-indolyl-β-D-glucopyranoside, 5-Bromo-4-chloro-3-indolyl-β-D-glucuronide;

ii) wherein each test composition comprises a different antimicrobial;

iii) said control composition comprises the same semi solid microbial growth medium and the same chromogenic substrates as said test composition, but does not comprise an antimicrobial as defined in i);

and wherein said semi solid microbial growth medium comprises tryptophan and one or more inducers; and wherein said platform comprises a control composition.
 
2. The platform according to claim 1, wherein the antimicrobial in the test composition is selected from the group consisting of aminoglycosides, ansamycins, beta-lactam antibiotics, glycopeptides, macrolides, lincosamides, polypeptides, quinolones, sulphonamides, tetracyclines, cyclic lipopeptides, glycylcyclines, oxazolidinones, diaminopyrimidines, nitrofurans, rifamycins, antibiotic peptides, amphenicols, nitroimidazoles, streptogramins and phosphomycins.
 
3. The platform according to claim 1 or claim 2, wherein said composition is present in each of said compartments in an amount corresponding to from 25-45% of the volume of the compartment.
 
4. The platform according to any one of claims 1 to 3, said platform having 7 or more compartments.
 
5. The platform according to any one of claims 1 to 4, wherein said compartments are separated by dividing members that have been treated to prevent diffusion of an antimicrobial between the compartments.
 
6. The platform according to any one of claims 1 to 5, wherein said solid support is manufactured from a plastic/polymer substrate, from a glass substrate or from a metal substrate.
 
7. The platform according to any one of claims 1 to 6, wherein said solid support is a Petri dish, optionally having a diameter of from 80 to 100 mm.
 
8. The platform according to any one of claims 1 to 7, wherein each of said compartments comprising a test composition has an area of from 4-9 cm2 and/or wherein said compartment comprising a control composition has an area of from 15-25 cm2.
 
9. The platform according to any one of claims 1 to 8, wherein one or more inducers are present in said test composition and in said control composition in amounts of 0.001 to 1.0 g/l.
 
10. The platform according to any one of claims 1 to 9, wherein said one or more inducers are selected from the group consisting of 4-aminophenyl-β-D-galactopyranoside, isopropyl -β-D-thiogalactopyranoside, 1-O-Methyl-β-D-galactopyranoside, Methyl-β-D-thiogalactopyranoside, 1-o-Methyl-α-D-galactopyranoside, Isopropyl-β-D-thioglucopyranoside, 1-O-methyl-β-D-glucopyranoside, Isopropyl-β-D-thioglucouronic acid, and 1-O-Methyl-β-D-glucouronic acid, sodium salt.
 
11. The platform according to any one of claims 1 to 10, wherein

i) colonies of E. coli, if present, will appear Salmon-red/pink;

ii) colonies of Citrobacter sp. , if present, will appear greenish-blue;

iii) colonies of Klebsiella, Enterobacter or Citrobacter spp. will appear dark blue;

iv) colonies of Proteus mirabilis/Morganella morganii, if present, will appear light brown;

v) colonies of Proteus vulgaris, if present, will appear dark green;

vi) colonies of Enterococcus faecalis or Enterococcus faecium, if present, will appear greenish or blue;

vii) colonies of Staphylococcus saprophyticus, if present, will appear red or salmon-red;

viii) colonies of Staphylococcus or Psecsdorraonas aeruginosa, if present, will appear white or yellow; and

ix) colonies of Candida spp. , if present, will appear white.


 
12. The platform according to any one of claims 1 to 11, wherein said antimicrobial is selected from the group consisting of Amikacin, Amoxicillin, Amoxicillin-clavulanic acid, Amphothericin-B, Ampicillin, Ampicllin-sulbactam, Apramycin, Azithromycin, Aztreonam, Bacitracin, Benzylpenicillin, Caspofungin, Cefaclor, Cefadroxil, Cefalexin, Cefalothin, Cefazolin, Cefdinir, Cefepime, Cefixime, Cefmenoxime, Cefoperazone, Cefoperazone-sulbactam, Cefotaxime, Cefoxitin, Cefpirome, Cefpodoxime, Cefpodoxime-clavulanic acid, Cefpodoxime-sulbactam, Cefprozil, Cefquinome, Ceftazidime, Ceftibutin, Ceftiofur, Ceftobiprole, Ceftriaxon, Cefuroxime, Chloramphenicole, Florfenicole, Ciprofloxacin (or other fluoroquinolone), Clarithromycin, Clinafloxacin, Clindamycin, Cloxacillin, Colistin, Cotrimoxazol (Trimthoprim/sulphamethoxazole), Dalbavancin, Dalfopristin/Quinopristin, Daptomycin, Dibekacin, Dicloxacillin, Doripenem, Doxycycline, Enrofloxacin, Ertapenem, Erythromycin, Flucloxacillin, Fluconazol, Flucytosin, Fosfomycin, Fusidic acid, Garenoxacin, Gatifloxacin, Gemifloxacin, Gentamicin, Imipenem, Itraconazole, Kanamycin, Ketoconazole, Levofloxacin, Lincomycin, Linezolid, Loracarbef, Mecillnam (amdinocillin), Meropenem, Metronidazole, Mezlocillin, Mezlocillin-sulbactam, Minocycline, Moxifloxacin, Mupirocin, Nalidixic acid, Neomycin, Netilmicin, Nitrofurantoin, Norfloxacin, Ofloxacin, Oxacillin, Pefloxacin, Penicillin V, Piperacillin, Piperacillin-sulbactam, Piperacillin-tazobactam, Rifampicin, Roxythromycin, Sparfloxacin, Spectinomycin, Spiramycin, Streptomycin, Sulbactam, Sulfamethoxazole, Teicoplanin, Telavancin, Telithromycin, Temocillin, Tetracyklin, Ticarcillin, Ticarcillin-clavulanic acid, Tigecycline, Tobramycin, Trimethoprim, Trovafloxacin, Tylosin, Vancomycin, Virginiamycin, Voriconazole, preferably selected from the group consisting of Amoxicillin, cluvulanic acid/ampicillin, sulbactam, a fluoroquinolone, Sulphamethoxazole, trimethoprim, an oral cephalosporin, Nitrofurantoin and Fosfomycin (fosfomycin-trometerole) or is selected from the group consisting of trimethoprim, sulfamethoxazole, ampicillin, nitrofurantoin and mecillinam (amdinocillin) and combinations of these.
 
13. The platform according to claim 16, wherein said fluoroquinolone is Ciprofloxacin and/or wherein said oral cephalosporin is selected from the group consisting of Cefalexin, cefuroxime, cefadroxil and cefaclor.
 
14. The platform according to any one of claims 1 to 13, wherein said semi solid microbial growth medium is selected from the group consisting of:

i) A medium composed of: 11 g/l Hydrolysed casein, 3 g/l Peptones, 2 g/l Glucose, 3 g/l Sodium Chloride, 1 g/l soluble starch, 2 g/l Disodium hydrogen Phosphate, 1 g/l Sodium Acetate, 0.2 g/l Magnesium glycerophosphate, 0.1 g/l Calcium gluconate, 0.001 g/l cobaltous sulphate, 0.001 g/l Cupric sulphate, 0.001 g/l Zinc sulphate, 0.001 g/l Ferrous Sulphate, 0.002 g/l Magnesium Chloride, 0.001 g/l Menadione, 0.001 g/l Cyanobalamin, 0.02 g/l L-Cysteine hydrochloride, 0.02 g/l Tryptophan, 0.003 g/l pyridoxine, 0.003 g/l pantothenate, 0.003 g/l nicotinamide, 0.0003 g/l Biotin, 0.00004 g/l Thiamine, 0.01 g/l Adenine, 0.01 g/l Guanine, 0.01 g/l Xanthine, 0.01 g/l Uracil, 8 g/l agar, in distilled water;

ii) A medium composed of: 2 g Na2HPO4 · 12 H2O, 625 g tryptone, 250 g starch, 833.6 g Potassium Chloride, 2.5 g detergent, 74.8 g meat broth (Oxoid CM975K), 800g D(+)Glucose-monohydrate, 1.75 g Xanthin, 1.75 g Guanin, 17.5 g Magnesium Sulphate 7 H2O, 19.2 g CaCl2 · 2 H2O, 2,720 g Agar, 5 N HCl to pH 7.4, solution of vitamins, and 12.5 1 horse blood per 250 liter distilled water;

iii) A medium comprising: 14.5 g/l Peptone, 2 g/l glucose, 5.5 g/l salt mix, 1 g/l Soluble starch, 1.5 g/l chromogenic mix, and 8 g/l Agar; and

iv) A medium comprising: 2 g/l Beef extract powder/beef extract, 17.5 g/l Acid Digest of Casein, 1.5 g/l starch and 17 g/l Agar;


 
15. The platform according to any one of claims 1 to 14, wherein said 3 or more chromogenic substrates comprise 5-Bromo-4-chloro-3-indolyl-β-D-galactopyranoside, 6-Chloro-3-indolyl-β-D-galactopyranoside, 5-Bromo-4-chloro-3-indolyl-β-D-glucopyranoside.
 
16. The platform according to any one of claims 1 to 14, wherein said three or more chromogenic substances is selected from the group consisting of 6-Chloro-3-indolyl-β-D-galactopyranoside, 5-Bromo-4-chloro-3-indolyl-β-D-glucopyranoside, 5-Bromo-6-chloro-3-indolyl-phosphate, 5-Bromo-4-chloro-3-indolyl-β-D-glucuronide and 5-Bromo-4-chloro-3-indolyl-β-D-galactopyranoside.
 
17. The platform according to any one of claims 1 to 14, wherein said test and said control composition comprises 6-Chloro-3-indolyl-β-D-galactopyranoside in combination with 5-Bromo-4-chloro-3-indolyl-β-D-glucopyranoside, 5-Bromo-4-chloro-3-indolyl-β-D-glucuronide, 5-Bromo-4-chloro-3-indolyl-β-D-galactopyranoside and 5-Bromo-6-chloro-3-indolyl phosphate.
 
18. The platform according to any one of claims 1 to 14, wherein said three or more chromogenic substances includes the group of 5-bromo-6-chloro-3-indolyl-beta-D-galactopyranoside, 5-bromo-4-chloro-3-indolyl-beta-D-glucopyranoside and 6-chloro-3-indolyl-beta-D-galactopyranoside.
 
19. The platform according to any one of claims 1 to 14, wherein said three or more chromogenic substances includes the group of 5-Bromo-4-chloro-3-indolyl-beta-D-galactopyranoside, 6-chloro-3-indolyl-beta-D-galactopyranoside, 5-Bromo-4-chloro-3-indolyl-beta-D-glucopyranoside and 5-Bromo-4-chloro-3-indolyl-beta-D-glucuronide.
 
20. The platform according to any one of claims 1 to 19, wherein said test composition and said control composition comprise four chromogenic substrates.
 
21. The platform according to any one of claims 1 to 20, wherein said semi solid microbial growth medium comprises 0.25-3.0 g/l tryptophan is present.
 
22. The platform according to any one of claims 1 to 21, wherein said solid support is a Petri dish divided into 5 test compartments and 1 control compartment.
 
23. A kit comprising a platform according to any one of claims 1 to 22.
 
24. The kit according to claim 23, said kit further comprising a standard illustrating the amount of growth on the platform, which results from contacting said platform with a suspension having a predetermined titre of a microbial reference strain.
 
25. The kit according to claim 24 wherein said standard is a photographic or printed reproduction of the platform.
 
26. The kit according to claim 24 wherein said standard has been generated by contacting the platform with a reference strain of E. coli bacteria (optionally E. coli ATCC 29522), and/or a reference strain of Staphylococcus aureus (optionally Staphylococcus aureus ATCC 25913).
 
27. The kit according to claim 25, said kit further comprising one or more separately packaged antimicrobials.
 
28. Use of a platform of according to any one of claims 1 to 22 in detection and/or diagnosis of infections selected from the group consisting of urinary tract infections, skin and soft tissue infections, infections with Staphylococcus aureus (including methicilin resistant Staphylococcus aureus), infections with meningococcy infection with gonococci, infections with streptococci including infections with pneumococci.
 


Ansprüche

1. Plattform, umfassend einen festen Träger, wobei der feste Träger eine Vertiefung umfasst, die dazu in der Lage ist, als Aufnahmebehälter für eine Probe mit einer möglichen mikrobiellen Infektion oder Kontamination zu fungieren, wobei die Vertiefung in drei oder mehr Kammern unterteilt ist, wobei jede der Kammern eine Testzusammensetzung oder eine Kontrollzusammensetzung sowie ein oder mehrere integrierte Unterteilungselemente zur Unterteilung der Vertiefung in die voneinander getrennten Kammern enthält, wobei

i) die Testzusammensetzung ein halbfestes Medium für mikrobielles Wachstum, ein antimikrobielles Mittel und
drei oder mehr chromogene Substrate umfasst, die ausgewählt sind aus der Gruppe bestehend aus 5-Brom-6-chlor-3-indolylphosphat, 5-Brom-4-chlor-3-indolyl-β-D-galactopyranosid, 5-Brom-6-chlor-3-indolyl-β-D-galactopyranosid, 6-Chlor-3-indolyl-β-D-galactopyranosid, 5-Brom-4-chlor-3-indolyl-β-D-glucopyranosid, 5-Brom-4-chlor-3-indolyl-β-D-glucuronid;

ii) wobei jede Testzusammensetzung ein unterschiedliches antimikrobielles Mittel umfasst;

iii) die Kontrollzusammensetzung das gleiche halbfeste Medium für mikrobielles Wachstum und die gleichen chromogenen Substrate wie die Testzusammensetzung, jedoch kein wie in i) definiertes antimikrobielles Mittel umfasst;

und wobei das halbfeste Medium für mikrobielles Wachstum Tryptophan und einen oder mehrere Induktoren umfasst; und wobei die Plattform eine Kontrollzusammensetzung umfasst.
 
2. Plattform nach Anspruch 1, wobei das antimikrobielle Mittel in der Testzusammensetzung ausgewählt ist aus der Gruppe bestehend aus Aminoglykosiden, Ansamycinen, Beta-Lactam-Antibiotika, Glykopeptiden, Makroliden, Lincosamiden, Polypeptiden, Chinolonen, Sulfonamiden, Tetracyclinen, cyclischen Lipopeptiden, Glycylcyclinen, Oxazolidinonen, Diaminopyrimidinen, Nitrofuranen, Rifamycinen, antibiotischen Peptiden, Amphenicolen, Nitroimidazolen, Streptograminen und Phosphomycinen.
 
3. Plattform nach Anspruch 1 oder Anspruch 2, wobei die Zusammensetzung in jeder der Kammern in einer Menge vorliegt, die von 25% bis 45% des Volumens der Kammer entspricht.
 
4. Plattform nach einem der Ansprüche 1 bis 3, wobei die Plattform 7 oder mehr Kammern aufweist.
 
5. Plattform nach einem der Ansprüche 1 bis 4, wobei die Kammern durch Unterteilungselemente voneinander getrennt sind, die derart behandelt wurden, dass sie die Diffusion eines antimikrobiellen Mittels zwischen den Kammern unterbinden.
 
6. Plattform nach einem der Ansprüche 1 bis 5, wobei der feste Träger aus einem Kunststoff-/Polymersubstrat, aus einem Glassubstrat oder aus einem Metallsubstrat hergestellt ist.
 
7. Plattform nach einem der Ansprüche 1 bis 6, wobei es sich bei dem festen Träger um eine Petrischale handelt, die gegebenenfalls einen Durchmesser von 80 bis 100 mm aufweist.
 
8. Plattform nach einem der Ansprüche 1 bis 7, wobei jede der Kammern, die eine Testzusammensetzung umfassen, eine Fläche von 4 bis 9 cm2 aufweist und/oder wobei die Kammer, die eine Kontrollzusammensetzung umfasst, eine Fläche von 15 bis 25 cm2 aufweist.
 
9. Plattform nach einem der Ansprüche 1 bis 8, wobei in der Testzusammensetzung und in der Kontrollzusammensetzung ein oder mehrere Induktoren in einer Menge von 0,001 bis 1,0 g/l vorliegen.
 
10. Plattform nach einem der Ansprüche 1 bis 9, wobei der eine oder die mehreren Induktoren ausgewählt sind aus der Gruppe bestehend aus 4-Aminophenyl-β-D-galactopyranosid, Isopropyl-β-D-thiogalactopyranosid, 1-0-Methyl-β-D-galactopyranosid, Methyl-β-D-thiogalactopyranosid, 1-0-Methyl-α-D-galactopyranosid, Isopropyl-β-D-thioglucopyranosid, 1-0-Methyl-β-D-glucopyranosid, Isopropyl-β-D-thioglucuronsäure sowie dem Natriumsalz der 1-O-Methyl-β-D-glucuronsäure.
 
11. Plattform nach einem der Ansprüche 1 bis 10, wobei

i) Kolonien von E. coli, sofern vorhanden, durch eine lachsrote bzw. lachsrosa Färbung angezeigt werden;

ii) Kolonien von Citrobacter sp., sofern vorhanden, durch eine grünlich-blaue Färbung angezeigt werden;

iii) Kolonien von Klebsiella, Enterobacter oder Citrobacter spp., sofern vorhanden, durch eine dunkelblaue Färbung angezeigt werden;

iv) Kolonien von Proteus mirabilis oder Morganella morganii, sofern vorhanden, durch eine hellbraune Färbung angezeigt werden;

v) Kolonien von Proteus vulgaris, sofern vorhanden, durch eine dunkelgrüne Färbung angezeigt werden;

vi) Kolonien von Enterococcus faecalis oder Enterococcus faecium, sofern vorhanden, durch eine grünliche oder blaue Färbung angezeigt werden;

vii) Kolonien von Staphylococcus saprophyticus, sofern vorhanden, durch eine rote oder lachsrote Färbung angezeigt werden;

viii) Kolonien von Staphylococcus oder Pseudomonas aeruginosa, sofern vorhanden, durch eine weiße oder gelbe Färbung angezeigt werden; und

ix) Kolonien von Candida spp., sofern vorhanden, durch eine weiße Färbung angezeigt werden.


 
12. Plattform nach einem der Ansprüche 1 bis 11, wobei das antimikrobielle Mittel ausgewählt ist aus der Gruppe bestehend aus Amikacin, Amoxicillin, Amoxicillin-Clavulansäure, Amphotericin-B, Ampicillin, Ampicillin/Sulbactam, Apramycin, Azithromycin, Aztreonam, Bacitracin, Benzylpenicillin, Caspofungin, Cefaclor, Cefadroxil, Cefalexin, Cefalotin, Cefazolin, Cefdinir, Cefepim, Cefixim, Cefmenoxim, Cefoperazon, Cefoperazon/Sulbactam, Cefotaxim, Cefoxitin, Cefpirom, Cefpodoxim, Cefpodoxim/Clavulansäure, Cefpodoxim/Sulbactam, Cefprozil, Cefquinom, Ceftazidim, Ceftibuten, Ceftiofur, Ceftobiprol, Ceftriaxon, Cefuroxim, Chloramphenicol, Florfenicol, Ciprofloxacin (oder weitere Fluorchinolone), Clarithromycin, Clinafloxacin, Clindamycin, Cloxacillin, Colistin, Cotrimoxazol (Trimethoprim/Sulfamethoxazol), Dalbavancin, Dalfopristin/Quinupristin, Daptomycin, Dibekacin, Dicloxacillin, Doripenem, Doxycyclin, Enrofloxacin, Ertapenem, Erythromycin, Flucloxacillin, Fluconazol, Flucytosin, Fosfomycin, Fusidinsäure, Garenoxacin, Gatifloxacin, Gemifloxacin, Gentamicin, Imipenem, Itraconazol, Kanamycin, Ketoconazol, Levofloxacin, Lincomycin, Linezolid, Loracarbef, Mecillinam (Amdinocillin), Meropenem, Metronidazol, Mezlocillin, Mezlocillin/Sulbactam, Minocyclin, Moxifloxacin, Mupirocin, Nalidixinsäure, Neomycin, Netilmicin, Nitrofurantoin, Norfloxacin, Ofloxacin, Oxacillin, Pefloxacin, Penicillin V, Piperacillin, Piperacillin/Sulbactam, Piperacillin/Tazobactam, Rifampicin, Roxythromycin, Sparfloxacin, Spectinomycin, Spiramycin, Streptomycin, Sulbactam, Sulfamethoxazol, Teicoplanin, Telavancin, Telithromycin, Temocillin, Tetracyclin, Ticarcillin, Ticarcillin/Clavulansäure, Tigecyclin, Tobramycin, Trimethoprim, Trovafloxacin, Tylosin, Vancomycin, Virginiamycin, Voriconazol, bevorzugt aus der Gruppe bestehend aus Amoxicillin, Clavulansäure/Ampicillin, Sulbactam, einem Fluorchinolon, Sulfamethoxazol, Trimethoprim, einem oralen Cephalosporin, Nitrofurantoin und Fosfomycin (Fosfomycin/Trometamol) oder ausgewählt ist aus der Gruppe bestehend aus Trimethoprim, Sulfamethoxazol, Ampicillin, Nitrofurantoin und Mecillinam (Amdinocillin) sowie Kombinationen aus diesen.
 
13. Plattform nach Anspruch 12, wobei es sich bei dem Fluorchinolon um Ciprofloxacin handelt und/oder wobei das orale Cephalosporin ausgewählt ist aus der Gruppe bestehend aus Cefalexin, Cefuroxim, Cefadroxil und Cefaclor.
 
14. Plattform nach einem der Ansprüche 1 bis 13, wobei das halbfeste Medium für mikrobielles Wachstum ausgewählt ist aus der Gruppe bestehend aus:

i) einem Medium, das sich zusammensetzt aus: 11 g/l hydrolysiertem Casein, 3 g/l Peptonen, 2 g/l Glucose, 3 g/l Natriumchlorid, 1 g/l löslicher Stärke, 2 g/l Dinatriumhydrogenphosphat, 1 g/l Natriumacetat, 0,2 g/l Magnesiumglycerophosphat, 0,1 g/l Calciumgluconat, 0,001 g/l Kobaltsulfat, 0,001 g/l Kupfersulfat, 0,001 g/l Zinksulfat, 0,001 g/l Eisensulfat, 0,002 g/l Magnesiumchlorid, 0,001 g/l Menadion, 0,001 g/l Cyanobalamin, 0,02 g/l L-Cysteinhydrochlorid, 0,02 g/l Tryptophan, 0,003 g/l Pyridoxin, 0,003 g/l Pantothenat, 0,003 g/l Nicotinamid, 0,0003 g/l Biotin, 0,00004 g/l Thiamin, 0,01 g/l Adenin, 0,01 g/l Guanin, 0,01 g/l Xanthin, 0,01 g/l Uracil, 8 g/l Agar, in destilliertem Wasser;

ii) einem Medium, das sich zusammensetzt aus: 2 g Na2HPO4 · 12 H2O, 625 g Trypton, 250 g Stärke, 833,6 g Kaliumchlorid, 2,5 g Tensid, 74,8 g Fleischbrühe (Oxoid CM975K), 800 g D (+) Glucosemonohydrat, 1,75 g Xanthin, 1,75 g Guanin, 17,5 g Magnesiumsulfat 7 H2O, 19,2 g CaCl2 · 2 H2O, 2,720 g Agar, 5 N HCl bis pH 7,4, einer Lösung aus Vitaminen und 12,5 1 Pferdeblut pro 250 Liter an destilliertem Wasser;

iii) einem Medium, das umfasst: 14,5 g/l an Pepton, 2 g/l an Glucose, 5,5 g/l eines Salzgemischs, 1 g/l löslicher Stärke, 1,5 g/l eines chromogenen Gemischs und 8 g/l Agar; und

iv) einem Medium, das umfasst: 2 g/l an Rindfleischextrakt/pulverförmigem Rindfleischextrakt, 17,5 g/l Säureverdau von Casein, 1,5 g/l an Stärke und 17 g/l Agar.


 
15. Plattform nach einem der Ansprüche 1 bis 14, wobei die drei oder mehr chromogenen Substrate 5-Brom-4-chlor-3-indolyl-β-D-galactopyranosid, 6-Chlor-3-indolyl-β-D-galactopyranosid und 5- Brom-4-chlor-3-indolyl-β-D-glucopyranosid umfassen.
 
16. Plattform nach einem der Ansprüche 1 bis 14, wobei die drei oder mehr chromogenen Substanzen ausgewählt sind aus der Gruppe bestehend aus 6-Chlor-3-indolyl-β-D-galactopyranosid, 5-Brom-4-chlor-3-indolyl-β-D-glucopyranosid, 5-Brom-6-chlor-3-indolylphosphat, 5-Brom-4-chlor-3-indolyl-β-D-glucuronid und 5-Brom-4-chlor-3-indolyl-β-D-galactopyranosid.
 
17. Plattform nach einem der Ansprüche 1 bis 14, wobei die Testzusammensetzung und die Kontrollzusammensetzung 6-Chlor-3-indolyl-β-D-galactopyranosid in Kombination mit 5-Brom-4-chlor-3-indolyl-β-D-glucopyranosid, 5-Brom-4-chlor-3-indolyl-β-D-glucuronid, 5-Brom-4-chlor-3-indolyl-β-D-galactopyranosid und 5-Brom-6-chlor-3-indolylphosphat umfassen.
 
18. Plattform nach einem der Ansprüche 1 bis 14, wobei die drei oder mehr chromogenen Substanzen die Gruppe von 5-Brom-6-chlor-3-indolyl-β-D-galactopyranosid, 5-Brom-4-chlor-3-indolyl-β-D-glucopyranosid und 6-Chlor-3-indolyl-β-D-galactopyranosid einschließen.
 
19. Plattform nach einem der Ansprüche 1 bis 14, wobei die drei oder mehr chromogenen Substanzen die Gruppe von 5-Brom-4-chlor-3-indolyl-β-D-galactopyranosid, 6-Chlor-3-indolyl-β-D-galactopyranosid, 5-Brom-4-chlor-3-indolyl-β-D-glucopyranosid und 5-Brom-4-chlor-3-indolyl-β-D-glucuronid einschließen.
 
20. Plattform nach einem der Ansprüche 1 bis 19, wobei die Testzusammensetzung und die Kontrollzusammensetzung vier chromogene Substrate umfassen.
 
21. Plattform nach einem der Ansprüche 1 bis 20, wobei das halbfeste Medium für mikrobielles Wachstum 0,25 bis 3,0 g/l an Tryptophan umfasst.
 
22. Plattform nach einem der Ansprüche 1 bis 21, wobei es sich bei dem festen Träger um eine Petrischale handelt, die in 5 Testkammern und eine Kontrollkammer unterteilt ist.
 
23. Kit umfassend eine Plattform nach einem der Ansprüche 1 bis 22.
 
24. Kit nach Anspruch 23, des Weiteren umfassend einen Standard, der die Menge des Wachstums auf der Plattform verdeutlicht, das aus dem Kontaktieren der Plattform mit einer Suspension resultiert, die eine vorbestimmte Konzentration eines mikrobiellen Referenzstamms aufweist.
 
25. Kit nach Anspruch 24, wobei es sich bei dem Standard um eine photographische oder gedruckte Abbildung der Plattform handelt.
 
26. Kit nach Anspruch 24, wobei der Standard dadurch erzeugt worden ist, dass die Plattform mit einem Referenzstamm von E. coli-Bakterien (gegebenenfalls mit E. coli ATCC 29522) und/oder einem Referenzstamm von Staphylococcus aureus (gegebenenfalls mit Staphylococcus aureus ATCC 25913) in Kontakt gebracht wurde.
 
27. Kit nach Anspruch 25, des Weiteren umfassend ein oder mehrere getrennt voneinander verpackte antimikrobielle Mittel.
 
28. Verwendung einer Plattform nach einem der Ansprüche 1 bis 22 bei der Detektion und/oder der Diagnose von Infektionen, die ausgewählt sind aus der Gruppe bestehend aus Infektionen der Harnwege, Infektionen der Haut und des Weichgewebes, Infektionen mit Staphylococcus aureus (einschließend mit gegen Methicillin resistentem Staphylococcus aureus), Infektionen mit Meningokokken, Infektionen mit Gonokokken, Infektionen mit Streptokokken, einschließend Infektionen mit Pneumokokken.
 


Revendications

1. Plateforme comprenant un support solide dans laquelle ledit support solide comprend une indentation capable de faire office de réceptacle pour un échantillon présentant une infection ou une contamination microbienne éventuelle, ladite indentation étant divisée en trois compartiments ou plus, chaque compartiment contenant une composition de test ou une composition témoin et un ou plusieurs éléments de division intégrés pour diviser ladite indentation en lesdits compartiments séparés, dans laquelle

i) ladite composition de test comprend un milieu de croissance microbienne semi-solide, un antimicrobien
et
trois substrats chromogènes ou plus choisis dans le groupe comprenant le phosphate de 5-bromo-6-chloro-3-indolyle, le 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside, le 5-bromo-6-chloro-3-indolyl-β-D-galactopyranoside, le 6-chloro-3-indolyl-β-D-galactopyranoside, le 5-bromo-4-chloro-3-indolyl-β-D-glucopyranoside, le 5-bromo-4-chloro-3-indolyl-β-D-glucuronique ;

ii) dans laquelle chaque composition de test comprend un antimicrobien différent ;

iii) ladite composition témoin comprend le même milieu de croissance microbienne semi-solide et les mêmes substrats chromogènes que ladite composition de test mais ne comprend pas d'antimicrobien tel que défini au point i) ;

et dans laquelle ledit milieu de croissance microbienne semi-solide comprend du tryptophane et un ou plusieurs inducteurs ; et dans laquelle ladite plateforme comprend une composition témoin.
 
2. Plateforme selon la revendication 1, dans laquelle l'antimicrobien dans la composition de test est choisi dans le groupe comprenant des aminoglycosides, des ansamycines, des antibiotiques bêta-lactame, des glycopeptides, des macrolides, des lincosamides, des polypeptides, des quinolones, des sulfonamides, des tétracyclines, des lipopeptides cycliques, des glycylcyclines, des oxazolidinones, des diaminopyrimidines, des nitrofuranes, des rifamycines, des peptides antibiotiques, des amphénicols, des nitro-imidazoles, des streptogramines et des phosphomycines.
 
3. Plateforme selon la revendication 1 ou la revendication 2, dans laquelle ladite composition est présente dans chacun desdits compartiments en une quantité correspondant à de 25 à 45% du volume du compartiment.
 
4. Plateforme selon l'une quelconque des revendications 1 à 3, ladite plateforme ayant 7 compartiments ou plus.
 
5. Plateforme selon l'une quelconque des revendications 1 à 4, dans laquelle lesdits compartiments sont séparés en divisant des éléments qui ont été traités pour empêcher la diffusion d'un antimicrobien entre les compartiments.
 
6. Plateforme selon l'une quelconque des revendications 1 à 5, dans laquelle ledit support solide est fabriqué à partir d'un substrat en plastique/polymère, d'un substrat en verre ou d'un substrat en métal.
 
7. Plateforme selon l'une quelconque des revendications 1 à 6, dans laquelle ledit support solide est une boîte de Pétri ayant éventuellement un diamètre de 80 à 100 mm.
 
8. Plateforme selon l'une quelconque des revendications 1 à 7, dans laquelle chacun desdits compartiments comprenant une composition de test a une surface de 4 à 9 cm2 et/ou dans laquelle ledit compartiment comprenant une composition témoin a une surface de 15 à 25 cm2.
 
9. Plateforme selon l'une quelconque des revendications 1 à 8, dans laquelle un ou plusieurs inducteurs sont présents dans ladite composition de test et dans ladite composition témoin en quantités de 0,001 à 1,0 g/l.
 
10. Plateforme selon l'une quelconque des revendications 1 à 9, dans laquelle lesdits un ou plusieurs inducteurs sont choisis dans le groupe comprenant la 4-aminophényl-β-D-galactopyranoside, l'isopropyl-β-D-thiogalactopyranoside, le 1-O-méhyl-β-D-galactopyranoside, le méthyl-β-D-thiogalactopyranoside, le 1-O-méthyl-α-D-galactopyranoside, l'isopropyl-β-D-thioglucopyranoside, le 1-O-méthyl-β-D-glucopyranoside, l'acide isopropyl-β-D-thioglucuronique et le sel de sodium de l'acide 1-O-méthyl-β-D-glucuronique.
 
11. Plateforme selon l'une quelconque des revendications 1 à 10, dans laquelle

i) des colonies d'E. coli, si elles sont présentes, apparaîtront en rouge saumon/rose ;

ii) des colonies de Citrobacter sp., si elles sont présentes, apparaîtront en bleu verdâtre ;

iii) des colonies de Klebsiella, Enterobacter ou Citrobacter spp. si elles sont présentes, apparaîtront en bleu foncé ;

iv) des colonies de Proteus mirabilis/Morganella morganii, si elles sont présentes, apparaîtront en brun pâle ;

v) des colonies de Proteus vulgaris, si elles sont présentes, apparaîtront en vert foncé ;

vi) des colonies d'Enterococcus faecalis ou d'Enterococcus faecium, si elles sont présentes, apparaîtront en verdâtre ou bleu ;

vii) des colonies de Staphylococcus saprophyticus, si elles sont présentes, apparaîtront en rouge ou rouge saumon ;

viii) des colonies de Staphylococcus ou de Pseudomonas aeruginosa, si elles sont présentes, apparaîtront en blanc ou jaune ; et

ix) des colonies de Candida spp., si elles sont présentes, apparaîtront en blanc.


 
12. Plateforme selon l'une quelconque des revendications 1 à 11, dans laquelle ledit antimicrobien est choisi dans le groupe comprenant l'amikacine, l'amoxicilline, l'amoxicilline-acide clavulanique, l'amphotéricine B, l'ampicilline, l'ampicilline/sulbactam, l'apramycine, l'azithromycine, l'aztréonam, la bacitracine, la benzylphénicilline, la caspofungine, le céfaclor, le céfadroxil, la céfalexine, la céfalotine, la céfazoline, le cefdinir, la céfépime, la céfixime, la cefménoxime, la céfopérazone, le céfopérazone/sulbactam, la céfotaxime, la céfoxitine, le cefpirome, la cefpodoxime, le cefpodoxime-acide clavulanique, le cefpodoxime-sulbactam, le cefprozil, le cefquinome, la ceftazidime, le ceftibutène, le ceftiofur, le ceftobiprole, le ceftriaxon, le cefuroxime, le chloramphénicol, le florfénicol, la ciprofloxacine (ou autres fluoroquinolones), la clarithromycine, la clinafloxacine, la clindamycine, la cloxacilline, la colistine, le cotrimoxazol (trimthoprim/sulfaméthoxazole), la dalbavancine, la dalfopristine/quinupristine, la daptomycine, la dibekacine, la dicloxacilline, le doripénem, la doxycycline, l'enrofloxacine, l'ertapénem, l'érythromycine, la flucloxacilline, le fluconazol, la flucytosine, la fosfomycine, l'acide fusidique, la garenoxacine, la gatifloxacine, la gémifloxacine, la gentamicine, l'imipénem, l'itraconazole, la kanamycine, le kétoconazole, la lévofloxacine, la lincomycine, le linézolide, le loracarbef, le mécillinam (amdinocilline), le méropénem, le métronidazole, la mezlocilline, le mezlocilline-sulbactam, la minocycline, la moxifloxacine, la mupirocine, l'acide nalidixique, la néomycine, la nétilmicine, la nitrofurantoïne, la norfloxacine, l'ofloxacine, l'oxacilline, la péfloxacine, la pénicilline V, la pipéracilline, le pipéracilline-sulbactam, le pipéracilline-tazobactam, la rifampicine, la roxythromycine, la sparfloxacine, la spectinomycine, la spiramycine, la streptomycine, le sulbactam, le sulfaméthoxazole, la teicoplanine, la télavancine, la télithromycine, la témocilline, la tétracycline, la ticarcilline, le ticarcilline-acide clavulanique, la tigécycline, la tobramycine, le triméthoprim, la trovafloxacine, la tylosine, la vancomycine, la virginiamycine, le voriconazole, choisi de préférence dans le groupe comprenant l'amoxicilline, l'acide clavulanique/ampicilline, le sulbactam, une fluoroquinolone, le sulfaméthoxazol, le triméthoprim, une céphalosporine orale, la nitrofurantoïne et la fosfomycine (fosfomycine/trométamol) ou est choisi dans le groupe comprenant le triméthoprim, le sulfaméthoxazole, l'ampicilline, la nitrofurantoïne et le mécillinam (amdinocilline) et leurs combinaisons.
 
13. Plateforme selon la revendication 12, dans laquelle ladite fluoroquinolone est la ciprofloxacine et/ou dans laquelle ladite céphalosporine orale est choisie dans le groupe comprenant la céfalexine, la céfuroxime, le céfadroxil et le céfaclor.
 
14. Plateforme selon l'une quelconque des revendications 1 à 13, dans laquelle ledit milieu de croissance microbienne semi-solide est choisi dans le groupe comprenant :

i) un milieu composé de 11 g/l de caséine hydrolysée, de 3 g/l de peptones, de 2 g/l de glucose, de 3 g/l de chlorure de sodium, de 1 g/l d'amidon soluble, de 2 g/l d'hydrogénophosphate disodique, de 1 g/l d'acétate de sodium, de 0,2 g/l de glycérophosphate de magnésium, de 0,1 g/l de gluconate de calcium, de 0,001 g/l de sulfate de cobalt, de 0,001 g/l de sulfate cuprique, de 0,001 g/l de sulfate de zinc, de 0,001 g/l de sulfate ferreux, de 0,002 g/l de chlorure de magnésium, de 0,001 g/l de ménadione, de 0,001 g/l de cyanobalamine, de 0,02 g/l de chlorhydrate de L-cystéine, de 0,02 g/l de tryptophane, de 0,003 g/l de pyridoxine, de 0,003 g/l de pantothénate, de 0,003 g/l de nicotinamide, de 0,0003 g/l de biotine, de 0,00004 g/l de thiamine, de 0,01 g/l d'adénine, de 0,01 g/l de guanine, de 0,01 g/l de xanthine, de 0,01 g/l d'uracile, de 8 g/l de gélose, dans de l'eau distillée ;

ii) un milieu composé de 2 g de Na2HPO4. 12 H2O, de 625 g de tryptone, de 250 g d'amidon, de 833,6 g de chlorure de potassium, de 2,5 g de détergent, de 74,8 g de bouillon de viande (Oxoid CM975K), de 800 g de D(+)glucose monohydraté, de 1,75 g de xanthine, de 1,75 g de guanine, de 17,5 g de sulfate de magnésium 7 H2O, de 19,2 g de CaCl2 . 2 H2O, de 2720 g de gélose, de 5 N HCl à pH 7,4, d'une solution de vitamines et de 12,5 1 de sang de cheval pour 250 litres d'eau distillée ;

iii) un milieu comprenant : 14,5 g/l de peptone, 2 g/l de glucose, 5,5 g/l de mélange de sels, de 1 g/l d'amidon soluble, de 1,5 g/l de mélange chromogène et de 8 g/l de gélose ; et

iv) un milieu contenant : 2 g/l de poudre d'extrait de boeuf/ extrait de boeuf, de 17,5 g/l de composé acide de caséine, de 1,5 g/l d'amidon et de 17 g/l de gélose.


 
15. Plateforme selon l'une quelconque des revendications 1 à 14, dans laquelle lesdits 3 substrats chromogènes ou plus comprennent le 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside, le 6-chloro-3-indolyl-β-D-galactopyranoside, le 5-bromo-4-chloro-3-indolyl-β-D-glucopyranoside.
 
16. Plateforme selon l'une quelconque des revendications 1 à 14, dans laquelle lesdites 3 substances chromogènes ou plus sont choisies dans le groupe comprenant le 6-chloro-3-indolyl-β-D-galactopyranoside, le 5-bromo-4-chloro-3-indolyl-β-D-glucopyranoside, le 5-bromo-6-chloro-3-indolyl-phosphate, le 5-bromo-4-chloro-3-indolyl-β-D-glucuronide et le 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside.
 
17. Plateforme selon l'une quelconque des revendications 1 à 14, dans laquelle ladite composition de test et ladite composition témoin comprennent le 6-chloro-3-indolyl-β-D-galactopyranoside en combinaison avec le 5-bromo-4-chloro-3-indolyl-β-D-glucopyranoside, le 5-bromo-4-chloro-3-indolyl-β-D-glucuronide, le 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside et le 5-bromo-6-chloro-3-indolyl-phosphate.
 
18. Plateforme selon l'une quelconque des revendications 1 à 14, dans laquelle lesdites 3 substances chromogènes ou plus comprennent le groupe comprenant le 5-bromo-6-chloro-3-indolyl-β-D-galactopyranoside, le 5-bromo-4-chloro-3-indolyl-β-D-glucopyranoside et le 6-chloro-3-indolyl-β-D-galactopyranoside.
 
19. Plateforme selon l'une quelconque des revendications 1 à 14, dans laquelle lesdites 3 substances chromogènes ou plus sont choisies dans le groupe comprenant le 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside, le 6-chloro-3-indolyl-β-D-galactopyranoside, le 5-bromo-4-chloro-3-indolyl-β-D-glucopyranoside et le 5-bromo-4-chloro-3-indolyl-β-D-glucuronide.
 
20. Plateforme selon l'une quelconque des revendications 1 à 19, dans laquelle ladite composition de test et ladite composition témoin comprennent quatre substrats chromogènes.
 
21. Plateforme selon l'une quelconque des revendications 1 à 20, dans laquelle ledit milieu de croissance microbienne semi-solide comprend 0,25 à 3,0 g/l de tryptophane.
 
22. Plateforme selon l'une quelconque des revendications 1 à 21, dans laquelle ledit support solide est une boîte de Pétri divisée en 5 compartiments de test et 1 compartiment témoin.
 
23. Kit comprenant une plateforme selon l'une quelconque des revendications 1 à 22.
 
24. Kit selon la revendication 23, ledit kit comprenant en outre une référence illustrant la quantité de croissance sur la plateforme, qui résulte de la mise en contact de ladite plateforme avec une suspension ayant un titre prédéterminé d'une souche microbienne de référence.
 
25. Kit selon la revendication 24, dans lequel ladite référence est une reproduction photographique ou imprimée sur la plateforme.
 
26. Kit selon la revendication 24, dans lequel ladite référence a été générée par la mise en contact de la plateforme avec une souche de référence d'une bactérie E. coli (éventuellement E. coli ATCC 29522) et/ou une souche de référence de Staphylococcus aureus (éventuellement Staphylococcus aureus ATCC 25913).
 
27. Kit selon la revendication 25, ledit kit comprenant en outre un ou plusieurs antimicrobiens conditionnés séparément.
 
28. Utilisation d'une plateforme selon l'une quelconque des revendications 1 à 22, dans la détection et/ou le diagnostic d'infections choisies dans le groupe comprenant des infections des voies urinaires, des infections de la peau et des tissus mous, des infections à Staphylococcus aureus (comprenant Staphylococcus aureus résistant à la méthiciline), des infections à méningocoques, des infections à gonocoques, des infections à streptocoques comprenant des infections à pneumocoques.
 




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REFERENCES CITED IN THE DESCRIPTION



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Patent documents cited in the description




Non-patent literature cited in the description