Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/02—Food
  • G01N33/04—Dairy products
• • 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
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/02—Food
  • G01N33/12—Meat; Fish
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/801—Anerobic cultivation

Definitions

• the present invention is broadly concerned with an improved assay for the determination of motile facultative anaerobic pathogens, and especially __ monocytogenes. in samples such as meat. More particularly, it is concerned with such an assay which makes use of a substance such as an oxygen-reactive enzyme (e.g., oxyrase enzyme) for enhancing the growth rate of a target pathogen in a selected growth medium, in order to materially lessen the amount of time required for completing the assay. Assays for determining the presence of L. monocytogenes in meat samples can be completed in times many hours shorter than previous standard assays.
• an oxygen-reactive enzyme e.g., oxyrase enzyme
• Listeria monocytogenes is a motile facultative anaerobic pathogen which can cause various diseases in man, including meningoen- cephalitis, low-grade septicemia, infectious mononucleosis-like syndrome, pneumonia, endocarditis, bacterial aortic aneurysm, localized abscesses, papular or pustular cutaneous lesions, conjunctivitis and urethritis. It is known that the pathogen can be transmitted from the eating of infected meat or milk. Accordingly, because of the pernicious effects of this pathogen, and its increasing incidence in human foods, there has been a significant increase in the concern expressed about __ monocytogenes and its effects on human health.
• the accepted present-day assay for L. monocytogenes is described in "FSIS Method for the Isolation and Identification of Listeria monocytogenes From Processed Meat and Poultry Products", Laboratory Communication No. 57, May 24, 1989, distributed by the USDA and others; this publication is incorporated by reference herein.
• the FSIS assay involves placing a sample of meat in UVM Broth followed by incubation at 30°C for 24 hours. A 0.1 ml. aliquot of the incubated mixture is then placed in 10 ml. of Fraser Broth and incubated at 30°C for 24-48 hours.
• the liquid is swabbed onto a modified Oxford agar plate, which is then incubated at 35°C for 24- 48 hours.
• the incubated plates are then examined for L. monocytogenes colomes exhibiting characteristic surrounding black zones resulting from hydrolyzed esculin.
• Suspected colonies of h_ monocytogenes are then gently touched with an inoculation needle and are streaked for isolation onto a Horse Blood Overlay Agar plate. These plates are incubated overnight at 35°C. Thereafter, the plates are examined under a fluorescent lamp and translucent colonies are then further screened and subjected to conventional confirming tests.
• the prior L. monocytogenes assay involves a total time of from 56-72 hours. This represents a real difficulty for the food processor, in that food otherwise ready for shipment must be held pending the completion of assay screening. Accordingly, there is a real need in the art for an effective assay for L. monocytogenes (or other motile facultative anaerobic pathogens) which can be successfully performed in a significantly shorter period of time.
• the present invention overcomes the problems outlined above and provides an improved assay for motile facultative anaerobic pathogens which in the case of L. monocytogenes can be completed in a total time of from about 24-36 hours.
• the assay of the invention is designed to determine the presence of target pathogens in a product sample (e.g. beef or other meats) capable of supporting the growth of such pathogens.
• the assay includes the steps of first incubating the sample in a growth medium containing an agent such as esculin which will change the color of the medium in the presence of the target pathogen. If such a characteristic color change is observed, the motility characteristics of the pathogen for the color change is determined.
• the specific improvement of the invention involves contacting the product sample during the incubation step with an effective amount of a substance which enhances the growth rate of the target pathogen, this substance being selected from the group consisting of growth-stimulating effective reducing agents and oxygen-reactive enzymes.
• the growth medium is Fraser Broth, a known medium commercialized by, inter alia. Becton Dickinson Microbiology Systems of Cockeysville, Maryland. A copy of a Becton Dickinson "Product Information Sheet" relative to the Fraser Broth is incorporated by reference herein. While Fraser Broth is the preferred medium, those skilled in the art will appreciate that other media can also be employed, so long as the media contains the described color- change agent.
• the most preferred growth-enhancing substance is oxyrase enzyme, known to be an effective oxygen-reducing enzyme used to produce anaerobic conditions.
• the oxyrase enzyme is described in a technical bulletin "Properties of the Oxyrase Enzyme System Used to Isolate and Cultivate Anaerobic Microorganisms", distributed by Oxyrase, Inc. of
• the initial incubation step of the assay invention is carried out for a period of about 5-30 hours at a temperature of from about 25°- 35°C. If the target pathogen is present in the sample, and is incubated in an esculin-containing medium, the initial incubation will yield a dark brown or blackened color, because of hydrolysis of the esculin. If no such darkening occurs, the sample is deemed free of the target pathogen.
• a small aliquot (e.g., 1 ml.) of the darkened liquid resulting from the initial incubation is placed within one leg of a previously prepared U-shaped KIMAX culture tube.
• the tube has a central bight section as well as a pair of spaced, upstand ⁇ ing, capped legs.
• a quantity of semisolid modified Oxford agar is placed within the central section of the tube, and 1 ml. portions of Fraser Broth supplemented with a small amount of oxyrase enzyme are placed atop the spaced ends of the agar.
• the modified Oxford agar is a known product and is described in a Becton Dickinson Microbiology Systems "Product Information Sheet" for the modified agar. A copy of this publication is incorporated by reference herein.
• This Oxford agar is specially formulated as a selective medium for the isolation of L. monocytogenes from specimens containing a mixed flora.
• L. monocytogenes exhibits a peculiar motility and, in the modified Oxford agar, will gradually move through the agar and eventually darken the Fraser Broth sample in the opposite leg of the culture tube.
• incubation is carried out at a temperature from about 30° to 40°C for a period of about 3-15 hours. If such darkening occurs, it is very probable that the starting sample is contaminated with L. monocytogenes.
• the sample is then assayed using conventional confirming assays, such as those described in Bergev's Manual of Systematic Bacteriology. Vol. 2, Sec.
• a preferred assay in accordance with the invention involves the screening determination of the presence of L. monocytogenes in beef
• this target pathogen may be assayed from samples of other meats such as poultry (e.g. chicken), fish or dairy products.
• other pathogens can also be assayed using the principles of the invention.
• pathogens would include the group consisting of Listeria spp., Salmonella spp., E. coli, E. coli 0157.H7, Campylobacter spp., Campylobacter jejuni. Yersinia spp., Campylobacter jejuni. Yersinia spp., Yersinia enterocolitica. and all members of the Family
• reducing agents which are effective for creating anaerobic reaction conditions and otherwise enhance the growth of target pathogens in a growth medium.
• exemplary reducing agents include L-cvsteine ⁇ Cl and titanium (IIP citrate.
• Fig. 1 is a schematic process flow drawing illustrating the preferred assay for the determination of motile facultative anaerobic pathogens
• Figs. 2-4 are respectively graphs illustrating the growth characteristics of different strains of L. monocytogenes in growth media and in the presence of oxyrase enzymes;
• Fig. 5 is a graph similar of that of Figs. 2-4 but illustrating the effect of oxyrase enzyme on S. tvphimurium suspended in TSB medium;
• Fig. 6 is a graph illustrating the effect of oxyrase enzyme on a strain of E. coli suspended in BHI medium
• Fig. 7 is a graph illustrating the growth characteristics of heat injured L. monocytogenes contacted with oxyrase enzyme in a growth medium.
• Fig. 8 is a graph illustrating the growth characteristics of L. monocytogenes in various growth media supplemented with esculin and iron.
• a conventional anaerobic Stomacher bag 10 (see Fig. 1), along with 0.25 units of oxyrase enzyme and 225 ml. of Fraser Broth.
• the Broth is first added to the bag, then the enzyme and finally the meat pieces.
• the bag 10 is sealed as at 12, and is placed into an incubator at 30°C. The mixture is incubated at that temperature for a period of from about 20-25 hours.
• a U-shaped culture tube 14 (such as the
• KIMAX tube depicted in Fig. 1 having a U-shaped central section 16 and spaced, upstanding capped legs 18, 20) is prepared.
• a quantity of the semisolid Oxford modified agar 22 is placed within the section 16, and 1 ml.
• portions 24, 26 of Fraser Broth supplemented with 0.1 units of oxyrase enzyme are placed atop the agar 22 as growth and confirming liquids respectively.
• the ends of the tube 14 are then capped to ensure anaerobic conditions.
• the tube is then placed within an incubator at 35°C for a period of 5-11 hours.
• L. monocytogenes exhibits a type of end-over-end tumbling motility which enables it to travel through the liquid 24 and agar 22.
• liquid 24 contains esculin
• the agar contains ferric ammonium citrate and esculin
• such characteristic motility may be readily observed by a blackening of liquid 24, progressive blackening of agar 22, and ultimately blackening of liquid 26.
• the following Table II sets forth assay reaction times observed using the above-described technique on beef samples inoculated with varying concentrations of L. monocytogenes strains.
• the first-stage reaction in the Stomacher bag involved approximately 20 hours incubation time, and thereafter 1 ml. samples of the liquid were placed in the described U-tube filled with agar and liquid.
• the "Pre-Enrich Time” for each sample was the incubation until a black color was observed in the growth liquid above the agar (i.e., liquid 24 of Fig. 1).
• the 'Thru-Agar Time” was the incubation time observed for the pathogen to pass through the agar.
• the "Post-Enrich Time” was the time observed until the confirming liquid 26 was blackened in the U-tube. In all instances, the times listed are cumulative.
• EXAMPLE 2 A series of tests were undertaken to determine the effect of oxyrase enzyme upon the growth characteristics of various L. monocyto ⁇ genes strains. In all instances the tests were directly comparative, and involved placing identical concentrations of the respective strains in identical quantities of growth medium, followed by incubation at 37°C for 50 hours. In particular, each strain was tested by placing 10 6 CFU/ml. of the strain in 100 ml. of Fraser Broth; a comparative test was undertaken using the same concentration of pathogen in the same quantity of Fraser Broth, but with the addition of 10 units of oxyrase enzyme.
• Figs. 2-4 graphically depict the results of these comparative tests, and demonstrate significantly faster growth of the L. monocytogenes strains in the presence of oxyrase.
• EXAMPLE 3 In this test, the effects of oxyrase on the growth of S. typhimurium suspended in TSB (Tryptic Soy Broth) and incubated at 37°C for 20 hours.
• TSB Tryptic Soy Broth
• the test was directly comparative, and involved placement of 10 5 CFU/ml. of S, typhimurium in 100 ml. of TSB, followed by incubation for 20 hours.
• a comparative sample was identically produced, save for the addition of 10 units of oxyrase enzyme in the TSB.
• the results of this test are set forth graphically in Fig. 5.
• EXAMPLE 5 In order to determine the effect of oxyrase enzyme on heat injured L. monocytogenes. the following experiment was undertaken. Specifically, Brain Heart Infusion Broth (BHI, pH 7.4; 200 g calf brain, 250 g beef heart, 10 g proteose peptone, 2 g dextrose, 5 g NaCl. and 2.5 g disodium phosphate per liter of distilled water) in a 10 ml. test tube was preheated at 60°C for 1 hour in a circulating water bath. One ml. of a 10 8 suspension of L. monocytogenes was added to the preheated BHI, shaken, and held in the 60°C water bath for exactly 15 minutes. The broth was then rapidly cooled in an ice bath.
• BHI Brain Heart Infusion Broth
• EXAMPLE 6 In this test the assay of the present invention was directly compared with the prior art FSIS method for the isolation and identification of L. monocytogenes.
• samples of ground beef were inoculated with varying concentrations of the LM 103M strain, and these samples were subjected to the prior art assay and that of the present invention, the latter being described in Example 1.
• Table III sets forth the results of these tests, and will be seen that in all instances the U-tube technique of the present invention gave significantly faster assay results, as compared with the prior art method.
• EXAMPLE 7 The assay of the invention is highly selective for the determination of motile facultative anaerobic pathogens such as _ monocytogenes.
• motile facultative anaerobic pathogens such as _ monocytogenes.
• a large number of potential competitive pathogens set forth in Table IV below have been determined to exhibit no growth in Fraser Broth. Hence, the presence of these pathogens in a given sample would not detract from the accuracy of the assay.
• Enterobacter cloacae EXAMPLE 8 In this Example a number of other selective enrichment broths supplemented with esculin and iron were tested and compared with Fraser Broth, insofar as growth of L. monocytogenes therein is concerned.

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• 1990
  • 1990-10-09 US US07/594,647 patent/US5187070A/en not_active Expired - Fee Related
• 1991
  • 1991-03-05 TW TW080101782A patent/TW197474B/zh active
  • 1991-10-08 WO PCT/US1991/007415 patent/WO1992006214A1/en not_active Ceased
  • 1991-10-08 EP EP19910919522 patent/EP0552279A4/en not_active Withdrawn
  • 1991-10-08 AU AU88653/91A patent/AU8865391A/en not_active Abandoned
• 1993
  • 1993-09-10 US US08/119,457 patent/US5405773A/en not_active Expired - Fee Related

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