WO2004050675A1 - Tests concurrents d'identification de micro-organismes et de sensibilite a partir d'un bouillon - Google Patents

Tests concurrents d'identification de micro-organismes et de sensibilite a partir d'un bouillon Download PDF

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Publication number
WO2004050675A1
WO2004050675A1 PCT/US2002/038543 US0238543W WO2004050675A1 WO 2004050675 A1 WO2004050675 A1 WO 2004050675A1 US 0238543 W US0238543 W US 0238543W WO 2004050675 A1 WO2004050675 A1 WO 2004050675A1
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Prior art keywords
kit
media
broth
antimicrobial
microorganisms
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PCT/US2002/038543
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English (en)
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Read Robert Taintor
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Read Robert Taintor
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Priority to CA002549213A priority Critical patent/CA2549213A1/fr
Priority to EP02797169A priority patent/EP1594881A4/fr
Priority to AU2002362043A priority patent/AU2002362043A1/en
Priority to PCT/US2002/038543 priority patent/WO2004050675A1/fr
Publication of WO2004050675A1 publication Critical patent/WO2004050675A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING 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
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C12Q1/045—Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/12—Well or multiwell plates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
    • C12M33/02—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus by impregnation, e.g. using swabs or loops
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
    • C12M33/04—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus by injection or suction, e.g. using pipettes, syringes, needles
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor

Definitions

  • TITLE CONCURRENT MICROORGANISM IDENTIFICATION AND SUSCEPTIBILITIES FROM BROTH
  • This invention relates to the concurrent identification (ID) and antimicrobial susceptibility testing (AST) of an unknown microorganism or microorganisms manually determined using one kit from specimen to results in normally 24 hrs.
  • antibiotic-resistant bacteria multi-resistant bacteria
  • Antibiotics are indiscriminately used, and this has contributed to the rise of antibiotic resistance in a variety of bacteria, including species of Enterococcus, Staphylococcus, Pseudomonas, and the Enterobacteriaceae family.
  • the emergence of antibiotic-resistant organisms is partly the result of the over-use of broad-spectrum antibiotics.
  • There is also concern that inappropriate veterinary use of antibiotics may lead to development of antibiotic resistant bacteria, which could in turn infect humans.
  • BACKGROUND- Prior Art-Specimen Collection and Transport The specimen must be material from the actual infection site.
  • the transport system consists of a protective container, transport medium and the culture swab (FIG 7).
  • a problem with the use of a holding or transport medium is that it may jeopardize the recovery of certain strains.
  • a major task is to reduce the time delay between collection of specimens and inoculation onto microbiological culture media.
  • the transport container is constructed to minimize hazards to specimen handlers. It is best to minimize adverse environmental conditions, such as rapid changes in pressure, exposure to extremes of heat and cold or excessive drying.
  • the transport of fluid specimens to the laboratory must be done as quickly as possible. It is recommended that a 2-hr maximum time limit be imposed between collection and delivery of specimens to the laboratory. This limit poses a problem for specimens collected any distance from a clinical microbiology laboratory.
  • a satisfactory microbiological culture medium must contain available sources of water, vitamins, inorganic phosphate and sulfur, trace metals, carbon and nitrogen. These needs are supplied from a number of sources. In addition, there are agents that manipulate what organisms can grow and others that enhance identification. The following is a list of common media constituents with their sources in parenthesis: (1) Amino-nitrogen (peptone, protein hydrolysate, infusions and extracts), (2) Growth factors (blood, serum, yeast extract or vitamins, NAD), (3) Energy sources (sugar, alcohols, and carbohydrates), (4) Buffer salts (Phosphates, acetates and citrates), (5) Mineral salts and metals (phosphate, sulfate, magnesium, calcium, iron), (6) Selective agents (chemicals, antimicrobials and dyes), (7) Indicator dyes (phenol red, neutral red), and (8) Solidifying agents (agar, gelatin, alginate, silica gel, etc.).
  • Amino-nitrogen peptone, protein hydrolys
  • the media can be In a liquid or a solid form.
  • Solid media provides for the isolation of microorganisms contained in a mixture of different microorganisms.
  • Liquid media referred to as "Broth”
  • Broth can provide a nutritionally rich environment which is more accessible to the individual cells than solid media. This allows the microorganisms to grow rapidly but they are not isolated from each other.
  • Brain Heart Infusion Broth is a rich media supplying many of the compounds that the cell would otherwise have to synthesize. This allows the cell to devote more of its energy to growth, which is another reason for their faster growth in liquid media.
  • a selection of the appropriate solid culture media for microbiological test(s) is made according to the particular specimen type. Several hundred culture media are commercially available.
  • ENRICHED MEDIA have special additives to support pathogens having fastidious growth needs. Examples of media include sheep blood agar and Brain Heart Infusion Broth. DIFFERENTIAL MEDIA allows differentiating of groups of microorganisms based on indicator color changes (such as pH) in the culture medium that take place as a result of biochemical reactions associated with microorganism growth. Separating organisms that ferment the sugar lactose, for example, from those that do not, is one example of differential media.
  • SELECTIVE MEDIA support the growth of certain microorganisms of interest, while suppressing the growth of others.
  • Azide blood agar is an example. Gram-positive organisms grow on this media whereas gram-negative organisms do not.
  • DIFFERENTIAL-SELECTIVE MEDIA combine the last two characteristics, to allow the selective growth and rapid differentiation of major groups of bacteria. These media are widely used for gram-negative
  • the equipment required for the primary inoculation of specimens consists of several microbiological agar-based media plates and a nichrome or platinum inoculating wire or loop (see FIG 8B-8E).
  • the plates generally have a shelf life of from one to two months. Streaking out the specimen spreads the microorganisms across the surface of the culture medium. This results in isolated colonies.
  • the first step is to touch and roll the tip of the swab 84 containing the specimen 116 on the surface of the medium (FIG.
  • ID is the culture requirements, which includes the atmospheric needs of the organism as well as nutritional requirements and ability to grow on different kinds of media.
  • a further basis of ID in regards to the biochemical characteristics includes the mode of carbohydrate utilization, catalase reactions of gram-positive bacteria and oxidase reactions of gram-negative bacteria. ID to the
  • 125 species level is based on a set of physiological and biochemical characteristics including the degradation of carbohydrates, amino acids, and a variety of other substrates.
  • kits perform a number of various biochemical reactions. The results of these reactions can reveal unique patterns for ID. Some systems are automated and others are manual. A problem with manual systems is the limited scope in terms of the organisms they target for ID. Additionally it is 130 necessary to first isolate the organism of interest from other microorganisms in an 18 to 24hr isolation step as described above before applying the organism to the manual or automated ID system.
  • the manufacturer bioMerieux Vitek® markets the following manual systems (listing the target organisms): API 20C AUX (yeasts), API 20E (Enterobacteriaceae and non-fermenting gram-negative bacteria), API 30 Strep.
  • the prior art calls for initial isolation and identification of the organism first and then, if deemed appropriate, i.e. where a pathogen is identified, performing an antimicrobial susceptibility test.
  • the analyst must decide which microorganism is responsible for the clinical disease in mixed cultures.
  • AST antimicrobial susceptibility testing
  • Two of them are disk-diffusion and micro dilution. In recent years, there has been a trend toward the use of
  • the initial isolation step results in colonies formed from a single microorganism.
  • the analyst then transfers like colonies into growth broth.
  • the broth Is incubated at 35°C for 2 to 8 hr until growth reaches
  • McFarland 0.5 standard 94 160 the turbidity at or above that of a McFarland 0.5 standard 94. This turbidity is equivalent to 1.5 x 10 8 colony forming units (CFU)/ml.
  • McFarland standards are prepared using different amounts of barium sulfate in water. This salt is insoluble in water and forms a very fine suspension when shook. Within 15 minutes of adjusting turbidity, a cotton swab 85 transfers this inoculum to a Standard Susceptibility Dish 122. The entire surface of the Mueller-Hinton plate is swabbed three times, rotating the plate
  • FIG. 12A The plate stands for 3 to 15 minutes before AST disk 124 is applied. Apply to the agar surface with a dispenser or manually with sterile forceps. Apply gentle pressure to ensure complete contact of the disk with the agar. (FIG. 12B showing one disk added). Incubate for 16 to 18 hours at 35°C in an ambient-air incubator.
  • Fig. 12C illustrates the basic principle of the disk-diffusion method of AST. As soon as the antibiotic-impregnated 170 AST disk 124 is exposed to the moist agar surface, water is absorbed into the filter paper and the antibiotic 128 diffuses into the surrounding medium.
  • the rate of extraction of the antibiotic out of the disk is greater than its outward diffusion into the medium, so that the concentration immediately adjacent to the disk may exceed that in the disk itself. As the distance from the disk increases, however, there is a logarithmic reduction in the antibiotic concentration. If the plate has been previously inoculated with a
  • MIC inhibitory concentration
  • the Minimal inhibitory concentration (MIC) is the lowest concentration of a chemotherapeutic agent that will prevent growth of the test microorganisms.
  • the disk-diffusion test that has become standard in the United States is based on the work of Bauer, Kirby and coworkers.
  • the zone size observed in a disk-diffusion test has no meaning in and of itself.
  • the interpretative standards provided by the NCCLS show the correlation between zone sizes and MICs of
  • FIG. 13 shows a poorly prepared AST plate with objectionable overlapping of the zones of growth inhibition from adjacent disks.
  • FIG 14 shows a poorly streaked AST plate with uneven growth. The zone margins are indistinct, compromising accurate measurement.
  • the present method and kit relates to the ID of microorganisms and concurrent or consecutive antimicrobial susceptibility testing (AST).
  • AST antimicrobial susceptibility testing
  • the process is novel and unconventional because 210 the testing is done directly from an initial broth culture with no isolation step needed.
  • the method and kit offers quick characterization of microorganisms, in one-third the time of standard manual methods.
  • the Kit employs a disposable multi-chambered plate (kit plate) with enriched, differential, selective, and differential-selective media in addition to AST medium.
  • Broth medium is provided for growing up the microorganisms for eventual dilutions and inoculation onto the kit plate.
  • AST disk-quarters are included
  • the shelf life of the kit is at least 5 months from date of manufacture when stored at 4°C
  • the Kit comes complete to perform the testing, except for an incubator and a simple microscope.
  • a portable incubator can be operated from any direct current source such as an automobile battery.
  • Microorganism is understood to mean, in particular, microbes, bacteria and yeasts.
  • the kit is well suited 225 in areas where microbiology laboratories are scarce or unavailable.
  • the kit serves to obtain rapid AST information.
  • Microorganisms such as Anthrax (Bacillus anthracis) can be determined concurrently with drug susceptibility testing within 24hrs.
  • AST is useful and important for the common microorganism species that are not predictably susceptible to drugs of choice because of acquired resistance mechanisms (e.g., members of the Enterobacteriaceae, the Pseudomonas species, Staphylococcus species, Enterococcus species, Streptococcus pneumoniae, Haemophilus influenzae, and Neisseria gonorrhoeae).
  • acquired resistance mechanisms e.g., members of the Enterobacteriaceae, the Pseudomonas species, Staphylococcus species, Enterococcus species, Streptococcus pneumoniae, Haemophilus influenzae, and Neisseria gonorrhoeae.
  • an ideal specimen from the site of infection or a microorganism- containing sample can be immediately applied to broth culture media.
  • the use of transport media is unnecessary. Therefore, the specimen is not subjected to time delays; possible adverse environmental 245 conditions or excessive drying that would compromise its integrity. Fluid specimens can also be immediately processed. In addition, a more rapid result is realized with this system due to immediate inoculation of the specimen to broth culture.
  • the microorganism sample is ready for dilution and inoculation onto the multi-chambered kit plate within 4 to 8 hrs following broth culture incubation. 250
  • a method and kit comprising a multi-chambered, easily visualized culture kit plate comprising a battery of different media with diagnostic functionality.
  • the miniaturization of the media is very cost effective.
  • the multi-kit plate media performs the ID and AST of gram-negative and gram-positive organisms.
  • One chamber is devoted to fungi determination. Any errors or misjudgments in the prior art of media selection do not exist with the present system. An appropriate selection of medium is already
  • kits A special dilution method utilized with the kit simplifies the inoculation of the microorganism sample to the various media of the kit plate. Time is of the essence with an infection. The prior art of streaking each
  • the present kit utilizes a liquid dilution to a standard equivalent from a starting broth culture. A further dilution added to the kit results in individual colonies in the various media test chambers. A magnifying lens such as a microscope 10X objective turned backwards provides a good view of the morphology and chemistry of the microorganism's colony on the various media. This makes the prior art isolation step unnecessary.
  • Isolation and ID takes place together in the same chamber at the same time.
  • the method is very reproducible and the faster growing organisms such as members of the Enterobacteriaceae family can produce discernable colonies within 12 to 18 hrs.
  • a modified Kirby-Bauer disk-diffusion method used with this kit allows for flexibility in terms of choice of antimicrobial agents.
  • the method of placing the antibiotics into the AST test chambers is quick, using a novel method.
  • the resultant zone size is measured as radius whereas in a prior art standard method, zone size is measured as diameter which is
  • test chambers containing the AST media are well covered with a uniform inoculum of bacteria and produce a lawn of microorganism growth unlike the larger dishes that are prone to unevenness.
  • each antimicrobial is in its own chamber, there is no overlapping of the zones of growth inhibition from adjacent disks.
  • 285 is manifest when there is more than one organism on the kit plate. When more than one zone is evident, morphology of the more resistant organism (inner zone) can to observed by taking a sample of inner zone bacteria and observing microscopically. It has been observed that routine cultures that grow three or more organism types should be discounted. Specimens obtained from non-sterile sites most commonly represent colonization or contamination.
  • kits and methods for ID and AST that can yield results in one-third the time of the prior art methods.
  • This method and kit can accomplish both rapid, straightforward ID and AST of an unknown microorganism or microorganisms from a single sample, where a prior isolation step is not required. Therefore, the patient can start on the correct antibiotic by the next day and avoid having to take an 295 incorrect empirical antibiotic for a 3-day period, as would be the case in the prior art. Where the infection is life threatening, it is a powerful approach to the problem.
  • kit plate component has a shelf life of at least 5 months when stored at 4°C.
  • FIG. 1 shows a perspective view of the preferred embodiment of the present invention, a multi- chambered kit plate with square test chambers. Eight of the test chambers show antimicrobial disk- quarters in the corners of the test chambers.
  • Fig. 2 shows preferred embodiment kit plate and lid with various selective, differential and non- selective agar-based solid media.
  • the lower right eight test chambers contain Mueller Hinton 310 agar.
  • Fig. 3 shows the preferred embodiment of the method of inoculation of Brain Heart infusion broth from a specimen and subsequent incubation called the initial broth culture
  • Fig. 4 shows three McFarland turbidity standards with a dilution of the incubated broth.
  • Fig. 5 shows the preferred embodiment method of applying a 0.5 McFarland equivalent dilution of 315 incubated microorganisms to the bottom three rows of the kit plate.
  • Fig. 6 shows the preferred embodiment device used to apply the antimicrobial disk-quarters to the susceptibility test chambers and a depiction of a set of antimicrobial disk-quarters.
  • Fig. 7 shows a typical culture transport system
  • Fig. 8A to 8E shows the prior art addition of a microorganism sample to a standard solid media plate 320 and subsequent streaking process which dilutes out the microorganism.
  • Fig. 9 shows a Bunsen burner
  • Fig. 12A to 12D shows the method and principle of the prior art AST Kirby-Bauer disk-diffusion test.
  • 325 Fig. 13 shows a poorly prepared Kirby-Bauer disk diffusion test with overlapping zones from prior art.
  • Fig. 14 shows a poorly prepared AST test with under applied bacteria from prior art.
  • Fig. 15 shows one embodiment of the inoculation of Thioglycolate broth and subsequent incubation.
  • Fig. 16A to 16D shows the preferred embodiment method of preparing dilutions directly from initial broth culture (without an isolation step) for inoculating the kit plate.
  • Fig. 17 shows the preferred embodiment method of applying a further microorganism suspension dilution (1 to 1000 of the 0.5 McFarland equivalent dilution) of to the top two rows of the kit plate.
  • Fig. 18 shows the preferred embodiment method of applying antimicrobial disk-quarters to the corners of the Mueller Hinton test chambers using the placement device.
  • Fig. 19A and 19B shows the principle of the standard Kirby-Bauer disk-diffusion test and illustrates 335 how the preferred embodiment system measures exactly one-half of the measurement of the prior art standard method.
  • Fig. 20A to 20D shows the preferred embodiment method for determining nitrate reductase activity as part of the kit.
  • Fig. 21 A to 21 C shows the preferred embodiment method for determining cytochrome oxidase activity 340 as part of the kit.
  • Fig. 24A to 24C CONVERTED TO TABLE I, for identifying non-fastidious gram-negative bacteria using kit results.
  • 345 Fig. 25 shows a blank kit plate before addition of microorganisms.
  • Fig. 26 shows an example of a kit plate incubated for 16 hrs following inoculation with the gram- positive organism Enterococcus faecalis ATCC 29212
  • Fig. 27 shows an example of the preferred embodiment kit plate incubated for 20 hrs following inoculation with the gram-positive organism Streptococcus pyogenes ATCC 19615 350
  • Fig. 28 shows an example of the preferred embodiment kit plate incubated for 16 hrs following inoculation with the gram-positive organism Staphylococcus epidermidis ATCC 12228
  • Fig. 29 shows an example of the preferred embodiment kit plate incubated for 16 hrs following inoculation with the gram-positive organism Staphylococcus aureus ATCC 25923
  • Fig. 30 shows an example of the preferred embodiment kit plate incubated for 16 hrs following 355 inoculation with the gram-positive organism Staphylococcus aureus ATCC 29213
  • Fig. 31 shows an example of the preferred embodiment kit plate incubated for 16 hrs following inoculation with the gram-negative organism E.coli ATCC 25922
  • Fig. 32 shows an example of the preferred embodiment kit plate incubated for 16 hrs following inoculation with the gram-negative organism Klebsiella pneumoniae ATCC 13883 360
  • Fig. 33 shows an example of the preferred embodiment kit plate incubated for 24 hrs following inoculation with the gram-negative organism Pseudomonas aeruginosa ATCC 27853
  • Fig. 34 shows an example of the preferred embodiment kit plate incubated for 20 hrs following inoculation with the gram-negative organism Proteus vulgaris ATCC 13315
  • Fig. 35 shows an example of the preferred embodiment kit plate incubated for 16 hrs following 365 inoculation with the gram-negative organism Salmonella typhimurium ATCC 14028
  • Fig. 36 shows an example of the preferred embodiment kit plate incubated for 48 hrs following inoculation with the fungus Candida albicans ATCC 14053
  • Fig. 37A shows an example of an incubated kit plate consisting of a mixture of E.coli and Staphylococcus aureus.
  • 370 Fig. 37A to 37C illustrates the microorganisms in Fig.37A being differentiated on the preferred embodiment kit plate in terms of ID as well as AST.
  • Fig. 38 shows a gram stained slide of the two microorganisms from Fig. 37 separated by their difference in AST to two antimicrobial agents.
  • Fig. 39A to 39E illustrates two microorganisms being differentiated on the preferred embodiment kit plate in terms of differences in medium selectivity, fermentation and production of fluorescent product formation.
  • Fig. 1 shows a perspective view of the preferred embodiment of a portion of the present invention, an ethylene oxide sterilized polypropylene multi-chambered kit plate77 with square test chambers. Eight of the test chambers (67-70' and 72 -75') show antimicrobial disk-quarters 76 in the corners of the test chambers. Quartering Standard Kirby-Bauer AST disks using a plastic jig and a razor blade is one way of preparing the disk-quarters.
  • Fig. 2 depicts a view of the preferred embodiment kit plate before antimicrobial disk-quarters are applied to the kit plate 80. Lid 78 also shown.
  • Fig. 3 illustrates Brain Heart infusion broth (BHIB) 82 and a specimen containing culture swab84. The 395 breakaway cap 86 and stopper 88 are associated with the broth 82 container. Incubator 135 and Inoculated incubated Brain Heart infusion broth (IIBHIB) 90 are also illustrated.
  • BHIB Brain Heart infusion broth
  • IIBHIB Inoculated incubated Brain Heart infusion broth
  • Fig. 4 illustrates a dilution 96 of the Inoculated incubated Brain Heart infusion 90, to a concentration that is equivalent to a 0.5 McFarland turbidity standard 94. Other standards are zero McFarland turbidity standard 92 and 1 McFarland turbidity standard 98. 400 Fig. 5 illustrates the preferred embodiment kit plate 80 ready to receive dilution 96, one of two dilutions used for kit plate inoculation. Disposable sterile pipette 100 is also shown.
  • Fig. 6 shows a device 102A shown in the charging position used in the placement of antimicrobial disk-quarters 110.
  • the device comprises two parts: a quilter's pin 104 and a push-off slider 106 made from a small pipette tip.
  • the antimicrobial storage container 108 is also shown.
  • Fig.18 illustrates the 405 placing device in the discharge position 102B
  • Fig. 37A illustrates an incubated kit plate containing two microorganisms and will be described in more detail later on.
  • Fig. 15 illustrates Thioglycolate broth (Thio) 132 and a culture swab containing an initial specimen sample 84.
  • the breakaway cap 86 and stopper 88 are associated with the broth 132 container.
  • Incubator 410 135 and Inoculated incubated Thioglycolate broth 134 is also illustrated.
  • Fig. 16A-16D illustrates components for preparation of dilutions from the Inoculated incubated Brain heart infusion broth (IIBHIB) 90: pipette 100, sterile diluent 136, cap 142, McFarland turbidity standards.'O" Standard 92, "0.5” Standard 94, and "1.0” Standard 98 and additional diluents 138 and 140.
  • Fig. 17 illustrates the preferred embodiment kit plate 80, ready to receive dilution140, second of two 415 dilutions. Also shown is a disposable sterile pipette 100.
  • Fig. 18 shows a cut away view of a multi-chambered kit plate 77' with antimicrobial disk-quarters76 placed in Mueller Hinton containing test chambers (67', 68', 69', 70', 72', 73', 74', 75').
  • the antimicrobial disk-quarter placement device is at discharge position 102B. Also shown is device in charge position 102A, a set of antimicrobial disk-quarters 110 (representing 8 different antimicrobial agents), container 420 108, and finger and thumb placement positions for manipulating the placement device: position 129T, position 129F, position 131T, and position 131F.
  • Fig. 19A-19B illustrates the equivalence between prior art and an AST chamber 144 from the kit multi- chambered kit plate 77.
  • the principle of the standard Kirby-Bauer disk-diffusion AST test is illustrated in Fig. 19A (review Figs 12C-12D).
  • This illustration shows how the preferred embodiment system (Fig. 19B, 425 zone radius 142) measures exactly one-half of the measurement of zone diameter 141 , the prior art standard method.
  • the zone of inhibition radius measurement 142 is measured from the corner of the chamber containing disk-quarter 76 to margin 126.
  • Fig. 20A-20D shows the components of a modified nitrate reductase assay: reagent vial lid 145, 430 Griess reagent sulfanilamide 146, Griess reagent N- (1-napthyl) ethylenediamine 148, and phosphoric acid diluent 150. When the three are combined, they make up the Griess working reagent 152.
  • Fig. 20C illustrates a positive griess reaction 154 or a negative griess reaction 156.
  • Fig. 20D illustrates the addition of zinc powder 158 and either a positive griess reaction after zinc 160 or a negative griess reaction after zinc 162. 435 Fig.
  • 21A-21C shows the components of a cytochrome oxidase assay: applicator 164, oxidase test paper 166, and water 168.
  • Fig. 21 B shows sample 170 addition and
  • Fig. 21 C illustrates a positive oxidase test 172 or a negative oxidase test 174.
  • Fig. 25-Fig. 36 show results of incubations with a number of different microorganisms.
  • the OPERATION OF INVENTION section will describe the details of ID and AST for each kit plate.
  • 445 Fig. 25 shows preferred embodiment blank kit plate before addition of microorganisms.
  • Fig. 26 shows an example of preferred embodiment kit plate inoculated and incubated for 16 hrs with the gram-positive organism Enterococcus faecalis ATCC 29212
  • Fig. 27 shows an example of preferred embodiment kit plate inoculated and incubated for 20 hrs with the gram-positive organism Streptococcus pyogenes ATCC 19615 450
  • Fig. 28 shows an example of preferred embodiment kit plate inoculated and incubated for 16 hrs with the gram-positive organism Staph. epidermidis ATCC 12228
  • Fig. 29 shows an example of preferred embodiment kit plate inoculated and incubated for 16 hrs with the gram-positive organism Staph. aureus ATCC 25923
  • Fig. 30 shows an example of preferred embodiment kit plate inoculated and incubated for 16 hrs with 455 the gram-positive organism Staph. aureus ATCC 29213
  • Fig. 31 shows an example of preferred embodiment kit plate inoculated and incubated for 16 hrs with the gram-negative organism E.coli ATCC 25922
  • Fig. 32 shows an example of preferred embodiment kit plate inoculated and incubated for 16 hrs with the gram-negative organism Klebsiella pneumoniae ATCC 13883 460
  • Fig. 33 shows an example of preferred embodiment kit plate inoculated and incubated for 24 hrs with the gram-negative organism Pseudomonas aeruginosa ATCC 27853
  • Fig. 34 shows an example of preferred embodiment kit plate inoculated and incubated for 20 hrs with the gram-negative organism Proteus vulgaris ATCC 13315
  • Fig. 35 shows an example of preferred embodiment kit plate inoculated and incubated for 16 hrs with 465 the gram-negative organism Salmonella typhimurium ATCC 14028
  • Fig. 36 shows an example of preferred embodiment kit plate inoculated and incubated for 48 hrs with the fungus Candida albicans ATCC 14053
  • Fig. 37A to 37C illustrates the growth of two microorganisms, Staph. aureus and E.coli, on the same preferred embodiment kit plate.
  • Fig. 37B and 37C are two different views of an enlargement of the AST
  • test chambers (67-70' and 72'-75').
  • Fig 37B viewed with back lighting.
  • Fig. 37C viewed with front lighting.
  • Two AST test chambers are featured in these figures: Mueller Hinton agar plus antimicrobial Cephalothin chamber 70'and Mueller Hinton agar plus antimicrobial Enrofloxicin chamber 73'.
  • Two margins are observable in each chamber: E coli & cephalothin margin180 and Staph. aureus & cephalothin margin181 for Cephalothin chamber 70'; and Staph. aureus & Enrofloxicin margin182 and
  • E.coli & Enrofloxicin margin183 for Enrofloxicin chamber 73'.
  • the region 176A is between the E.coli margin 180 and Staph. aureus margin 181 in Cephalothin chamber 70'.
  • the region 178A is between the Staph. aureus margin182 and the E.coli margin 183 in Enrofloxicin chamber 73'.
  • Illustrated in Fig. 38 are two gram 176B and 178B of a sample from regions 176A and 178A respectively.
  • Fig. 39A to 39E is an example of a preferred embodiment kit plate inoculated with a mixture of two
  • Fig. 39A shows the preferred embodiment kit plate and the result of growth and biochemistry of the organisms on the various media.
  • Fig. 39B is a 10X magnification view of Blood agar chamber 51. The view shows two different types of colonies based on size: An E. coli colony 184 and a Salmonella typhimurium colony 186.
  • Fig. 39C is a
  • Fig. 39D shows two colony types in the Lactose MacConkey chamber 53, differentiated in terms of their ability to ferment lactose.
  • the pink centered lactose fermenting, E.coli 188 is contrasted to the non-lactose fermenting Salmonella 190.
  • Fig. 39E illustrates the fluorescence in the MUG MacConkey chamber 65, due to the action of a specific enzyme found in E.coli.
  • Fig.1 as indicated in the description of the preferred embodiment is comprised of Enriched, Differential, Selective, Differential-Selective and Single purpose media.
  • kits various media used in this kit are available from the Eleventh edition of the Difco Manual.
  • One modification to those formulas may comprise the incorporation of iota carrageenan to the agar-based media for reducing the watering out of the hydrocolloid agar gel (syneresis) as well as increasing stability and shelf life. I however do not wish to be bound by this observation.
  • the multi-chambered kit plate 80 and associated lid 78 are packed under nitrogen atmosphere in a low oxygen-permeable sealed bag to
  • kit plate media preparation follows standard practices of sterile technique. Envisioned but not illustrated is a process that could be used to produce the kit plates in an efficient fashion.
  • the system conceptually would comprise a temperature-regulated box with lid, large enough to hold the individual kit plate chamber medium vessels at 50°C plus. The distribution of that media to the test
  • 505 chambers of the multi-chambered kit plate would be accomplished by using a dispensing pump able to dispense the correct amount of media into each test chamber.
  • the pump would drive a multi-channeled pump head with the same number of channels as the number of test chambers in the kit plate allowing for a relatively simple method
  • a description of the diagnostic usefulness of each of the medium of the multi-chambered kit plate is as 510 follows:
  • Blood agar 51 is used in the isolation of a wide variety of microorganisms. All non-fastidious gram-negative and gram-positive organisms will grow on this medium. The majority of the aerobic gram-positive and gram-negative bacterial pathogens of domestic animals and man will grow on blood agar when incubated in air at 35°C. Blood agar also allows for determination of
  • hemolytic patterns adjacent to bacterial colonies are classified as non- hemolytic (gamma hemolysis), complete (beta-hemolysis), and partial (alpha-hemolytic).
  • Bile esculin azide agar 56 is used for isolating, differentiating and presumptively identifying group D streptococcus and Enterococcus. These organisms cause the formation of a dark brown or black complex in the agar.
  • Mannitol salt agar 61 allows staphylococci to grow while the growth of most other bacteria is inhibited.
  • Tellurite Glycine agar 66 permits the isolation of coagulase positive staphylococci whereas coagulase negative staphylococci and other bacteria are completely inhibited. Coagulase positive staphylococci reduce tellurite and produce black colonies.
  • Littman oxgall agar 71 is used for the isolation of fungi and is suitable for growth of pathogenic fungi. Incubation is for several days. Molds and yeasts form non-spreading, discrete colonies.
  • Azide blood agar 52 is used in the isolation of gram-positive organisms from clinical and non- clinical specimens. Azide suppresses the growth of gram-negative bacteria and is useful in determining hemolytic reactions.
  • Pseudomonas agar F 64 is used for differentiating Pseudomonas aeruginosa from other pseudomonads based on fluorescein production and is visible with UV lamp at 365nm.
  • Pseudomonas agar P 64 is used for differentiating Pseudomonas aeruginosa from other pseudomonads based on the production of pyocyanin, a non-fluorescent blue pigment.
  • Hektoen enteric agar 60 is used to isolate and differentiate Salmonella. Colonies are greenish blue, with black centers.
  • MUG MAC 65 is a MacConkey agar with lactose plus an added substrate 4- 540 methylumbelliferyl-b-D-glucuronide (MUG). MUG becomes fluorescent when E.coli is present.
  • the E.coli beta-glucuronidase enzyme cleaves the colorless MUG to a fluorescent product detected with UV light at365nm.
  • MacConkey agar which contains bile salts, is a selective media for the majority of gram-negative pathogens. The media inhibits gram-positive bacteria and a few gram-negative pathogens.
  • MAC media contain six different sugars to allow for ID of microorganisms based on their fermentation patterns:
  • Lactose MAC 53 is MacConkey agar with lactose, a selective and differential medium for growing 550 gram-negative bacilli. Lactose fermenting strains grow as red or pink colonies.
  • Glucose MAC 54 is MacConkey agar with glucose, a selective and differential medium for growing gram-negative bacilli. Glucose fermenting strains grow as red or pink colonies.
  • Mannitol MAC 55 is MacConkey agar with mannitol, a selective and differential medium for growing gram-negative bacilli. Mannitol fermenting strains grow as red or pink colonies.
  • Inositol MAC 57 is MacConkey agar with inositol, a selective and differential medium for growing gram-negative bacilli. Inositol fermenting strains grow as red or pink colonies.
  • Sucrose MAC 58 is MacConkey agar with sucrose, a selective and differential medium for growing gram-negative bacilli. Sucrose fermenting strains grow as red or pink colonies.
  • Arabinose MAC 59 is MacConkey agar with arabinose, a selective and differential medium for 560 growing gram-negative bacilli. Arabinose fermenting strains grow as red- pink colonies.
  • Mueller Hinton agar (67-70, 72-75) is considered the best media for routine AST of non-fastidious bacteria. Eight test chambers are set-aside for this purpose.
  • Fig. 3 illustrates a crucial part of the method and kit. Instead of the 24hr prior art method of streaking 565 to isolate individual organisms, a short 4 to 8 hr incubation in Brain-Heart Infusion broth is utilized. A swab specimen 84 inoculates the broth for growth to a stationary phase 90 in incubator 135.
  • Thioglycolate broth is also included for growth of potential anaerobic microorganisms134.
  • the isolation of the microorganisms is concurrent with a 12 to 20hr. ID testing on the kit plate.
  • kits plate Specifically, isolated colonies become visible in the top two rows of the kit plate. These rows received the 570 higher dilution (lower concentration) of microorganism(s) 140. The bottom three rows of the kit plate seeded with a higher concentration of microorganisms 96 performs the AST in addition to other tests as described below.
  • Fig. 16A, 16B, 16C, and 16D show the process of preparing the two dilutions (dilution 96 and dilution
  • a sterile-forty microliter/drop-pipette100 removes a portion of the grown up (usually to stationary phase) microorganisms (IIBHIB90).
  • Five drops into sterile diluent 136 results in a 1 to 11 dilution.
  • the dilution96 created is generally close to a 0.5 McFarland turbidity standard as shown in Fig.
  • Dilution 96 and standard 94 are compared against a black lined background and adjustments made so dilution 96 will be close in turbidity to standard 94.
  • the 0.5 McFarland equivalent dilution 96 is in turn
  • Fig. 5 shows an addition of dilution 96 to the first of fifteen test chambers of the bottom three rows of the kit plate. Eighty milliliters of this dilution is added per chamber.
  • Fig. 17 illustrates the first chamber of the top two rows inoculated using dilution140. Eighty micro liters of dilution 140 is added per chamber to the ten upper test chambers. Following the additions of the 2 dilutions, the microorganisms are spread
  • kits 590 out on the surface of the media by briefly shaking the kit plate in a back and forth motion in both directions.
  • the excess liquid is removed by tapping the kit plate upside down into the lid containing an absorbent tissue.
  • the kit plate is allowed to air-dry for approximately 10 minutes before the addition of antimicrobial disk-quarters 76.
  • FIG. 19A-19B illustrates the equivalence between prior art (Fig.19A) and an AST chamber 144 from the kit plate.
  • Fig. 18 illustrates the placement of disk-quarters 76 into the test chambers using
  • the thumbs and index fingers of both hands hold the device as shown by the position 129T(right thumb), 129F(right-index finger), 131T(left thumb), and 131 F(left -index finger).
  • the hands can be switched if desired.
  • the disk-quarter 76 is picked up with the placement device 102A using a piercing motion into the disk-quarter.
  • the disk-quarter 76 is rotated and placed in the corner of the test chamber.
  • the disk-quarter is then removed by pushing off with the slider as shown by device discharge
  • the kit also includes a modified Nitrate Reductase determination system Fig. 20A-20D as well as a Cytochrome oxidase test as shown in Fig. 2 A-21C.
  • a modified Nitrate Reductase determination system Fig. 20A-20D as well as a Cytochrome oxidase test as shown in Fig. 2 A-21C.
  • TABLE I shows a database table for identifying non-fastidious gram-negative bacteria using kit results.
  • This database is supplied with the kit and can be searched. After filling in the criteria, search the database forthe best match. In some cases, the result is unique. Other cases result in several presumptive choices. However, if other criteria are included, such as colony morphology or cellular
  • kits plate medium allow for definitive ID such as Hektoen enteric agar for Salmonella, MUG MAC for E. coli and Pseudomonas agar F for the expression of fluorescein in identifying Pseudomonas aeruginosa.
  • definitive ID such as Hektoen enteric agar for Salmonella, MUG MAC for E. coli and Pseudomonas agar F for the expression of fluorescein in identifying Pseudomonas aeruginosa.
  • the criteria that are utilized forthe above database include the following: Citrate utilization; arabinose fermentation; glucose fermentation; inositol
  • the database is filtered using a spreadsheet software program.
  • the database can be manually searched.
  • Motility of bacteria is an important characteristic in the ID of unknown bacteria.
  • a drop of incubation broth is placed on a clean glass slide, a cover slip is added, and the cells are viewed directly for motility.
  • Three types of motion are seen under a microscope: (1) Brownian motion, which is
  • Fig. 25-Fig. 36 show results of incubations with a number of different single microorganisms.
  • Fig. 37A-37C, Fig. 38, and Fig. 39A-39E illustrate results where more than one
  • Fig. 25 shows a blank kit plate with media before the addition of microorganisms. This figure provides the initial baseline for appearance and color reactions that take place in the kit plate with various organisms.
  • Fig. 26 shows an example of preferred embodiment kit plate inoculated and incubated for 16 hrs at
  • Blood agar chamber 51 supports the growth of this organism as well as all non-fastidious gram-negative and gram-positive organisms. Shown are non-hemolytic colonies. Azide blood agar chamber 52 also supports the growth of this organism and is non-hemolytic. Gram-negative organisms do not grow on this medium. Bile
  • esculin azide agar chamber 56 shows the formation of a dark brown or black complex in the agar, which is unique to the growth of group D streptococcus and Enterococcus organisms.
  • Test chambers (67'-70 ⁇ 72'-75') are the Mueller Hinton AST test chambers containing eight different antimicrobial agents (see the Drawings-reference numerals for the antimicrobial used). The pattern of AST is only faintly evident from this depiction and will not be discussed here.
  • FIG. 27 shows an example of preferred embodiment kit plate inoculated and incubated for 20 hrs with the gram-positive organism Streptococcus pyogenes ATCC 19615.
  • Blood agar chamber 51 reveals beta- hemolytic small colonies.
  • Azide blood agar chamber 52 also supports growth.
  • Fig. 28 shows an example of preferred embodiment kit plate inoculated and incubated for 16 hrs with the gram-positive organism Staph. epidermidis ATCC 12228. Note the growth in the Blood agar chamber
  • 660 only permits the growth of coagulase positive Staphylococcus.
  • the coagulase-negative staph. and other bacteria are completely inhibited on this medium.
  • the coagulase-positive staph. reduce tellurite and produce black colonies when present.
  • Pseudomonas agar F chamber 63 and Pseudomonas agar P chamber 64 support growth of the Staph. epidermidis but no pigment is produced.
  • Fig. 28 chamber 68' which contains the antimicrobial agent Amoxicillin/Clavulanic acid (Clavamox or Augmentin). Then refer to Fig. 19B which show a schema of a typical endpoint antimicrobial-containing Mueller Hinton chamber.
  • a measurement 142 in millimeters using ruler or caliper, is made from the disk-quarter 76 chamber corner to the margin 126 (the interface between the growing 125 and inhibited 127 microorganisms).
  • the microorganisms 670 are Resistant, Intermediate, or Susceptible to the antimicrobial agent, match the measured value to the value listed in the Modified Interpretive Standards Table (TABLE II).
  • Fig. 29 and Fig. 30 show examples of two preferred embodiment kit plates incubated for 16 hrs following inoculation with Staph. aureus ATCC 25923 and Staph. aureus ATCC 29213 respectively.
  • One primary difference between these two strains is their difference in AST to Ampicillin. Note Ampicillin test
  • Fig. 33 which is the Pseudomonas aeruginosa inoculated kit plate, contains the only pseudomonas F agar test chamber 63 where green pigment is produced (fluorescein). Additionally, irradiation of fluorescein by UV light at 365nm produces fluorescence (not shown). Note the Hektoen agar test chambers 60 showing growth in Fig. 32, and color
  • the preferred embodiment kit plate contains six test chambers (53,54,55, 57, 58, and 59) designed to. measure the ability of the test organism to ferment a particular carbohydrate.
  • kits 695 are respectively Lactose, Glucose, Mannitol, Inositol, Sucrose and Arabinose.
  • MacConkey agar-based media used in the kit plate is selective forthe growth of gram-negative organisms only. When fermentation takes place, the medium becomes acidified resulting in red to pink colonies of the bacteria. For example, fermentation is obvious in Fig. 31 test chambers 53, 54, 55, and 59. Contrast this result to Fig. 31 test chambers 57 and 58 where there is growth but no fermentation. TABLE IV lists the results of
  • Fig. 36 shows an example of the preferred embodiment kit plate incubated for 48 hrs after inoculation with the fungus Candida albicans ATCC 14053. Perhaps most obvious is the lack of inhibition by any of the eight antimicrobial agents. None of these agents is active against fungus. Note the growth of
  • Fig. 37A to 37C illustrates the growth of two microorganisms, Staph. aureus and E. coli, together on the same preferred embodiment kit plate. Both organisms, from stock culture, inoculate the BHIB 82.
  • kits plate If there are more than two types of microorganisms on the kit plate, consider the following: Although polymicrobic infections do occur, particularly when mixed bacterial species are recovered from deep wounds or visceral organs, this same mixture of organisms from culture of urine, the respiratory tract, or superficial skin wounds or ulcers must be interpreted differently. R.C. Bartlett (Am. J. Clinical.
  • the third step in the ID of the hypothetical unknown in Fig. 37A is to perform a cytochrome oxidase assays on the colonies growing on the MacConkey media.
  • the results, from testing several colonies from the MacConkey test chambers, are negative for cytochrome oxidase.
  • a nitrate reductase test on the IIBHIB 90 show that nitrate is reduced during the incubation.
  • the forth step involves observing the test chambers.
  • Fig. 37A reveals organisms growing on the azide
  • step 5 it is found that the MacConkey media shows only one type of colony. Since the organism is growing on the MacConkey media, it is gram negative (also recall the above gram stain results). The organism ferments the following sugars in the respective test chambers: Lactose53, glucose 54, Mannitol I55, and arabinose 59. The organism does not ferment inositol 57 or sucrose 58. Step 6 takes the
  • E. coli is the most common gram-negative microbe isolated and identified in clinical microbiology laboratories.
  • Methylumbelliferyl-beta-D-glucuronide (MUG) is a substrate of the E.coli enzyme beta- glucuronidase. MUG becomes fluorescent when this enzyme cleaves it. Incorporating MUG directly into
  • kit plate incorporates this medium (MUG-MAC test chamber 65.
  • Step 7 involves irradiating test chamber 65 with UV light at 365nm. Fluorescence is observed in Fig.37A, MUG-MAC test chamber65 (not specifically illustrated but see Fig39E for example). The fluorescence confirms E. coli as the other microorganism present
  • the AST portion of the kit plate shown in Fig. 37A involves the following test chambers: Ampicillin test chamber 67', Augmentin test chamber 68', Amikacin test chamber 69', Cephalothin test chamber 70', Doxycycline test chamber 72', Enrofloxicin test chamber 73', Gentamicin test chamber 74', and Septra test chamber 75'. Since there are two microorganisms on the above kit plate, they will both be present in the AST chambers. In the event that both organisms are pathogens, the choice would be to treat with the
  • Fig. 37B and 37C provide two different enlarged views of the Fig. 37A AST chambers (67'-70' and 72'-75').
  • Fig 37B is a view with back lighting.
  • Fig. 37C is a view with front lighting.
  • Two AST chambers featured in these figures are the Mueller Hinton agar plus antimicrobial Cephalothin test chamber 70'and the Mueller Hinton agar plus antimicrobial Enrofloxicin test chamber 73'.
  • Two margins are observable in each test chamber: E coli margin180 and Staph. aureus
  • 770 margin181 forthe Cephalothin test chamber 70' and Staph. aureus margin182 and E.coli margin183 for the Enrofloxicin test chamber 73'.
  • the region 176A is found to consist of E. coli and the region 178A is found to consist of Staph. aureus as confirmed by gram staining (Fig. 38) both regions.
  • the cellular morphology is different enough, it would be sufficient to do wet mounts of the regions between the
  • Fig. 39A is an example of a preferred embodiment kit plate inoculated with a mixture of two microorganisms, E. coli and Salmonella typhimurium. The sets of
  • Fig. 39A shows the preferred embodiment kit plate and the result of growth and biochemistry of the organisms on the various media. The antimicrobial susceptibilities appear to be quite similar. There is no growth in the Azide test chamber 52, which illustrates gram-negative organisms only. Citrate is utilized but it may not clear which organism is utilizing it.
  • Fig. 39A Hektoen test chamber 60 indicates Salmonella
  • Fig. 39E illustrates fluorescence in the MUG MacConkey test chamber 65, due to the action of E. coli beta-glucuronidase enzyme on the substrate 4-methylumbelliferyl-beta-D-glucuronide.
  • E. coli is
  • FIG. 39D supports the ID's.
  • E. coli colony 188 is fermenting lactose whereas Salmonella typhimurium colony 190 is not fermenting lactose.
  • the method and kit is adaptable for the ID and AST of a broad number of microorganisms comprising gram-positive bacteria, gram-negative bacteria, higher bacteria and Mycoplasma, and fungi.
  • the choice of broth used for the initial inoculation may be selected from a number of media that support the growth of the specific type of microorganism in question.
  • the specimen may be inoculated into any number of growth media and not necessarily a broth type medium. In certain circumstances, such as
  • the broth may be rendered selective at the onset with the addition of any number of agents.
  • a specimen possibly containing the gram-positive Bacillus anthracis (Anthrax) needs ID and AST.
  • the broth be selective such as Brain-Heart infusion broth plus 50 units/ml of Polymyxin B. This initial incubation media will inhibit most of the gram-negative microorganisms that could be present in a specimen, and favor growth of gram-positive microorganisms..
  • the multi-chambered kit plate media can include several selective and differential media useful for Bacillus: Bacillus cereus selective agar (BCA) and/or Phenylethanol agar with 5% defibrinated sheep blood.
  • Anthrax Bacillus anthracis
  • Bacillus cereus selective agar BCA
  • Phenylethanol agar with 5% defibrinated sheep blood BCA
  • Anthrax Bacillus anthracis
  • Anthrax Bacillus anthracis
  • Anthrax is differentiated from other gram-positive rods on culture by lack of hemolysis, lack of motility and by preferential lack of growth on Phenylethyl alcohol blood agar.
  • Other Bacilli are generally hemolytic, motile and grow on Phenylethyl alcohol blood agar.
  • Fig. 1 illustrates one of the kit components, a multi-chambered polystyrene plate having 25 square test chambers and ethylene oxide sterilized.
  • the kit plate it is possible forthe kit plate to have test chambers of any dimension and any composition of plastic material such as polypropylene where the plastic can be formed into a multi-chambered kit plate that can be sterilized.
  • the kit plate can be steam sterilized instead of ethylene oxide sterilized.
  • kit plate can be produced and utilized.
  • the kit plate can contain circular or rectangular test chambers or any shape of chamber with sufficient surface for observing bacterial growth.
  • AST media 835 Maintenance of Medical Bacteria. Jean F. MacFaddin (1985)
  • Other embodiments would comprise different combinations of the medium listed below as well as newly developed formulations.
  • Mueller Hinton medium used in the preferred embodiment, may be enriched with other nutrients in another embodiment.
  • Any other suitable AST medium can be used that will allow for reliable AST
  • an AST media can be utilized that comprises a selective agent to eliminate unimportant
  • An embodiment where anaerobic microorganisms are AST tested, would utilize a set of antimicrobial agents with clinical indications against anaerobic bacteria. Examples are Clindamycm, Imipenem, Ampicillin-Sublactam, and Metronidazole It is important to note, concerning anaerobes, that resistance among the B. fragilis group is increasing, while certain Clostridia species are highly resistant, and
  • AST of anaerobes is very desirable
  • additional wells or test chambers are utilized for AST with any available antimicrobial agent under any atmosphere.
  • kit plates These systems comprise a plastic see-through pouch and a paper gas-generating sachet
  • the paper sachet contains ascorbic acid and activated carbon that react on contact with air. Oxygen is rapidly absorbed and carbon dioxide produced When the paper sachet is placed in a sealed plastic pouch, the reaction creates ideal atmospheric conditions for the growth of anaerobes
  • the broth culture is diluted and inoculated onto the ID-AST kit plate 870
  • the antimicrobial portion shows visible results even by 8 hrs, with the faster growing
  • the kit can be used anywhere that an incubation temperature can be maintained (35°C-37°C).
  • the kit is versatile in that many different types of organisms are tested at the same time.
  • the AST portion of the kit is also novel and unique in that the end-of-incubation measurements correlate exactly (x1/2) to the standard Kirby-Bauer disk-diffusion AST system. Any set of antimicrobial agents can be tested and more than one microorganism can exist in the same test chamber and still be analyzed (see above). A paradigm in microbiology is that isolated colonies are required (i.e. "pure cultures") before any
  • lota carrageenan in conjunction the agar in the media at several different ratios, results in a stronger gel, . elastic and cohesive with little syneresis (watering out).
  • the gel is more stable to freeze-thaw
  • the type of incubation vessel can be any number of different materials.
  • the culture atmosphere can comprise any type and mixture of gas.
  • the way of determining and preparing the density of the bacterial growth for study can be by any number of methods from the McFarland standards to a spectrophotometric determination.
  • the method of inoculating the multi-chambered kit plate can also
  • kits plate 915 antigenic nature, or other molecular features of the specific microorganism.
  • the process of applying the antimicrobial agents on the kit plate can be done with other devices than the one shown in the preferred embodiment such as tweezers, forceps, vacuum devices, static electricity, air driven applicators or any other of placement.
  • the preferred embodiment disk quarter is unique in the shape of the antimicrobial agent carrier in terms of the equivalence to standard methods.
  • kits reagents and analytical papers forthe determination of nitrate reductase and cytochrome oxidase activity in the microorganisms growing from the specimen.
  • reagents and analytical papers forthe determination of nitrate reductase and cytochrome oxidase activity in the microorganisms growing from the specimen.
  • other reagents in various forms can be utilized in the method.
  • Other embodiment could utilize discs or similar material impregnated with various enzyme substrates, carbohydrates, or with various chemical agents for differentiating microorganisms on the identification section of the kit plate. Each of these differentiation
  • the carbohydrate discs are for the differentiation of microorganisms based on carbohydrate fermentation patterns.
  • an anaerobe differentiation disc set may be used in the presumptive identification of gram-negative anaerobic bacilli.
  • Databases can be developed for searching gram-positive microorganisms as is shown for gram-
  • the process of preserving the kit plates for later use comprise the packaging and storage under a nitrogen atmosphere performed in a glove box in a low permeability bag.
  • Other embodiments would be to

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Abstract

Procédé et kit d'essais concurrents d'identification et de sensibilité aux antimicrobiens à partir d'un bouillon de culture. Une incubation du bouillon s'étendant généralement sur 4 à 6 heures permet d'obtenir des quantités suffisantes de micro-organismes en vue de leur inoculation dans une plaque de titration à chambres multiples comprenant des milieux enrichis, différentiels, sélectifs, différentiel-sélectifs, uni-focntionnels et de sensibilité. A partir du milieu de culture, on prépare plusieurs dilutions pour inoculation sur la plaque de titration. La concentration plus diluée fournit des colonies individuelles de micro-organismes pour essai d'identification. L'isolement rend superflue une opération initiale d'isolememt. La dilution à concentration plus forte convient pour une inoculation faible en vue d'épreuves de sensibilité aux antimicrobiens (directement proportionnelle à une épreuve de sensibilité aux antimicrobiens de Kirby-Bauer) ainsi que d'autres essais d'identification. Les sensibilités aux antimicrobiens restent valables même si plusieurs micro-organismes différents coexistent dans la même chambre d'essai. Ce procédé est rapide dans le cas de bactéries et permet de recueillir des données d'identification et de sensibilité en l'espace de 24 heures. La trousse est complète (mais sans incubateur ni microscope). Ce procédé simple peut s'utiliser pratiquement n'importe où. De nouveaux milieux à base d'agar-agar préparés avec du iota-carrageenan permettent d'accroître sensiblement la durée de conservation des liquides d'essai.
PCT/US2002/038543 2002-12-03 2002-12-03 Tests concurrents d'identification de micro-organismes et de sensibilite a partir d'un bouillon WO2004050675A1 (fr)

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AU2002362043A AU2002362043A1 (en) 2002-12-03 2002-12-03 Concurrent microorganism identification and susceptibilities from broth
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US10266869B2 (en) 2013-11-28 2019-04-23 Viktor Veniaminovich Tets Device for determining the sensitivity of microorganisms to antimicrobial drugs
US10745662B2 (en) 2015-06-23 2020-08-18 Viktor Veniaminovich Tets Nutrient medium for cultivating bacteria
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WO2009026920A1 (fr) * 2007-08-31 2009-03-05 Statens Serum Institut Compositions et moyens permettant de diagnostiquer des infections microbiennes
US8753875B2 (en) 2007-08-31 2014-06-17 Statens Serum Institut Compositions and means for diagnosing microbial infections
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US10745662B2 (en) 2015-06-23 2020-08-18 Viktor Veniaminovich Tets Nutrient medium for cultivating bacteria
CN113039282A (zh) * 2018-07-18 2021-06-25 赛录科试诊断公司 抗微生物剂敏感性测试和微生物鉴定
WO2021188037A1 (fr) * 2020-03-20 2021-09-23 Kavalopoulos Nikolaos Détermination des effets de l'interaction entre agents
EP4368723A1 (fr) 2022-11-10 2024-05-15 bioMérieux Milieu de culture pour détecter des microorganismes comprenant un mélange d'agar et de kappa-carraghénane en tant qu'agent gélifiant
WO2024100070A1 (fr) 2022-11-10 2024-05-16 Biomerieux Milieu de culture pour détecter des microorganismes comprenant un mélange d'agar et de kappa-carraghénane en tant qu'agent gélifiant

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AU2002362043A1 (en) 2004-06-23
CA2549213A1 (fr) 2004-06-17
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EP1594881A1 (fr) 2005-11-16

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