WO1996040981A1 - Dispositif de controle mesurant la sensibilite des micro-organismes a des agents inhibiteurs - Google Patents

Dispositif de controle mesurant la sensibilite des micro-organismes a des agents inhibiteurs Download PDF

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Publication number
WO1996040981A1
WO1996040981A1 PCT/EP1996/002400 EP9602400W WO9640981A1 WO 1996040981 A1 WO1996040981 A1 WO 1996040981A1 EP 9602400 W EP9602400 W EP 9602400W WO 9640981 A1 WO9640981 A1 WO 9640981A1
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WIPO (PCT)
Prior art keywords
test device
microorganisms
matrix
test
matrices
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Application number
PCT/EP1996/002400
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English (en)
Inventor
William Otto Bergerson
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Bayer Aktiengesellschaft
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Filing date
Publication date
Application filed by Bayer Aktiengesellschaft filed Critical Bayer Aktiengesellschaft
Priority to AU62212/96A priority Critical patent/AU6221296A/en
Publication of WO1996040981A1 publication Critical patent/WO1996040981A1/fr

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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/18Testing for antimicrobial activity of a material

Definitions

  • the present invention is directed generally to an improved means for assessing the susceptibility of microorganisms to inhibitory agents in specimen samples, e.g., biological fluids. More specifically, the present invention combines semi-quantita ⁇ tive detection of microorganisms and their susceptibility to an inhibitory agent(s) into a single, simple reagent strip test.
  • the most conventional test for the determination of the susceptibility of microorganisms to various antibiotics is a two-step test. First, a sample containing the pathogen is incubated in a conventional culture medium and allowed to grow. Next, the antibiotic susceptibility of the pathogen is measured by placing multiple small paper discs impregnated with various antibiotics, or alternatively various concentrations of antibiotic, on the culture medium containing the pathogen. After a second incubation period with the discs, antibiotics efficacy is indicated by the degree of growth inhibition found around the discs. Depending upon the absolute magnitude of this so-called zone of inhibition, the microorganisms in the sample are classified as being either:
  • Ericcson (U.S. Patent No. 5,028,529) teaches a variation on the two-step disc test.
  • Ericcson's test uses a single rectangular carrier strip containing a gradient of a compound instead of multiple discs differing in concentration. The strip is placed on a culture medium inoculated with a cultured sample containing micro- organisms. Measurement of the growth inhibition zone's curve in the culture medium is used to determine the optimum antibiotic concentration necessary for treatment.
  • Another method utilized to test antibiotic efficacy against microorganisms, involves the use of a sample plate with channels containing different concentrations and types of antibiotics. An agar solution containing a microbial sample is added into the channels and the plate is incubated. Bacterial colonies growing in the channels are counted and compared to determined which antibiotics best inhibit microbial growth. While the latter method may be considered an improvement over the first described method because only one incubation period is required, the testing process still requires wet media, sterilized equipment, and greater technical skill than a one-step dry test would require. Also, the equipment must be cleaned and resterilized before each use.
  • the testing device of this invention provides a bibulous matrix impregnated with a nutrient medium containing test reagents, an inhibitory agent or several inhibitory agents immobilized in distinct areas within the matrix, and a culture-fixing agent.
  • a substrate and container may be provided to form an integral test device.
  • the impregnated matrix is dipped momentarily into, or otherwise saturated with, liquid to inoculate the matrix which absorbs a known volume of sample.
  • the inoculated device is then incubated in a sealable container for a period of up to forty-eight hours.
  • Microorganisms present on the matrix will be inhibited from propagating and forming distinct colonies if the test agent in the matrix is effective against those microorganisms. Efficacy is measured by comparing colony formation within impregnated regions against a control matrix or region of matrix containing no inhibitory agent.
  • This unitized system consisting of both growth medium and inhibitory agent co ⁇ existing in a single phase allows testing fluids not only for the presence of microorganisms, but also to detect efficacy of an inhibitory agent against said microorganisms for the purposes of their control / eradication. Furthermore, the present invention provides a simple and inexpensive test that is less generative of waste as compared to currently utilized procedures.
  • Fig. 1 is an elevational view of a test device of this invention enclosed within a container.
  • Fig. 2 is an enlarged fragmentary sectional view of the device of Fig. 1 removed from the container, said view being taken along the line 2 - 2 of Fig. 1, and Figs. 3, 4, 5, and 6 are plan views of device matrices showing colony locations in conjuntions therewith.
  • Fig. 7 is a plan view of a matrix in which no colonies have grown.
  • test device 10 generally includes absorbent control and test matrices lie & l it, both attached to a substrate 12 enclosable within a sealable container 13.
  • the present invention provides for a test device for analyzing sample specimens, such as liquids, for microorganisms and their susceptibility to inhibitory agents.
  • sample specimens such as liquids
  • microorganisms any specimen sample suspected of having microorganisms therein (e.g., food, tissue, ground water, cooling water, pharmaceutical products, sewage, etc.) can be assayed
  • the invention is particularly useful for bacterial detection in aqueous liquids, such as human and animal biological fluids (e.g., urine, spinal fluid, blood, milk and the like, as well as stool secretions) and suspensions of animal or human tissues.
  • the preferred biological fluid used in practicing this invention is urine (diluted or undiluted).
  • the matrices l ie & l it are formed from a flat bibulous material, such as absorbent filter paper or the like, impregnated with a suitable nutrient medium, a reagent, and a culture-fixing agent.
  • the impregnation mixture for matrix l it includes an inhibitory agent(s) as well.
  • the matrices are adapted to be inoculated by dipping them into the sample specimen to be assayed and absorbing a portion thereof, thereby obviating the necessity to measure and/or dilute the sample. It is necessary for the matrix to have a known constant absorption capacity for purposes of semi -quantitative determinations.
  • each matrix is known to absorb a known volume of the sample, i.e., 0.1 milliliter, the concentration of microorganisms in the sample and their susceptibility to the inhibitory agent(s) at a particular concentration can be easily determined, as hereinafter described.
  • microorganisms refers to the general class of microorganism comprising yeast, other fungi, bateria, protozoa, etc. present in the sample to be analysed.
  • colony refers to the "micro ⁇ organisms” subsequent to culturing.
  • colony location indicates the site of a colony as observed on the matrices and is synonymous, for the purpose of this disclosure, with the term colony.
  • the nutrient medium can be any conventional medium, or modification thereof, known to provide a suitable environmental for the selected test.
  • a general nutrient medium such as Bacto Brain Heart Infusion, com-bitally available from Difco Laboratories, Detroits, Michigan, USA is preferred.
  • the reagent preferably includes an indicator capable of providing a color change in the matrix in response to a positive culturing of microorganisms.
  • the reagent may be included in the nutrient medium and may comprise a simple pH indicator such as phenol red or the reagent may include a reduction-oxidation type indicator such as one of the various tetrazolium compounds, i.e., 2,3,5-triphenyltetrazolium chloride (colorless), which compound is reduced to a formazan, i.e., triphenyl- tetraformazan (intense red), by most microorganisms during their growth.
  • triphenyltetrazolium as an indicator is preferred, since the reduced product, triphenyltetraformazan, not only provides a bright visible color, it also has the characteristics of being insoluble.
  • the insoluble property combined with the hereinafter described culture-fixing agent, facilitates the formation of separate and distinct colony locations.
  • the addition of a culture-fixing agent to the impregnation formulations is neces ⁇ sary to localize motile microorganisms in the matrices subsequent to inoculation.
  • the ability of the culture-fixing agent to localize the microorganisms, and thereby enable the formation separate and distinct colonies in and on the matrix, is particularly important to enable the cultured colonies to be counted for purposes of performing meaningful semi-quantitative tests.
  • the fixing agent is used in the impregnated formulation in concentrations ranging from about 0.1 to about 5.0 percent by weight, with a range of about 0.5 to about 3.0 percent by weight preferred.
  • the fixing agent is characterized as being soluble in an aqueous solution to form a viscous colloidal suspension.
  • Fixing agents found to be suitable are inert gums, such as carrageens and various linear polysaccharides or alginates.
  • this invention is distinguishable from the prior art by virtue of one or more inhibitory component(s) added to the bibulous matrix.
  • the addition of the inhibitory component(s) allows its (their) suitability for the task at hand, e.g., antibiotic susceptibility, to be determined in one simple test.
  • antibiotic efficacy tests require long incubation periods, multiple phases and highly trained personnel.
  • the present invention incorporates such efficacy testing into a single, simple diagnostic test.
  • the term "inhibitory agent” refers to any compound capable of killing or inhibiting the growth of a microorganism.
  • Pre ⁇ ferred inhibitory agents include classical groups of antibiotics such as the amino- glycosides, beta-lactams, cephalosporins, chloramphenicols, macrolides, nitro- furanes, sulfonamides, tetracyclines, quinolones and any derivatives thereof, e.g., salts, adducts, etc.
  • inhibitory agents refers to any chemical substance which is active against microorganisms, such as disinfectants, anti-mycotics, anti ⁇ viral agents, and anti-tuberculosis agents. However, it should be understood that not all inhibitory agents are operable for this purpose.
  • the determination of whether a given agent is operable and within the scope of the present invention is dependent upon the agent's inherent stability characteristics and its compatibility with the growth medium selected and/or the indicator system employed. Because of their favorable characteristics, quinolone antibiotics, such as ciprofloxacin and enrofloxacin, are especially preferred for this application. When other types of antibiotics are employed, particular attention must be given to the aforementioned factors, among others.
  • one or more of the zones of the elements of this invention can contain a variety of other desirable, but optional, components, including buffers, surfactants, binders and/or (micro-)encapsulating agents, as is know in the art.
  • the impregnated agent(s) is (are) effective in inhibiting growth and would therefore be effective in combating the microorganism.
  • body fluids includes such fluids as blood, liquor, pus, sputum, blad ⁇ der content or urine, and any other secretion which may be isolated from an animal source in order to diagnose microbial infection.
  • the term "animal” refers to human beings as well as the wide arrays of types of animals normally treated by veterinarians including, but not limited to dogs, cats, rabbits, horses, cows and pigs.
  • One the matrices l ie &l lt are impregnated, they are then dried. Since a high temperature may affect some of the nutrient and test materials impregnated into the matrices, it has been found that drying the impregnated matrices in a vacuum or forced air oven at about 40°-60°C for 1 to 3 hours is preferred, although temperatures as high as 100°C for times as brief as 15 minutes have been success ⁇ fully employed.
  • the dried impregnated matrices l ie & l it are attached to a substrate or handle 12.
  • the substrate 12 is an elon ⁇ gated member preferably formed of a semi-stiff, insoluble material, such as a strip of polyethylene terephthalate, polystyrene, or the like. Since the matrices lie & l it are to be adapted for macrobiological purposes, special care must be taken to secure them to the substrate 12 with a microbiologically inert adhesive (not shown). Certain double-faced tapes, such as No. Y9494, commercially available from 3M Company, Minneapolis, Minnesota, USA, have been found suitable for this purpose.
  • the matrices lie & lit can also be readily attached to the substrate 12 by bonding without affecting them, the impregnated medium, the reagent or the inhibitory agent(s).
  • a thin polyethylene sheet 14 (Fig. 2) is disposed between the substrate 12 and each matrix l ie & l it, and sufficient heat is applied to the side 16 of the substrate 12 distal to each matrix lie & lit to cause the sheet 14 to melt and bond the matrices 1 lc & 1 It to the substrate 12.
  • the matrices l ie & l it (Fig.1) and attached substrate 12 are then placed in the sealable container 13, and the container 13 is sealed.
  • any conventional container 13 capable of being reseated has been found suitable for this purpose. It has been found that an envelope of polyethylene, or the like, which is sealed along it sides 17 & 18 and bottom 19 is acceptable for this purpose.
  • the top 21 of the container envelope 13 is preferably provides with a conventional interlocking seal lip 22 which frictionally seals the container 13 upon the application of pressure thereon and thus obviates the necessity for special sealing instruments.
  • the container 13 is formed of a clear transparent material which the container 13 without exposing either the operator or laboratory to the cultured microorganisms or to the inhibitory agent(s).
  • the contaner 13 is unsealed and the substrate 12 and attached matrices l ie & 1 It are removed therefrom.
  • the matrices are dipped into the liquid sample to be tested, such as urine, milk, water, or the like, to rehydrate and inoculate the matrices l ie & l it.
  • the inoculated matrices l ie & l it and attached substrate 12 are aseptically replaced in the container 13, and the container 13 is resealed and incubated at the optimum temperature, depending upon the suspected micro- organisms. Most microorganisms are optimumly incubated at temperatures ranging from about 4° to about 40°C, and preferably from 35° to about 39°C.
  • the matrices 1 lc & 1 It are visually observed and the results of the particular test are read. The number of colored spots, indicating colony locations, are counted and recorded, as hereinafter described.
  • the device 10 may be sterilized and discarded. It has been found, however, that sterilization does not affect most color changes formed in the matrices lie & l it as a result of the culturing of the microorganisms, and the device 10, or merely the matrices l ie & l it, may be retained as a permanent record if desired.
  • microbiological populations are often of such magnitude that it is difficult to express them easily or exactly.
  • the microbiologist uses exponents of 10.
  • a concentration of microorganisms per milliliter of specimen expressed as 10 2 is equivalent to 100 microorganisms per milliliter, 10 3 to 1,000, 10 4 to 10,000, and so on.
  • the exponent of 10 represents the number of zeros to the right of the digit 1. Since only the relative concentration of microorganisms in a sample is sufficient for microbiological purposes, this manner of expressing the number of micro ⁇ organisms per milliliter is not only adequate, but expedient.
  • the ability to semi-quantitatively assess the number of microorganisms in the sample being tested is an essential aspect of the device, since the concept of inhibitory agent(s) efficacy is only meaningful in relation to real and significant microbiological load.
  • the intention of the device is two-fold: First, it must demonstrate that the number of microorganisms present is significant enough to warrant further action. Provided that this condition is met, the second task of the device is to permit an assessment of the efficacy of a particular inhibitory agent or agents in combating the microorganisms present.
  • Urinary tract infection is usually associated with baterial counts of 100,000 (10 5 ) organisms per milliliter, a condition referred to as significant bacteriuria, and here it is crucial to be certain that a given antibiotic is effective against the pathogens at hand before selecting it for treatment.
  • Figs. 3-6 the significance of the culture-fixing agent can be best illustrated in Figs. 3-6.
  • the appearance of 4 locations 23a can be observed. This number would be indicative of 20 microorganisms per milliliter or a concentration expressed as 10 1 .
  • the locations 23b appearing on Fig. 4 would indicate a concentration of 10 2 .
  • the locations 23c and 23d on Figs 5 and 6, respectively, while clear and distinct, become too numerous to easily count and can best be interpreted by comparing same to a standard or chart showing the approximate number of locations that would appear on a corresponding matrix, and assigning concentrations of 10 3 and 10 4 to the Figs. 5 and 6, respectively.
  • test matrix 1 By comparing the appearance of the control and test matrices l ie & lit following incubation, it will be evident whether or not and, if so, to what extent the inhibitory agent(s) has (have) effectively suppressed the growth of and/or killed the microorganisms contained in the sample. Hence, the relative appearance of the test matrix 1 It will allow the microorganisms to be classified as either:
  • Test samples were prepared by propagating pure cultures of Escherichia coli, Enterobacter cloacae, Klebsiella pneumoniae, Proteus vulgaris, Serratia marcesens, Pseudomonas aeruginosa, Staphylococcus aureus and Streptococcus faecalis in brain heart infusion broth to a maximum turbidity of « 10 9 cells per milliliter.
  • Tenfold serial dilution of each culture were prepared using sterile saline as a diluent to provide serial bacterial suspensions of each culture at 10 3 , 10 s and 10 7 colony forming units (CFU) per milliliter.
  • the devices were withdrawn from the incu ⁇ bator and the matrices were observed through the transparent containers.
  • the reduction of the colorless triphenyltetrazolium to the magenta colored tri- phenyltetraformazan was visible in all of the control pads.
  • the physical distribution of this color formation stood in definite and distinct correlation to the concentration of microorganisms in the test suspensions.
  • the invention also contemplates the production of only the impregnated matrices.
  • conventional laboratory equipment such as forceps and slide mailers, can be used to replace the substrate 12 and container 13, respectively.
  • the dehydrated matrix could be held by the forceps and dipped into the samples to be analyzed and subsequently placed in a slide mailer and incubated.
  • the impregnated matrices l ie & l it may be rehydrated and inoculated, if desired, by adding a measured volume of the sample directly to each matrix, rather than by the immersion procedure hereinbefore described.
  • the device is adaptable for detecting the presence of microorganisms in samples other than liquids.
  • the matrix 11 can be rehydrated with sterile water, or the like, and inoculated by contact with the "solid" sample to be analyzed.
  • the present invention is not limited to the specific bibulous materials, nutrient media, reagents, inhibitory agents, or fixing agents described in the above examples, as one skilled in the art will recognize that a wide variety or combination of bibulous material, nutrient media, reagents, inhibitory agents and fixing agents can be employed to carry out the purpose of the novel test device.

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  • Chemical & Material Sciences (AREA)
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  • Health & Medical Sciences (AREA)
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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention décrit un dispositif amélioré de contrôle pour détecter les micro-organismes et leur sensibilité à des agents inhibiteurs, à savoir des antibiotiques, se trouvant dans des spécimens échantillons. Le dispositif est conçu pour subir une inoculation par immersion dans un spécimen, et ensuite pour contenir la culture des micro-organismes, par incubation dans un récipient scellé hermétiquement. Généralement, le dispositif comporte une matrice absorbante imprégnée avec un milieu nutritif contenant des réactifs d'examen, un agent inhibiteur ou plusieurs agents inhibiteurs différents situés dans des zones distinctes de la matrice, ainsi qu'un agent fixateur de culture servant à déterminer l'emplacement des colonies tout au long de la période de culture. Cette invention est particulièrement utile dans les cas impliquant des infections urinaires.
PCT/EP1996/002400 1995-06-07 1996-06-03 Dispositif de controle mesurant la sensibilite des micro-organismes a des agents inhibiteurs WO1996040981A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU62212/96A AU6221296A (en) 1995-06-07 1996-06-03 Testing device for determining the susceptibility of microor ganisms to inhibitory agents

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US48666795A 1995-06-07 1995-06-07
US08/486,667 1995-06-07

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WO1996040981A1 true WO1996040981A1 (fr) 1996-12-19

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AR (1) AR002368A1 (fr)
AU (1) AU6221296A (fr)
WO (1) WO1996040981A1 (fr)
ZA (1) ZA964728B (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999018232A1 (fr) * 1997-10-02 1999-04-15 Idexx Laboratories, Inc. Detection de microorganismes pathogenes et leur sensibilite aux antimicrobiens
EP1253203A1 (fr) * 2001-04-25 2002-10-30 Becton Dickinson and Company Germination, développement, immobilisation et détéction rapide des microorganismes
FR2985520A1 (fr) * 2012-01-10 2013-07-12 Biomerieux Sa Procede pour capturer et concentrer un microorganisme dans un echantillon biologique

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3881993A (en) * 1971-03-16 1975-05-06 Miles Lab Testing device for microorganisms
AU1157876A (en) * 1976-03-02 1977-09-08 Mc Donnell Douglas Corp Antibiotic susceptibility test and device for microorganisms biotics
EP0322591A2 (fr) * 1987-12-01 1989-07-05 Abbott Laboratories Essai pour tester la résistance aux antibiotiques
EP0550903A1 (fr) * 1992-01-10 1993-07-14 Bayer Ag Dérivés d'acides carboxyliques de quinolone- et de naphthyridone comme agents antibactériens

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3881993A (en) * 1971-03-16 1975-05-06 Miles Lab Testing device for microorganisms
AU1157876A (en) * 1976-03-02 1977-09-08 Mc Donnell Douglas Corp Antibiotic susceptibility test and device for microorganisms biotics
EP0322591A2 (fr) * 1987-12-01 1989-07-05 Abbott Laboratories Essai pour tester la résistance aux antibiotiques
EP0550903A1 (fr) * 1992-01-10 1993-07-14 Bayer Ag Dérivés d'acides carboxyliques de quinolone- et de naphthyridone comme agents antibactériens

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999018232A1 (fr) * 1997-10-02 1999-04-15 Idexx Laboratories, Inc. Detection de microorganismes pathogenes et leur sensibilite aux antimicrobiens
US6984499B2 (en) 1997-10-02 2006-01-10 Idexx Laboratories, Inc. Method and apparatus for concurrently detecting pathogenic organisms and antimicrobial susceptibility
EP1253203A1 (fr) * 2001-04-25 2002-10-30 Becton Dickinson and Company Germination, développement, immobilisation et détéction rapide des microorganismes
US7642060B2 (en) 2001-04-25 2010-01-05 Becton, Dickinson And Company Rapid resuscitation, growth, capture and detection of microorganisms
FR2985520A1 (fr) * 2012-01-10 2013-07-12 Biomerieux Sa Procede pour capturer et concentrer un microorganisme dans un echantillon biologique
WO2013104858A1 (fr) * 2012-01-10 2013-07-18 Biomerieux Procede pour capturer et concentrer un microorganisme dans un echantillon biologique

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AU6221296A (en) 1996-12-30
ZA964728B (en) 1997-01-07
AR002368A1 (es) 1998-03-11

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