Government of Canada

Gouvernement du Canada

HPB Method

MFHPB-21 September 2005

HEALTH PRODUCTS AND FOOD BRANCH OTTAWA ENUMERATION OF STAPHYLOCOCCUS AUREUS IN FOODS

Microbiological Methods Committee Microbiology Evaluation Division Bureau of Microbial Hazards, Food Directorate, Health Products and Food Branch, Health Canada Postal Locator: 2204A1 Ottawa, Ontario K1A 0L2 [email protected]

Rick A. Szabo Microbiology Evaluation Division Bureau of Microbial Hazards, Food Directorate, Health Products and Food Branch, Health Canada Postal Locator: 2204A1 Ottawa, Ontario K1A 0L2 [email protected]

1.

APPLICATION This method is applicable to the enumeration of Staphylococcus aureus in foods to determine compliance with the requirements of Sections 4 and 7 of the Food and Drugs Act. Where an Official Method for a food is specified, that method shall be followed. This revised method replaces MFHPB-21, dated November 2000, and Supplements to Method MFHPB-21, dated February 2002, August 2002, and December 2003.

2.

DESCRIPTION The method has been shown to produce satisfactory results with naturally-contaminated meats, fish, poultry, vegetables, cereals and dairy products, and artificially-contaminated foods in AOAC and HPB studies (8.18.10). This method can be used successfully for the detection of Staphylococcus aureus in other foods, food ingredients and environmental samples.

3.

PRINCIPLE Certain staphylococci produce enterotoxins which cause food poisoning. This ability to produce enterotoxins, with few exceptions, is limited to those strains that are coagulase-positive, and/or produce a heat-stable

Published on the Food Directorates (Health Canada's) website at http://www.hc-sc.gc.ca/fn-an/res-rech/analy-meth/microbio/index_e.html

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MFHPB-21 September 2005

nuclease (TNase). This method determines the presence of S. aureus by plating known quantities of (dilutions of) a food sample onto a selective agar. After incubation, presumptive staphylococcal colonies are selected, and subjected to confirmatory tests. From the results of these tests, the number of S. aureus per g or mL of the food is calculated. The numbers present may indicate a potential for the presence of enterotoxin, or they may also indicate a lack of adherence to Good Hygienic Practices. 4. DEFINITION OF TERMS See Appendix A of Volume 2. 5. COLLECTION OF SAMPLES See Appendix B of Volume 2. 6. MATERIALS AND SPECIAL EQUIPMENT The Laboratory Supervisor must ensure that the analysis described in this method is carried out in accordance with the International Standard referred to as ISO/IEC 17025:2005 (or latest version): General Requirements for the Competence of Testing and Calibration Laboratories.

NOTE:

The media listed below are commercially available and are to be prepared and sterilized according to the manufacturer's instructions. See also Appendix G of Volume 1 for the formulae of individual media. NOTE: If the analyst uses any variations of the media listed here (either product that is commercially available or made from scratch), it is the responsibility of the analyst or Laboratory Supervisor to ensure equivalency.

6.1 6.2 6.3 6.4 6.5 6.6 6.7 6.8 6.9 6.10 NOTE :

Baird-Parker (BP) agar. BD CHROMagar Staph aureus (BD). Mannitol Salt Agar (MSA; BD). BP-RPF (Rabbit plasma + bovin fibrinogen) Agar (BIO-RAD). Rapid Staph (BIO-RAD) Brain Heart Infusion (BHI) broth. A non-selective agar; either Blood agar (BA), Nutrient agar (NA), or Trypticase Soy agar (TSA) TSA slants Peptone Water diluent Staphylococcus broth Use Staphylococcus broth when investigating food poisoning outbreaks and/or when sample size is very small. Stomach or macerate sample in 1:10 volume of broth and incubate at 35C 18-24 h. Streak onto selective agars and proceed as in Section 7.4. Sodium citrate, 2% Stomacher, blender or equivalent Vortex mixer or equivalent

6.11 6.12 6.13

-36.14 Control strains; use the following or equivalent strains. Positive Controls: S. aureus coagulase positive, e.g. ATCC 27154, 25923 S. aureus coagulase negative, e.g. ATCC 14990, 33501 Escherichia coli, e.g. ATCC 23509 Pseudomonas aeruginosa, e.g. ATCC 7700 S. epidermidis, e.g. ATCC 12228

MFHPB-21 September 2005

Negative Controls:

6.15 6.16

Crystal violet stain. Coagulase (Rabbit) Plasma. Follow manufacturer's instructions for reconstitution. Incubator capable of maintaining 35/C. Waterbath capable of maintaining 40-45/C. It is the responsibility of each laboratory to ensure that the temperature of the incubators or waterbaths are maintained at the recommended temperatures. Where 35/C is recommended in text of the method the incubator may be at 35 +/-1.0/ C. Similarly, lower temperatures of 30 or 25 may be +/- 1.0/C. However, where higher temperatures are recommended, such as 43 or 45.5/C, it is imperative that the incubators or waterbaths be maintained within 0.5/C due to potential lethality of the higher temperatures on the microorganism(s) being isolated. Supplies needed for confirmation: (The following supplies may be needed for confirmation; see 7.5) 6.19.1 Accuprobe (see MFLP-79) 6.19.2 Enterotoxin assay (see MFLP- 47, -67, -68, or -69) 6.19.3 Latex Agglutination kits, such as the Pastorex Staph-plus (BIO-RAD) 6.19.4 Rapid ID kits 6.19.5 Anaerobic utilization of glucose (Phenol red carbohydrate broth with 0.5% glucose, sterile paraffin oil) 6.19.6 Anaerobic utilization of mannitol (Phenol red carbohydrate broth with 0.5% mannitol, sterile paraffin oil) 6.19.7 Lysostaphin sensitivity (phosphate saline buffer, lysostaphin solution) 6.19.8 TNase (see MFHPB-28)

6.17 6.18 NOTE :

6.19

7.

PROCEDURE: Each sample unit shall be analyzed individually. The test shall be carried out in accordance with the following instructions:

7.1

Handling of Sample Units 7.1.1 During storage and transport, the following shall apply: with the exception of shelf-stable products, keep the sample units refrigerated. Sample units of frozen products shall be kept frozen. Thaw frozen samples in a refrigerator, or under time and temperature conditions which prevent microbial growth or death.

-47.1.2 7.2 Analyze the sample units as soon as possible after receipt at the laboratory.

MFHPB-21 September 2005

Preparation for Analysis 7.2.1 7.2.2 Have sterile diluent prepared. Clean the surface of the working area with a suitable disinfectant.

7.3

Preparation of sample 7.3.1 Combine portions from several locations within each solid sample unit, to ensure a representative analytical unit, or If the sample unit is a liquid or a free-flowing solid (powder), thoroughly mix each sample unit by shaking the container. Prepare a 1:10 dilution of the food by adding aseptically 11(10) g or mL (the analytical unit) to 99(90) mL of the appropriated diluent (see Table I). Shake, stomach or blend according to the type of food . Weight or volume in brackets indicates alternate procedure for making dilutions. Blend/stomach for the minimum time required to produce a homogeneous suspension; to avoid overheating, blending time should not exceed 2.5 min. With foods that tend to foam, use blender at low speed and remove aliquot from below liquid/foam interface. If the 1:10 dilution is to be mixed by shaking, shake the dilution bottle 25 times through a 30 cm arc in approximately 7 sec. The food homogenate (1:10 dilution) of dry foods should stand at room temperature for 15 min. In all other instances, the analysis should be continued as soon as possible. Prepare succeeding decimal dilutions in peptone water as required, using a separate sterile pipette for making each transfer. Shake all dilutions immediately prior to making transfers to ensure uniform distribution of the microorganisms present.

7.3.2

7.3.3

NOTE: 7.3.4

7.3.5

7.3.6

7.3.7

7.3.8

7.4

Enumeration of Presumptive S. aureus 7.4.1 Plating 7.4.1.1 Agitate each dilution to resuspend material that may have settled during preparation. Plating should be carried out within 15 min of preparing the dilutions. The amount plated on BP is determined by the tolerance limits specified by government standards or guidelines, or a laboratorys in-house specifications. When counts <25/g is an acceptable reported result for a commodity, then spread 0.2 mL of each dilution to be used onto duplicate BP agar plates. When <5/g is an acceptable reported result for a commodity, then spread 0.4 mL of the 1:10 dilution onto the surface of each of five BP agar plates. The liquid should not be spread right to the edge of the plate, since this causes confluent growth at the plate-agar interface which is difficult to count. Retain the plates in an upright position until the inoculum has been absorbed by the medium (approximately 10 minutes on properly dried plates). If the inoculum is not

7.4.1.2

7.4.1.3

7.4.1.4

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MFHPB-21 September 2005

readily absorbed by the medium, the plates may be placed in an upright position in an incubator for up to one hour. NOTE: It is recommended (but optional) that analysis now include two selective and differential agars (i.e. Baird-Parker (BP) and one other agar), as per standard methods for other pathogens in this Compendium. If using only one plating agar, equivalent media can be used in place of BP ONLY if validation data has been produced that shows equivalency.

7.4.2

Incubation 7.4.2.1 Invert the plates and incubate at 35/C. 7.4.2.1.1 BP - Plates should be observed at 24 to 30 h for possible overgrowth; presumptive colonies may be counted at this time but the count should be verified at 48 4 h. BD CHROMagar - Plates are incubated for 24 2 h. BP-RPF- Plates are incubated 24-48 h. Plates should be observed at 24 to 30 h for possible overgrowth; presumptive colonies may be counted at this time but the count should be verified at 48 4 h. MSA - Plates are incubated for 18-24 (to 48 h). Plates should be observed at 24 to 30 h for possible overgrowth; presumptive colonies may be counted at this time but the count should be verified at 48 4 h. Rapid Staph - Plates are incubated 24-48 h. Plates should be observed at 24 to 30 h for possible overgrowth; presumptive colonies may be counted at this time but the count should be verified at 48 4 h.

7.4.2.1.2 7.4.2.1.3

7.4.2.1.4

7.4.2.1.5

7.4.2.2

Avoid excessive crowding or stacking of plates in order to permit rapid equilibration of plates with incubator temperature.

7.4.3

Counting Colonies and Recording Results 7.4.3.1 BP - Observe the following four types of presumptive staphylococcal colonies: Type 1. Convex, entire, shiny black surrounded by clear zones extending into the opaque medium. Type 2. Convex, entire, shiny black without well defined clear zones. Type 3. Dark grey colonies similar to type 1. Type 4. Dark grey colonies similar to type 2. For the four types of colonies listed in this section, if obvious morphological differences or size differences exist within one type, count these types separately and confirm separately.

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MFHPB-21 September 2005

For types 3 and 4 (grey colonies with and without clear zones), only dark grey colonies are counted. Light grey colonies should not be considered as presumptive S. aureus colonies. For all types of presumptive S. aureus colonies, do not count pin point colonies. Each colony type may show grey-white margins around the colonies and/or opaque zones (double halos).Black mucoid colonies larger than 2 mm in diameter and swarmers should not be counted. Such colonies usually belong to the genus Bacillus. 7.4.3.1.1 Count the colonies of each type and record separately, but add together to give the total presumptive count.

7.4.3.2

BD CHROMagar - Presumptive S.aureus colonies appear light mauve to mauve (or pink). MSA - Presumptive coagulase-positive staphylococci produce yellow colonies with bright yellow zones while coagulase-negative staphylococci will produce small red colonies with no color change to the medium surronding them. BP-RPF agar - Similar to BP but with a whitish halo around the colonies as S. aureus converts the plasma fibrinogen to fibrin as a result of coagulase activity. Rapid Staph - Similar to BP but with convex entire, shiny black colonies surrounded by clear zones extending into opague medium.

7.4.3.3

7.4.3.4

7.4.3.5

7.4.4

Counting of five plates of the 1:10 dilution (solid food only) 7.4.4.1 If the number of all presumptive staphylococcal colonies per plate is fewer than 20, add separately the counts for each type from all five plates and record as the respective presumptive count. This is the count of one of the four types per 2 mL (0.2 g of food). Multiply each count by 5, and record as the respective presumptive count per g of food (C). Add the results, and report as the total presumptive count per g of food. If the number of all presumptive staphylococcal colonies per plate is between 20 and 200, select two plates at random, count separately the colonies of each type and compute the respective average presumptive count per plate (per 0.4 mL; which is equivalent to 0.04 g of food) (A/2). Multiply each count by 25 and record as the respective presumptive count per g of food (C). Add the results and report as the total presumptive count per g of food. If the number of presumptive staphylococcal colonies on some of the five plates is < 20, but on others is $ 20, proceed as in 7.4.4.1. above. If no plate containing 20-200 presumptive S. aureus is available, estimated counts may be made on plates giving presumptive counts outside this range.

7.4.4.2

7.4.4.3

7.4.4.4 7.4.5

Counting of duplicate plates (any dilution)

-77.4.5.1 7.4.5.2

MFHPB-21 September 2005

Select plates containing 20-200 presumptive staphylococcal colonies per plate consisting of the combined counts of all types. Compute the average presumptive count per plate for each type (A/2), multiply by five and by the appropriate dilution factor, and record as presumptive count per g or mL of food for each type (C). Add the results and report as the total presumptive count per g or mL of food. If plates from more than one dilution are used, the counts are to be averaged as shown below. If no plate containing 20-200 presumptive S. aureus is available, estimated counts may be made on plates giving presumptive counts outside this range. Report results as estimated counts when results are outside the range of 20-200. When an estimated count contributes to an average count, this average itself becomes an estimated value.

7.4.5.3 7.4.5.4

7.4.5.5

7.4.6

Averaging of counts over two dilutions If plates from two consecutive decimal dilutions contain counts within the range of 20-200 presumptive staphylococcal colonies per plate, the counts on all four plates should be used to arrive at the average count. In as much as the four different types of colonies are to be counted separately and it is quite possible that individual counts may be < 20, although the combined counts are within range, estimates and true values would have to be combined in order to arrive at an average value. This can be avoided by using the following formula:

Average colony count/g or mL

Total number of colonies counted Volume used ( per dilution 1 + 1 ) (Dilution1 Dilution2)

For an example of counting colonies, see Table II. 7.4.6.1 If no presumptive staphylococcal colonies are obtained, record presumptive counts as < 5 per g or mL for the five plates of the 1:10 dilution, or < 2.5 x the dilution factor for duplicate plates. These are estimated counts.

7.5

Confirmatory Tests For confirmation of S. aureus, perform the coagulase test (following manufacturers instructions) as an initial step. Run controls (positive and negative cultures as well as media controls) simultaneously when performing all confirmation tests. 7.5.1 Selection of Colonies 7.5.1.1 From the replicate plates counted, a number of each colony type observed is selected as follows to check for culture purity: When the total count per type for all the plates of a dilution is less than five, pick all colonies of that type. When the total count per type for all plates of a dilution is equal to or greater than five colonies, pick five colonies of that type at random. 7.5.1.2 If the colonies are typical and well isolated proceed to 7.5.1.5. If not well isolated,

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MFHPB-21 September 2005

streak each colony picked onto a non-selective medium, such as BA, NA or TSA to obtain discrete colonies. 7.5.1.3 7.5.1.4 Incubate at 35/C for 24 2 h. If the colonies are typical and well isolated, proceed to 7.5.1.5. Otherwise, make a smear from the growth of each isolate on the non-selective medium and stain with a simple stain (e.g., crystal violet). Observe microscopically for the presence of cocci. If an isolate is not pure, choose another colony at step 7.5.1.2 above and repeat colony isolation steps above. Transfer inoculum from each colony into a separate tube of BHI broth. Incubate the inoculated BHI broth tubes at 35/C for 18-24 h and observe for growth. Retain BHI broth cultures.

7.5.1.5 7.5.1.6 7.5.1.7 7.5.2 Coagulase Test 7.5.2.1 7.5.2.2

Transfer 0.2 mL of each BHI broth culture into sterile 13 x 100 mm tubes containing 0.5 mL certified coagulase plasma. Mix thoroughly. Incubate tubes at 35/C and examine after 1 h and after 4 h. Do not shake tubes during incubation. Negative tubes should be incubated overnight at room temperature and rechecked. A firm clot which does not move when the tube is tipped on its side (4+ coagulase reaction) is considered a positive test for S. aureus; no further confirmation is required. Run controls (positive and negative cultures as well as media controls) simultaneously when performing all confirmation tests. If the coagulase reaction is 3+, 2+ or 1+, perform at least two of confirmation tests listed in 7.5.3. If two of these tests are positive, then the isolate is considered S.aureus. If no clot is observed, it is considered as a negative test for S. aureus; no further confirmation is required. Refer to figure 2 for visual explanation.

7.5.2.3

7.5.3

Confirmatory Tests At least two of the following confirmatory tests may be done keeping in mind that Anaerobic utilization of glucose and Lysostaphin sensitivity must not be the only two tests performed. 7.5.3.1 7.5.3.2 Accuprobe method Perform the test according to the protocol contained in MFLP-79 Staphylococcal enterotoxin assays Perform the test according to the protocol contained in MFLP-47, MFLP-67, MFLP68 or MFLP-69. 7.5.3.3 7.5.3.4 7.5.3.5 Latex agglutination kits Perform the test as described in manufacturers instructions Rapid ID kits Perform the test as described in manufacturers instructions Anaerobic utilization of glucose. Inoculate culture to be tested into a tube of carbohydrate fermentation medium containing 0.5% glucose. Overlay with sterile paraffin oil and incubate at 35/C for 18-

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MFHPB-21 September 2005

24 h. Colour change indicating an acid reaction is a positive test for S. aureus. 7.5.3.6 Anaerobic utilization of mannitol. Same as for glucose utilization except that the source of carbohydrate is mannitol. S. aureus usually gives a positive reaction but some strains do not ferment mannitol. 7.5.3.7 Lysostaphin sensitivity Inoculate culture to be tested into 0.2 mL of phosphate saline buffer and emulsify.Transfer one half of the suspended cells to another tube (13 x 100 mm) and mix with 0.1 mL of phosphate saline buffer to serve as a negative control. Add 0.1 mL of lysostaphin solution to the original tube to give a concentration of 25 :g lysostaphin per mL of cell suspension. Incubate both tubes at 35/C for up to 2 h. If the turbidity clears in the tube containing cells plus lysostaphin, and there is no clearing in the control tube, the test is positive for S. aureus. If clearing has not occurred in 2 h, the test is negative. 7.5.3.8 Thermonuclease Test Perform the test for the presence of thermostable nuclease (TNase) according to the protocol contained in MFHPB-28. 7.5.3.9 If two confirmatory tests are positive, the isolate is considered to be S. aureus. On the basis of the confirmatory tests for each of the four types of cultures, record the total number of S. aureus per g or mL of food (NT). Total No. S. aureus per g or mL equals the sum of No. S. aureus types 1, 2, 3 and 4 (NT = N1 + N2 + N3 + N4)

No. S. aureus type 1 per g or mL (N1)

No. of colonies confirmed as S. aureus (P) X No. colonies tested (G)

presumptive colony count type 1 (C)

Same for types 2, 3 and 4. See Table II. 8. REFERENCES 8.1 American Public Health Association (APHA). 1992. Compendium of Methods for the Microbiological Examination of Foods. APHA. Washington, DC. Chapt. 33. pp:533-550. Association of Official Analytical Chemists (AOAC). 1999. Official Methods of Analysis of AOAC International. 16th. AOAC. Gaithersburg, Md. Vol. I, Chapt. 17. pp:32-34. Baird-Parker, A.C. 1962. An improved diagnostic and selective medium for isolating coagulase positive staphylococci. J. Appl. Bacteriol. 25:12-19. Baird-Parker, A.C. and E. Davenport. 1965. The effect of recovery medium on the isolation of Staphylococcus aureus after heat treatment and after storage of frozen or dried cells. J. Appl. Bacteriol. 28:390-402.

8.2

8.3

8.4

- 10 8.5

MFHPB-21 September 2005

Collins-Thompson, D.L., A. Hurst and B. Aris. 1974. Comparison of selective media for the enumeration of sublethally heated food-poisoning strains of Staphylococcus aureus. Can. J. Microbiol. 20:1072-1075. Crisley, F.D., J.T. Peeler and R. Angelotti. 1965. Comparative evaluation of five selective and differential media for the detection and enumeration of coagulase-positive staphylococci in foods. Appl. Microbiol. 13:140-156. Koskitalo, L.D. and M.E. Milling. 1969. Lack of correlation between egg yolk reaction in staphylococci medium 110 supplemented with egg yolk and coagulase activity of staphylococci isolated from cheddar cheese. Can. J. Microbiol. 15:132-133. Rayman, M.K., C.E. Park, J. Philpott and E.C.D. Todd. 1975. Reassessment of the coagulase and thermostable nuclease tests as means of identifying Staphylococcus aureus. Appl. Microbiol. 29:451-454. Selepak, S.T., and F.G. Witelsky. 1985. Inoculum size and lot-to-lot variation as significant variables in tube coagulase test for Staphylococcus aureus. J. Clin. Microbiol. 22(5): 835-837. Sperber, W.H. and S.R. Tatini. 1975. Interpretation of the tube coagulase test for identification of Staphylococcus aureus. Appl. Microbiol. 29:502-505.

8.6

8.7

8.8

8.9

8.10

- 11 TABLE I Preparation of the Initial Dilution

MFHPB-21 September 2005

Type of Food Product

Preparation

Liquids:

milk, water etc.

pipette directly into Petri dishes and/or into peptone water diluent

viscous liquids

weigh into peptone water diluent

Solids:

water soluble solids

weigh into peptone water diluent

powder, meats

weigh into peptone water diluent

spices

weigh into peptone water diluent

shellfish

weigh into peptone water diluent

cheese products

weigh into sodium citrate 2% (warmed to 45/C) diluent

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MFHPB-21 September 2005

TABLE II Example of Computing S. aureus Count per g or mL of Food Total No. of Colonies of No. of No. of Total No. of Colonies of No. of S. aureus from one of the four Types Isolates Isolates one of the four Types per one of the four Types on Duplicate Plates Tested Confirmed as g or mL. per g or mL "A" "G" S. aureus "C" "N" "P" C = 1/2AxD*5** N = (P/G)xC Fewer than 5 (e.g. 4) More than 5 (e.g. 18) All (4) 2 1,000 500

5(5)

4,500

3,600

Calculate N1, N2, N3 and N4 for each colony type to obtain total number of S. aureus. (NT) per g or mL NT = N1 + N2 + N3 + N4 e.g. if N1 = 1,000 and N2 = 100 and N3 = 0, and N4 = 0 NT = 1,000 + 100 = 1,100 per g * ** Dilution factor = 100 For duplicate plates, 0.2 mL per plate. Divide by 2 since "A" represents the total count of one of the four types on two duplicate plates. Likewise, when 5 plates of the 1:10 dilution are counted, divide by 5. Report total number of Staphylococcus aureus per g or mL of food to two significant figures

- 13 FIGURE 1 Flow Diagram for Confirmation Process

MFHPB-21 September 2005

Coagulase test

+4 Clot

+3, +2 or +1 CLot

No Clot

positive for S.aureus

other confirmatory tests (at least two)

negative for S.aureus

1. Accuprobe (MFLP-78) 2. Enterotoxin assay (MFLP-47, 67, 68, 69) 3. Latex agglutination 4. Rapid ID kits 5. Anaerobic utilization of glucose 6. Anaerobic utilization of mannitol 7. Lysostaphin sensitivity 8. Thermonuclease test

Note : 5 and 7 must not be the only two tests performed

If two of the confirmatory tests carried out are positive for S.aureus, report the confirmed cfu/g food.

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MFHPB-21 September 2005

FIGURE 2 COAGULASE TEST REACTION

Tube number

Intensity of reaction (degree of clotting)

NEGATIVE

MUST CONFIRM

POSITIVE