Comparison of methods for optimum detection of stressed and low levels of Listeria monocytogenes

Food

Microbiology,

1992,9,

127-l 45

Comparison of methods for optimum detection of stressed and low levels of Listeria monocytogenes Donald W. Warburton*l, Jeffrey M. Farbe+, Conrad Powell3, Narayan P. Tiwari4, Susan Reads, Robert PIantes, Tim Babiuk7, Patrick Laffey*, liiu Kauril, Paul Mayersl, Marie-Jo&e Champagneg, Tom Huntlo, Pierre LaCassell, Kathy Vietl2, Richard Smandol3 and Fran Coatesz ‘Microbiology Evaluation and 2Research Divisions, Bureau of Microbial Hazards, HPB, Health and Welfare Canada, Ottawa, Ontario KlA OL2, 3Fish Inspection Laboratory, Fisheries and Oceans Canada, St. John’s, Nfld. AIC 5X1,4Food Laboratory Services Branch, Alberta Agriculture, Edmonton, Alberta T6H 4P2, 5Health ofAnimals Laboratory, Agriculture Canada, Guelph, Ontario Nl G 3W4, “Service de l’environment, Communaute urbaine de Montreal, Montreal, Quebec H4N 2T2, 7Fish Inspection Laboratory, Fisheries and Oceans Canada, Burnaby, B.C. V5M 4L9, sFood Statistics and Operational Planning, HPB, Health and Welfare Canada, Ottawa, Ontario KlA OL2, sLaboratoire d’Hygi&ne veterinaire, Agriculture Canada, St-Hyacinthe, Quebec J2S 8E3, loManitoba Research Council, Portage la Prairie, Manitoba RlN 3J9, “Laboratoires d’expertises et d’analyses alimentaires de Saint-Foy, MAPAQ, St. Foy, Quebec GlP 3W8, 12Nova Scotia Research Foundation Corporation, Dartmouth, N.S. B2Y 327, and IsFish Inspection Laboratory, Fisheries and Oceans Canada, Winnipeg, Manitoba, R3T 2N6, Canada Received 20 September 1991 Twelve laboratories across Canada participated in this third part of a comparative study of modified versions of the O.O5) in their ability to isolate stressed and low levels of L. monocytogenes. The pH of the enrichment broths varied greatly after 24 and 48 h incubation. However, the ‘USDA’ enrichment broth maintained its pH closer to neutrality which may aid in the isolation of this micro-organism, especially injured *Corresponding author. 0740-0020/92/020127

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0 1992 Academic

Press

Limited

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D. W. Warburton

et al.

cells, from some foods. Modified Fraser broth (MFB) proved to be useful as a secondary enrichment broth, as shown by the significantly greater number (P~0.05) of isolates being recovered from this broth, even though it is not very selective with a 74% false-positive rate. This improved recovery may result from the combination of the use of MFB with the selective plating media. During qualitative testing, Oxford agar (0X4) proved to be the significantly better medium, with lithium chloridephenylethanol-moxalactam medium (LPM), modified Oxford agar (MOX) and Palcam agar (PAL,) being comparable in isolating stressed and low levels of this microorganism from a variety of samples. Quantitative counts of stressed and non-stressed cultures showed that LPM, OXA and PAL were comparable (not significantly different, P~0.05) in their ability to recover stressed cells of this micro-organism.

Introduction Certain environmental and chemical stresses applied at sublethal levels can produce various structural and physiological injuries in non-sporing microorganisms. Such stresses include heat, refrigeration, drying, irradiation, changes in nutritional environment, chemicals (such as sanitizers, food preservatives and acids), freeze drying, as well as freezing and thawing (Smith and Palumbo 1982, Mossel 1989). The potential exists for the injury of micro-organisms during food processing, and for the subsequent repair of these injured cells in foods during storage. There is abundant evidence that sublethally injured Gram-negative and Gram-positive bacterial cells maintain their pathogenic properties and virulence (Mossel 1989). The potential for. food poisoning caused by food products containing injured but repaired pathogenic micro-organisms is therefore very real (Smith and Palumbo 1982). Metabolic injury to micro-organisms often results in their inability to recover and form colonies on selective media that otherwise would support growth. On selective media containing bile salts, NaCl, antibiotics, etc, injured cells undergo additional stresses and fail to repair the initial damage; however, on non-selective media, injured cells can

repair the damage and subsequently grow. Thus, if a particular foodborne pathogen has been injured, the food microbiologist may either fail to detect it or will underestimate its numbers, particularly if selective agars are used directly (Smith and Archer 1988). Many Listeria spp. found in food products may be sublethally injured (Lovett 1988) and, although quite resistant to temperature stress, L. monocytogenes undergoes sublethal injury upon exposure to elevated (52-60°C) or reduced (-18°C) temperature (Golden et al. 198813). A recent study showed that spray dried cells of Listeria spp. exhibit signs of osmotic shock and heat injury (In’t Veld et al. 1991). Several investigations have also demonstrated that sublethally-injured bacteria typically have increased nutritional requirements (Golden et al. 1988a). Despite this knowledge, either the methods used to isolate L. monocytogenes from foods subjected to heating or freezing processes lack provisions for recovering injured cells, or the suitability of the procedures to recover uninjured cells is unknown (Golden et al. 1988a). Most media currently used for the enrichment and isolation of Listeria spp. from food samples contain inhibitory substances that prevent repair and subsequent colony formation by injured cells. The presence of phenylethanol, acriflavin, NaCl and

Methods

for optimum

polymyxin-acriflavin were all found to be detrimental to the recovery of heat-injured and non-stressed L. monocytogenes (Crawford et al. 1989, Leasor et al. 1990, Smith and Archer 1988, Warburton et al. 1991a,b, Werner and Lim 1990). In previous studies (Warburton et al. 1991a,b), the modified ‘USDA’ method proved to be slightly more efficient in isolating L. monocytogenes than the modified ‘FDA’ method. Also, the ‘USDA’ enrichment broth (LEB) gave a slightly better recovery of L. monocytogenes than the ‘FDA’ enrichment broth (EB) in artificially-contaminated cheese (G. A. Prentice, P. Neaves and A. D. Hitchins, unpubl. res.). Generally, the enrichment and isolation media as well as the two-stage enrichment protocol, used by the ‘USDA’ for isolation of L. monocytogenes from meat products were shown also to yield the best recoveries from dairy products (Lammerding and Doyle 1989, Prentice et al., unpubl. res.). Other authors have found both the ‘FDA’ and ‘USDA’ enrichment broths to be equally effective for recovery of nonstressed L. monocytogenes (Bailey et al. 1989, Crawford et al. 1989, Hitchins 1989, Lovett et al. 1991). However, the greater selectivity of the ‘USDA’ procedure may offer an advantage for isolating non-heat-stressed Listeria when the aerobic plate count of the product is high (Lovett et al. 1991). Skovgaard and Morgen (1988) also found the ‘USDA LEB #l to be suitable for detection of Listeria spp. in heavily contaminated material as well as in foods with low bacterial counts. In our previous studies (Warburton et al. 1991b), the main difference between the modified ‘FDA’ and ‘USDA’ methods was the enrichment broth, with the ‘USDA’ LEB maintaining constant pH in all samples. Buffering of the enrichment broth may improve the detection of a low number of injured cells (In’t

detection

of L. monocytogenes

129

Veld et al. 1991). Bailey et al. (19901 found the ‘USDA’ LEB to be the superior medium for recovery of heat-injured cells, while Crawford et al. (1989) found the ‘FDA’ method more efficient in detecting heat-injured L. monocytogenes in raw milk. Lovett et al. (1991) found the ‘FDA’ procedure isolated heated L. monocytogenes from seafoods at a lower level than did the ‘USDA’ method. As knowledge regarding optimum methods to isolate stressed L. monocytogenes cells from food products is important to food microbiologists, and as the results of previous studies were contradictory, we initiated this third part of a comparative study for several reasons. (1) To compare the overall efficacy of the modified ‘FDA’ and ‘USDA’ methods in isolating stressed and low levels of L. monocytogenes in samples. (2) To compare the effect of two different enrichment times (1 and 2 days) for isolating stressed and low levels of L. monocytogenes from a variety of samples. (3) To determine the most selective plating media [lithium chloride-phenylethanolmoxalactum agar (LPM), Oxford agar (OXA), Modified Oxford agar (MOX) and Palcam agar (PAL)] for isolating low levels and stressed L. monocytogenes from a variety of samples. (4) To further determine the reliability of the modified Fraser broth (MFB) as a screening tool. (5) To determine the effect of the pH of the enrichment broth on the isolation of stressed and low levels of L. monocytogenes. The overall objective was to provide uniform methodology for regulatory use across Canada for the detection of L. monocytogenes in food and environmental samples. Materials

and Methods

There are two methods presently employed by the Health Protection Branch (HPB), Health and Welfare Canada and other Cana-

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D. W. Warburton

et al. was used for all samples. Thus, all foods and environmental samples were analysed using either MFLP-59 (Warburton and Farber 1990a) - the modified ‘FDA method’ - or MFLP-60 (Warburton and Farber 1990blthe modified USDA Method’ (Table 11. The Bureau of Microbial Hazards laboratory, and several other laboratories tested all samples by both modified methods. All media and chemicals used were obtained from Difco Laboratories (Detroit, MI, USA), and Sigma Chemical Company (St Louis, MO, USA), respectively. Oxford agar was obtained from Unipath Ltd. (Ottawa, Ontario, Canada) and Palcam agar was obtained from BDH (Toronto, Ontario, Canada). Twelve federal, private and research laboratories across Canada participated in this third part of a comparative study. Threehundred and twenty foods and environmenanalysed using the tal samples were modified ‘FDA’ method (Table 2), while 269 samples were analysed using the modified ‘USDA’ method (Table 3). Of these, 165 sam-

dian agencies for the detection of L. monocytogenes in foods. The first method, MFLP-59 (Warburton and Farber 1990a), designed for the isolation of L. monocytogenes from all foods except meat, initially followed the ‘FDA’ method published in chapter 29 of the Bacteriological Analytical Manual (Lovett 1987, Lovett and Hitchins 1988). The other method, MFLP-60 (Warburton and Farber 1990b1, designed for the isolation of L. monoqtogenes from meat, was based on the ‘USDA’ method of McClain and Lee (McClain and Lee 1988, 1989). Both HPB Laboratory Procedures include the use of modified Fraser broth (MFB; McClain and Lee 1989), LPM (McClain and Lee 1989) and OXA (Curtis et al. 19891 media. Although the ‘FDA’ method is intended for analysing foods other than red meats and poultry, for the purpose of this comparative study it was used for all foods and environmental samples. Similarly the ‘USDA method is intended for analysing red meats and poultry, but for the purpose of this study Table

1. A comparison

of the modified Modified

Sample

types

Primary

enrichment

USDA

Blend or stomach in EB

Blend or stomach 24 and 48hb

Secondary

enrichment

MFBa incubated for 24-48h

tests

Modified

methods.

All foods*, including red meats and poultry, environmental samples

24 and 48h

Confirmation

‘USDA’

All foods, including red meats0 and poulty, and environmental samples

times

media

and modified

FDA

Incubation

Plating

‘FDA’

at 35°C

MFB incubated for 24-48hb

in LEB #l

at 35°C

LPM, MOXa, PALd and OXA from EB and MFB

LPMb, MOX, PALb and OXAb from LEB #lb and MFB

Catalase Hemolysis Motility Gram stain Sugars CAMP test MR-VP Serology

Catalase Hemolysis Motility Gram stain Sugars CAMP test MR-VP Serology

0 Differences from the original FDA method (Lovett and Hitchins 1988, Anon. 1990). b Differences from the original USDA method (McClain and Lee 1989). Abbreviations: EB, Enrichment broth; LEB, Listeria enrichment broth; MFB, modified Fraser LPM, Lithium chloride-phenylethanol-moxalactam agar; OXA, Oxford agar; MOX, modified agar; PAL, Palcam agar.

broth; Oxford

Methods

for optimum

ples were analysed using both modified methods (Table 4). Samples were categorized as follows: (a) environmental samples included swabs of drains, food cutting surfaces, and processing equipment, as well as soil; (b) dairy samples included soft and hard cheese, yoghurt and other dairy products; (c) meat samples included raw, further-processed and ready-toeat red meat products; (d) poultry samples included raw, cooked and further-processed products; (e) vegetable/fruit samples included raw or processed vegetable and fruit products; (f) seafood samples included raw or ready-to-eat marine and freshwater products and included shellfish and finfish; (g) ‘other’ samples included foods that either do not fit in the above categories (such as pastries) or contained more than one food category (such as meat and vegetable pies, or mixed meat Weiner&. Spiked samples are described later in this section.

Methodology 1. MFLP-59; the modified ‘FDA’ method (Table 1). An analytical unit of food (25 g) was blended or stomached in 225 ml of enrichment broth (EB; Lovett and Hitchins 1988). Environmental swabs were placed in 10 ml of EB in test tubes and vortexed. After 24 and 48 h at 30°C the pH of the EB was determined and 0.1 ml of EB culture was inoculated into MFB (McClain and Lee 1989). At 24 and 48 h, the EB was streaked onto OXA (Curtis et al. 19891, LPM and MOX (McClain and Lee 1989) or PAL (van Netten et al. 1989). The MOX, OXA and PAL plates were incubated at 35°C for 24-48 h and LPM plates at 30°C for 24-48 h. All plates were examined at 24 h and reincubated for an additional 24 h if no typical colonies were present. After 24 h incubation at 35”C, any ‘blackened’ MFB was streaked onto the plating media described above. Negative or ‘straw-coloured’ MFB were reincubated for another 24 h and then streaked onto plates if positive (‘blackened’ or darker than the original straw-colour). Up to five suspect colonies from each plate were subcultured and purified on Tryptic soy agar with 0.6% yeast extract (TSA-YE) for confirmation. Suspect or typical appearing L. monocytogenes colonies were stabbed into corresponding grid patterns on horse blood agar plates and carbohydrate agar plates

detection

of L. monocytogenes

131

(Hunt and Sado 1989) for screening purposes. (Carbohydrate agar plates consisted of 16 g agar, 16 g purple broth base, 1 ml of 1.6% Bromcresol purple dye in 950 ml distilled H,O. After autoclaving, 50 ml of 20% filter-sterilized mannitol, rhamnose or xylose was added.) Con6rmation tests, such as motility, catalase, hemolysis, Gram stain, sugar utilization, CAMP test, MR-VP, and serology were done as previously described (Warburton and Farber 1990a,b). 2. MFLP-60; the modified ‘USDA’ method (Table 1). An analytical unit of food (25 g) was blended or stomached in 225 ml of Listeria enrichment broth #l (LEB #l; McClain and Lee 1989). Environmental swabs were placed in 10 ml of LEB #l in test tubes and vortexed. After 24 and 48 h at 3O”C, the pH of the LEB #l was determined and O-1 ml of LEB #l was transferred into MFB (McClain and Lee 1989). MFB was incubated and streaked onto the plating media as described above. Also at 24 and 48 h, the LEB #l was streaked onto the plating media as above. Purification of colonies and confirmation tests were also done as described above. 3. Comparison studies of the modified ‘FDA’ method and the modified ‘USDA’ method (Table 1). A 50 g analytical unit of food was blended for 2 min with sufficient 0.1% (w/v) peptone water to obtain a homogeneous slurry. The slurry was then divided into two equal portions; one portion was analysed by the modified ‘FDA’ method (MFLP-59) while the other was analysed by the modified ‘USDA’ method (MFLPBO), as described above. 4. Bacterial cultures - spiked foods. Sixtyone strains (six serotype l/2, 20 serotype 1, 22 serotype 4 and 13 untyped) of L. monocytogenes maintained in the HPB culture collection were used. The following non-Listeria cultures from the HPB culture collection were used: Acetobacter liquefaciens, Aeromonas hydrophila (17 strains), A. caviae, A. salmonicida (three strains), Bacillus cereus, Enterobacter aerogenes, Enterococcus spp. (three strains), E. durans, E. faecalis (six strains), Escherichia coli (15 strains), Klebsiella pneumoniae, Lactobacillus acidophilus, Oerskovia sp., Pseudomonas aeruginosa (13 strains), P. diminuta, P. maltophilia, P. putrefaciens, Salmonella agona, Staphylococcus aureus (14 strains), and yeast (two strains).

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The following methods were used to stress L. monocytogenes cultures. Six-month-old cultures (TSB-YE at 21°C) were frozen in a refrigerator freezer at -20°C and are referred to as ‘frozen-old’. Seven-day-old cdtures (TSB-YE at 21°C) were frozen in a deep freezer at -80°C and are referred to as ‘frozen (-80°C)‘. Twenty-four hour (TSB-YE at 35°C) cultures were frozen in a refrigerator freezer at -2O”C,/and are called ‘frozen (-20°C)‘. ‘Heat-shocked’ cultures were obtained by heating 24 h stock cultures (TSBYE at 35°C) at 52-56°C for 1 h following the procedure of Smith and Hunter (1988). Some of the heat-shocked cultures were frozen at -80°C until analyses and are called ‘heatfrozen’. Spiked foods were obtained by adding stressed or stock cultures of L. monocytogenes to obtain initial counts of c102, 102-103, or >103 cfu g-1 food. To ensure high levels of competitors, 1 ml each of several non-listeria cultures (TSB-YE, 24 h, 35°C) that gave positive MFB reactions, or 2 g of soil (Listeria negative), were added to each sample. Analyses were done as described above. To test the selectivity of the plating media, the 85 non-Listeria stock cultures were grown in TSB-YE for 24 h at 35°C. After a second subculture in TSB-YE (24 h at 35”(Z), the cultures were streaked onto the plating media which were incubated as described above. To test the quantitative recovery by the various plating media, the 48 strains of L. monocytogenes from TSB-YE (24 h at 35°C) and stressed cultures [48 heat-shocked, 29 frozen (-BO”C), nine frozen (-2O”C), 26 frozen-old, 12 heat-frozen and 41 stock cultures] were serially diluted in 0.1% peptone water, and 0.1 ml of each dilution was spread onto each of the three plating media. To test the quantitative recovery of heatstressed cells, after heating at 52-56°C for 1 h, serial dilutions were made using the primary enrichments. After incubation at 30°C for 24 and 48 h, 0.1 ml was transferred from tubes demonstrating growth, into MFB and incubated as described above. MFB showing ‘positive’ reactions were also streaked onto the plating media. 5. Statistical analysis. The number of positive samples for L. monocytogenes by both modified methods in routine and spiked samples (Tables 2 and 31, the results obtained using both methods on the same sample in

the same laboratory (Table 4), and the comparison of the four plating media (Tables 8 and 10) were analysed using McNemar’s Chi-square test (Everitt 1977).

Results Recovery of L. monocytogenes ‘FDA’ and ‘USDA’ methods

by the

the 320 samples tested by the modified ‘FDA’ method, L. monocytogenes was recovered from most of the contaminated samples after 24 h of enOf

richment,

i.e. 131 of 139 (94.2%)

positive

samples were detected after 24 h of enrichment while another five (3.6%) were detected after 48 h of enrichment (Table 2). However, six samples that were positive at 24 h became negative after enrichment in the ‘FDA’ EB for 48 h. There was no significant difference in recovery of L. monocytogenes between 24 and 48 h for the modified ‘FDA’ method. When comparing the number of positives isolated from both the original and modified ‘FDA’ method, it was found that culturing both the EB and MFB for a total of 48 h can improve detection (Table 2). Subculturing the 24 and 48 h EB into MFB, and then plating both broths into LPM, OXA and PAL and/or MOX yielded L. monocytogenes from 139 samples, while plating the EB only (24 and 48 h into the three plating media) yielded 119 positive samples. The modified method has a significantly better recovery rate (PcO.05) than the original method. Thus, streaking both the EB and MFB at 24 and 48 h (the modified ‘FDA’ method) onto the plating media resulted in a 14.4% increase in L. monocytogenes isolation. Of the 269 samples tested by the modified ‘USDA’ method, 84 of 96 (87.5%) were positive for L. monocytogenes after 24 h of enrichment (Table 31, while another 11 (11.5%) became positive after 48 h of enrichment. Five sam-

0 h C d c

-.

136

5 20 12 1 9 30 25

11 5 15 3

48 h

139

5 20 13 1 9 30 27

11 5 15 3

one other)

Total no. positive

by the original

Includes positives from both EB and MFB. Original: 24 and 48 h EB onto LPM and OXA. Modified: 24 and 48 h EB and modified FB onto LPM, OXA, MOX or PAL. ND, Not done (i.e. both 24 and 48 h EB not streaked). The enrichment broth from six samples (one dairy, two meat, two vegetable/fruit,

131’

Totals

5 20 10

9 5 14 2

24 h _--.

ii 30 27

.--

by day

for L. monocytogenes

No. positives

positive

Spiked samples Environmental Dairy Meat Poultry Vegetable/fruit Seafood Other

Routine samples Meat Poultry Seafood Other

Sample types

Table 2. Number of samples enrichment times.

were

positive

119

; 28 25

139

5 20 13 1 9 30 27

11 5 15 3

Modified methodc

at 48 h.

320

6 24 21 5 11 45 31

44 5 65 63

-~ Total tested

and comparison

by method

method

at 24 h but negative

9 5 14 0 C2NDP 3 18 7

‘FDA’

No. positives

modified

____ -.-Original methodb

and

__-

of

Q b r d

5 20 12 2 9 25 84d

Spiked samples Environmental Dairy Meat Poultry Vegetable/fruit Other

Totals

Includes positives from both LEB and MFB. Original: 24 and 48 h LEB into modified FB and MOX. Modified: 24 and 48 h LEB and modified FB onto LPM, The Lisleria enrichment broth from five samples (three

0 6 5 0

24 h

OXA, MOX or PAL. meat, one vegetable/fruit,

96

z 26

ii 26 95

5 21 14

5 0

z

5 21 14

0 11

0

one other)

Total no. positive

by the original

11

48 h

by dap

for L. monocytogenes

No. positives

positive

Routine samples Meat Poultry Seafood Other

Sample types

Table 3. Number of samples of enrichment times.

were

positive

82

12 1

2:

0 5 5 0

Original methodb

‘USDA’

at 24 h but negative

92

5 21 14 5 8 26

0 8 5 0

and

at 48 h.

by method .

method

Modified methodc

No. positives

and modified

269

6 24 21 5 11 31

9 135 5 22

Total tested I

comparison

Methods Table 4. Comparison and spiked samples Sample types

Spiked sample& Environmental Dairy Meat Poultry Vegetable/fruit

Other Totalc

detection

of L. monocytogenes

of the modified ‘FDA’ and ‘USDA’ methods tested by both methods in the same laboratories. FDA+USDA+a

Routine samples Meat Poultry Other

for optimum

FDA+USDA-

FDA-USDA+

for

FDA-USDA-

135

all routine Total tested

6 5 0

1 0 0

4 0 0

23 0 22

34 5 22

5 20 12 1 9 32

0 0 1 0 0 1

0 1 2 4 0 0

1 3 6 0 2 4

6 24 21 5 11 37

90

3

11

61

165

0 FDA+USDA+ signifies that the modified FDA and USDA methods isolated L. monocytogenes; similarly the negative sign indicates that the method did not isolate 15. monocytogenes. b Listed in Table 5. c Total positive: modified FDA method, 93 samples (56.4%); modified USDA method, 101 samples (61.2%).

ples that were positive at 24 h became negative after enrichment in the ‘USDA LEB for 48 h. Recovery of the organism at 48 h was significantly greater V’
5. Comparison

of recovery FDA+USDA+=

Stressed cultures Heat-shocked Frozen (-80°C) Frozen (-20°C) Frozen-old Beat-frozen Stock cultures Low level.+ High level& Totale 0 h c d e

positive samples isolated from both the original and modified ‘USDA’ methods, it was found that culturing both the LEB #l and MFB improved detection by 10% (Table 3). Subculturing the 24 and 48 h LEB #l into MFB, and then plating

of L. monocytogenes FDA+USDA-

in spiked

FDA-USDA+

samplesa. FDA-USDA-

Total

19 15 19 6 9

0 0 0 1 0

0 5 0 0 0

1 3 0 3 0

20 23 19 10 9

5 6

1 0

2 0

9 0

17 6

79

2

7

16

104

These spiked samples are shown in Table 4 by sample type. Explanation of symbols in Table 4. lOOO. Total positive: modified FDA method, 81 samples; modified USDA

method,

86 samples

136

D. W. Warburton

et al.

both broths at 24 and 48 h on LPM, OXA, and PAL and/or MOX yielded L. monocytogenes from 92 samples, while plating the 24 h MFB onto MOX (the original ‘USDA’ method) resulted in only 82 positives. Again, the modified method was significantly better at recovering L. monocyJogene.5 (P
broths

A comparison of the effect of the enrichment broth and the incubation time, on the recovery of L. monocytogenes is shown in Table 6. The inclusion of MFB

significantly (PcO.05) enhanced the recovery of L. monocytogenes in both the modified ‘FDA’ and ‘USDA’ methods. A comparison was also done to see if the pH of the enrichment broths at the time of streaking would affect the isolation of this micro-organism (Table 7). It is evident that the pH of the enrichment broths varied considerably on both days 1 and 2 for each method. More samples remained in the neutral range in the ‘USDA’ method. Interestingly, at day 2 a total of nine samples from both methods were positive even though the pH was between 4.5 and 4.9. Six samples found positive by the modified ‘FDA’ and five samples positive by the modified ‘USDA’ method from the 24 h enrichment broth were negative from the enrichment broth at 48 h. All of the negative ‘FDA’ EB, while only two of the negative ‘USDA’ LEB, had pH values below 5.7 (Table 7). The false-positive rate for the MFB (McClain and Lee 1989) approached 74% for all routine and 27% for spiked samples (data not shown). Many nonListeria stock cultures inoculated into MFB gave false-positive reactions including A. caviae, A. hydrophila (six

Table 6. Comparison of the effect of enrichment broths and incubation times the recovery of L. monocytogenes from positive routine and spiked samples. FDA

USDA

EB

MFB

Sample tested by both methods in same lab Day 1 Day 2

59 56

72 77

Sample tested by one method only Day 1 Day 2

38 51

47 53

Abbreviations: EB, FDA enrichment broth; LEB#l, USDA enrichment broth. 0 Six samples were positive at 24 h but negative at 48 h. h Five samples were positive at 24 h but negative at 48 h.

LEB#l

MFB

606 63

broth;

MFB,

78 88

modified

Fraser

on

Methods Table 7. Effect samples positive

for optimum

of length of incubation for L. monocytogenes. Total no. of positive samples

detection

times

of L. monocytogenes

on pH of the

enrichment

137 broths

in

pH ranges 4.0-4.4

4.5-4.9

5.0-5.4

5.5-5.9

6.0-6.4

6.5-6.9

7.0-7.4

Sample tested by both methods in same lab: FDA

USDA

Different different FDA

EB Day 1 Day2

56 59a

0 0

0 7

7 19

9 18

12 7

20 4

11 1

LEB#l Day 1 Day 2

60 636

0 0

0 2

0 3

2 7

2 11

26 33

30 7

0 0

0 0

0 4

23 40

4 6

7 3

5 0

samples labs: EB Day 1 Day 2

by

39 53

a Six samples that were positive at day 1 were negative at day 2, had the following pH values on day 2: 44,&I, 4.1, 5.1,5.3 and 5.7. h Five samples that were positive at day 1 were negative at day 2, had the following pH values on day 2: 4.3, 4.8, 6.4, 6.5 and 6.6.

strains). A. liquifaciens, A. salmonicida, P. aeruginosa (five strains), P. maltiphila and P. putrefaciens. Interestingly, all these non-Listeria bacteria needed an initial inoculum of >107 cfu ml-l to give a ‘positive’ (blackening) reaction (data not shown). Fourteen heat-shocked cultures (as described above) were serially diluted into both EB and LEB #l. After 24 h incubation at 3O”C, 28.6% of the cultures showed no growth in the ‘FDA’ EB, while 100% had recovered in the ‘USDA LEB #l. After 48 h, recovery occurred up to the 10-S dilution for all cultures in both broths (data not shown). Plating media Eighty-five non-Listeria strains were plated onto the selective media and growth was observed. Twelve S. aureus cultures grew on OXA, while only nine

of the 12 grew on PAL (six with weak growth), with only two being MFB positive. Oerskouia sp., yeast (two strains), and one strain of each of E. coli, B. cereus, A. salmonicida, and P. dimunata, grew on PAL. Only one yeast strain grew on OXA and was MFB positive. One strain of P. fluorescens grew on OXA and one strain of A. hydrophila grew on LPM. Seven Aeromonas strains, six Pseudomonas strains, five Enterococcus strains, K. pneumoniae, S. agona, and L. acidophilus were MFB positive, but did not grow on any of the plating media. A comparison done by the same lab, on the same sample of selective mediaused in both methods (‘FDA’ and ‘USDA’), showed LPM, OXA and PAL to be almost equivalent in efficiency of recovery (Table 8). Interestingly, for a limited number of fish samples, MOX agar recovered considerably more L. monocy-

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D. W. Warburton

et al.

togenes as compared to LPM and OXA. One hundred and sixty-five stressed and stock cultures were also examined to see if there were any recovery differences on LPM, OXA and PAL (data not shown). These quantitative tests show-ed all three media to be comparable in their recovery of both stressed and nonstressed strains of L. monocytogenes. Freezing at -20 and -80°C for 35 and 5 months, respectively, resulted in only a l-2 log reduction in the counts of L. monocytogenes on all selective media. The effects of freezing the g-month-old cultures at -20°C for 3.5 months resulted in a reduction in counts of 4-5 logs. Freezing the ‘heat-shocked cultures at -20°C for 1 week did not result in any changes in viable counts other than that which would be attributed to the heat-stress (data not shown). Discussion

and Conclusions

Up to 94% of the positive samples were detected after 24 h of primary enrichTable

8. The

effects

of

selective

media

ment (Tables 2 and 31. The observation that most positive samples (82-92%) were detected after 24 h of primary enrichment was discussed previously (Warburton et al. 1991a,bl, i.e. only another 8-18% of positive samples were detected after 48 h of primary enrichment. In previous studies, subculturing both the EB and MFB after 24 and 48 h incubation (the modified ‘FDA’ method) onto the three plating media, resulted in a 2-11% increase in L. monocytogenes recovery as compared to the original ‘FDA’ method (Warburton et al. 1991a, bl. This increase can be accounted for by the subculturing of both the primary and secondary broths, together with the multiple plating. In this study, up to 5.2% and in our previous studies, up to 18% of the samples that were positive at 24 h, became negative after a further 24 h of enrichment in the ‘FDA’ EB (Warburton et al. 1991a,bl, possibly because some competitors outgrew and masked the L. monocytogenes (Dallas et al. 19901. and

method

on

the

recovery

of L.

monocytogenes.

Number of positive plates

Samesampleby both methodsin samelab All sampletypes FDA USDA Meat samples FDA USDA Different samples in one lab by one method Fish samples FDA

LPM

OXA

PAL

MOX

243 263

256 (1 NDP 272 (1 ND)

249 (9 ND) 265 (9 ND)

NAb NA

20 44

20 44

NA

NA

20 44

87

65

NA

107

0 ND, Not done. h NA, Not applicable (i.e. not used in these tests). These data are included in Table 10.

Methods

for optimum

Competition, when it occurs, may be due to specific bacterial competitors, rather than bacterial numbers, among the food’s normal microflora that are resistant to the selective agent(s) used (Tran et al. 1990). Wisteria spp. are inhibited by some Enterococcus, Lactococcus, Leuconostoc, Lactobacillus and Pediococcus species, and can be stimulated by Pseudomonas spp. (Tran et al. 1990). The inability to isolate L. monocytogenes after 24 h may also be due to the fact that further enrichment of the ‘FDA’ EB beyond 48 h is accompanied by a substantial decrease in the pH of the broth for most foods tested (Lammerding and Doyle 1989, Bailey et al., 1990). This agreed with the results of our previous study, where in most cases the pH dropped to between 4.4 and 5.5 after 24 to 48 h of incubation, respectively (Warburton et al. 1991b). The pH decrease has been attributed to a lack of sufficient buffering capacity combined with the presence of the fermentable carbohydrate, glucose (Bailey et al. 1990). In contrast, the pH of ‘USDA’ enrichment broth (LEB #l) was l-2 pH units higher than that of the EB for the same sample (Warburton et al. 1991b). Recovery of L. monocytogenes can be reduced when the pH drops below 5.5 (Schaack and Marth 1988, Bailey et al. 1990, Connor et al. 1990). In the present study, six positive samples isolated from 24 h ‘FDA’ EB and two positive samples isolated from 24 h ‘USDA’ LEB #l became negative at 48 h (Table 71, possibly due to a drop in pH below 5.5. Three other positive meat samples from 24 h LEB #l also became negative at 48 h. In these samples the pH values ranged from 6.4 to 6.6. Bailey et al. (1990) stated that the low recovery of heat-injured cells of L. monocytogenes from ‘FDA’ EB, as compared to the ‘USDA’ LEB #l, is most likely due to the low pH to which the medium falls in the

detection

of L. monocytogenes

139

presence of the food’s indigenous microflora. The normal formulation of EB resulted in the medium having an initial pH of 5.2-5.7, which is in the range where low concentrations of heatinjured cells of L. monocytogenes will not be recovered (Bailey et al. 1990). Commercial versions of this broth have an initial pH close to 7.0. In this study, there was no significant difference (P>O.O5) between either modified method in the recovery of L. monocytogenes from spiked and naturally contaminated samples. Nor was there any significant difference (P = 0.06) in the recovery of stressed and low numbers of cells. In previous studies (Warburton et al. 1991a,b), 106 (34.9%) positive samples were obtained by the modified ‘FDA’ method and 109 (35.9%) positive samples by the modified ‘USDA method. A summary of the testing of a total of 469 samples from the present and previous studies by both methods is shown in Table 9. Based on this collective data, 199 (42.4%) and 210 (44.8%) samples were found positive by the modified ‘FDA’ and ‘USDA’ methods, respectively. Both modified methods thus appear to be comparable (i.e. not significantly different, P = 0.14) in their overall recovery of L. monocytogenes. A two-stage selective-enrichment procedure has been shown to improve the detection of Listeria spp. in foods and environmental samples (Lee and McClain 1986, Fernandez-Garayzabal and Genigeorgis 1990, Warburton et al. 1991a,b) by inhibiting the growth of other micro-organisms during the enrichment of such foods as dairy prod- _ ucts, that contain high numbers of competing micro-organisms (Lammerding and Doyle 1989). The use of the MFB at days 1 and 2 in both the modified ‘FDA and ‘USDA’ methods greatly improved recovery of L. monocytogenes (Table 6). When compared to the performance of

140

D. W. Warburton

Table

9. A summary

Sample types Environmental Dairy Meat Poultry Vegetable/fruit Seafood Other Spiked foods/ controls Totalc

et al. of the comparison

FDA+USDA@

of the modified

FDA+USDA-

‘FDA’

FDA-USDA+

and ‘USDA’ FDA-USDA-

method@. Total tested

0 1 5 3 1 1 0

0 1 12 2 2 0 0

12 15 59 20 16 27 43

14 26 102 30 23 34 44

128

7

12

49

196

181

18

29

241

469

2 9 26 5 4 6 1

0 Samples tested

by both methods in the same laboratory as completed by Warburton et al. (1991a,b) and this study (Table 4). h FDA+USDA+ signifies that the modified FDA and USDA methods isolated L. monocytogenes; similarly the negative sign indicates that the method did not isolate L. monocytogenes. c Total positive: modified FDA method, 199 samples (42.4%); modified USDA method, 210 samples (44+3%).

both enrichment broths, streaking of the MFB usually resulted in pure Listeria cultures. Despite this fact, the number of false-positives (blackened tubes) did approach 74%, although most did not show growth on the plating media when subcultured. In our previous studies (Warburton et al. 1991b), the false-positive rate for the MFB approached 60% for all foods and environmental samples. No false-negatives were noted in any of our previous studies. This parallels the main disadvantage associated with the original FB (Fraser and Sperber 19881, i.e. a high incidence of falsepositives which can range from 39 to 71% (Fraser and Sperber 1988, Warburton et al. 1991a). Despite its high false-positive rate, MFB can be used as a screening medium. It was found that 31 strains of non-Listeria (including Aeromonas spp., Bacillus spp., Pseudomonas spp., S. aureus, E. coli and Oerskovia sp.) that could grow on LPM, OXA, PAL and MOX, were screened out by their negative MFB reactions. This parallels our

previous findings (Warburton et al. 1991b), that indicated the need or usefulness of the MFB as a screening medium. Twenty-one out of 33 cultures that gave positive MFB reactions had ‘no growth’ on the plating media. This may indicate that the combinations of selective agents in the MFB and plating media are needed to suppress competitors. In a variety of studies, LPM has been more effective than other media in recovering L. monocytogenes from meat, dairy products, processed chicken, vegetable and mastitis samples (Lee and McClain 1986, Bailey et al. 1989, Cassiday et al. 1989a,b, Heisick et al. 1989a,b, Lammerding and Doyle 1989, Ralovich 1989, Fernandez-Garayzabal and Genigeorgis 1990). In addition, Cassiday et al. (1989a) found LPM better than other media for recovering frozen and heat-injured L. monocytogenes cells. In contrast, van Netten et al. (1989) reported that the better media for isolating L. monocytogenes was PAL, followed by OXA, then by LPM. Other re-

Methods

for optimum

searchers (Northolt 1989, Schiemann et al. 1990, Walker et al. 19901 have also found OXA to be superior to LPM for recovery of L. monocytogenes from dairy, meat and poultry products. In our previous studies (Warburton et al. 1991a,b), OXA was the most efficient plating medium in isolating L. monocytogenes from foods and environmental samples, then LPM, followed by MOX agar. The present study again found OXA to be the best medium in qualitative tests, with LPM, MOX and PAL being comparable to one another (Table 8). In our previous study (Warburton et al. 1991b), quantitative counts of cultures from MFB and TSB-YE (24 h and 2-month-old) showed no difference in the ability of LPM, MOX and OXA to recover this micro-organism. In this study, quantitative recovery of stressed and non-stressed stock cultures was comparable on all media (LPM, OXA and PAL). Therefore, quantitive tests showed all four media to be comparable. Based on the result from our present and earlier studies, it seems that overall, OXA is the most efficient plating medium in isolating L. monocytogenes from foods and environmental samples (Table 10). LPM, MOX and PAL agars Table media.

10. A comparison

of the isolation

detection

of L. monocytogenes

141

proved to be comparable in their efficacy. The components of selective agars may influence the growth of L. monocytogenes (Leasor et al. 1990, Werner and Lim 1990, Warburton et al, 1991a,b) and the effects may be strain dependent (C. Powell pers. comm., Tiwari and Aldenrath 1990, Warburton et al. 1991b). In this study, it was again apparent that there was a strain-dependent variation in quantitative counts and qualitative recovery on the various media (data not shown). Up to 6.5% of the L. monocytogenes isolates in our studies (Warburton et al. 1991a,b) were isolated by one medium and not by the other two. Even though OXA was the better medium, there were instances where L. monocytogenes were isolated only on LPM or MOX. Thus, the use of two or more plating media was recommended (Warburton et al. 1991b). Some researchers have found reductions in counts of some Listeria spp. of over 3.5 logs in media containing fosfomycin, such as Oxford (van Netten et al. 1989). Swaminathan et al. (1988) reported that growth of three laboratory strains of L. monocytogenes was inhibited on LPM agar while three clinical of L. monocytogenes

by the four

plating

No. of positive plates LPM

OXA

MOX

PAL

Source of data

1351 312 506

1449 301 528

ND 324

ND ND 514

a.6.c b,c c

ND

ND, Not done. (2 Warburton et al. 1991a. b Warburton et al. 1991b. This study had the following explanation). c This study (Table 4). d Those media underlined are not significantly different e ‘>’ means the recovery of L. monocytogenes was greater

results:

Significanced OXA > LPMe MOX LPM OXA OXA.PAL > LPM

OXA LPM MOX (see footnote

(P < 0.05) in their or significantly

‘d’ for

recovery ofL. monocytogenes. better on the one medium.

-

142

D. W. Warburton

et al.

isolates (serotype l/2a) were partially inhibited on LPM. They also indicated that it would be prudent to use at least two plating media (one highly selective and the other not) to insure isolation. In our previous study, using additional plating media increased the number of L. monocytogenes isolations by up to 456% (Warburton ‘et al. 1991a) - another reason for multiple plating. Both the ‘FDA’ (Anon. 1990) and Canadian (Warburton and Farber 1990a,b) methods require multiple plating. The ‘USDA’ method presently only uses MOX (McClain and Lee 1989). In our previous study (Warburton et al. 1991b) six cultures (E. coli, L. acidophilus, Oerskovia sp., E. faecalis and two yeast strains) grew on LPM, MOX and OXA media. Retesting of these cultures in this study showed that all cultures (except L. acidophilus) grew on PAL. The E. coli and Oerskovia sp. gave negative MFB reactions but grew on all the plating media. The two S. aureus strains that gave positive MFB reactions also grew on all plating media tested. Yeast and molds were problems (in some cases over-growing the plates) when some samples were plated on PAL. These results show that some nonListeria can interfere with direct-plating and culturing of MFB. Contrary to our results, van Netten et al. (1989) found that their hardiest S. aureus strain (THDG) was reasonably well suppressed on OXA and PAL, at a reduction of about 4.5 logs. Since processed foods will almost never contain S. aureus at a level exceeding 103 g-1, while in raw foods S. aureus should not occur at levels greater than 104 g-1, these researchers felt that S. aureus would not interfere with the enumeration of L. monocytogenes via direct plating on these media (van Netten et al. 1989). Many researchers (Lee and McClain 1986, Loessner et al. 1988,

Swaminathan et al. 1988, Brackett and Beuchat 1989) have found that only a few non-Listeria spp. grew on LPM. In contrast, van Netten et al. (1989) found LPM very poor at suppressing competitors, while PAL and OXA performed excellently in suppressing Enterococcus spp. and other micro-organisms. Our results demonstrate that freezestress does not appear to affect L. monocytogenes to the same extent as heat treatment. During frozen storage, little loss of viable L. monocytogenes cells of strains Scott A or V7 occurred, and low levels of frozen cells were more readily recovered than low levels of heat-injured cells (Lammerding and Doyle 1989). Data obtained by Golden et al. (1988b) indicate that this organism is quite resistant to die-off over a 14 day storage period at -18°C. The viable populations of the four strains tested decreased by only about 3-6%, although up to 82% of the viable population was injured. Palumbo and Williams (1989) found that L. monocytogenes survived freezing at -18°C for 8 weeks with decreases of only one to three log cycles in viable count. When frozen cells were plated on selective Listeria media there was no decrease in count when compared to nonselective media, indicating that L. monocytogenes was not injured during frozen storage (Palumbo and Williams 1989). Similarly, Golden et al. (1988a) found that freeze-injury did not appreciably affect the ability of L. monocytogenes to grow on selective media. Thus, quantitative recovery from frozen products can occur even when media selective for Listeria are used (Palumbo and Williams 1989). Our results agree with this. Based on the results of this work, we recommend the following method for the isolation of L. monocytogenes. An analytical unit of food (25 g) is blended or stomached in 225 ml of Lis-

Methods

for optimum

teria enrichment broth #l (LEB #l). Environmental swabs are placed in 10 ml of LEB #l in test tubes and vortexed. After 24 and 48 h incubation at 3O”C, 0.1 ml of LEB #l culture is inoculated into MFB and the LEB #1 is streaked onto OXA, and at least one additional plating medium, either LPM, MOX or PAL. After 24 h incubation at 35”C, the positive (blackened’ or darker than the original straw colour) MFB is streaked onto OXA, and at least one additional plating medium, either LPM, MOX or PAL. The MOX, OXA and PAL plates are incubated at 35°C for 24-48 h, and

detection

of L. monocytogenes

143

LPM plates at 30°C for 2448 h. All plates are examined at 24 h and reincubated for an additional 24 h if no typical colonies are present. Negative or ‘strawcoloured’ MFB are reincubated for another 24 h and then streaked onto plates if positive (blackened’). Up to five suspect or typical appearing colonies from each plate are subcultured and purified on TSA-YE for confirmation. Confirmation tests, such as motility, catalase, hemolysis, Gram stain, Sugar utilization, CAMP test, MR-VP, and serology are done as previously described (Warburton and Farber 1990a,b).

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Smith, J. L. and S. E. Hunter. (1988) Heat injury in Wisteria nolzocytogenes: prevention by solutes. Lebensm. Wiss. u. Technol. 21,307-311. Swaminathan, B., Hayes, P. S., Przybyszewski, V. A. and Plikaytis, B. D. (1988) Evaluation of enrichment and plating media for isolating Listeria monocytogenes. J. Assoc. Ofi Anal. Chem. 71,664-668. Tiwari, N. P. and Aldenrath, S. G. (1990). Isolation of Listeria monocytogenes from food products on four selective plating media. J. Food Protect. 53, 382-385. Tran, T. T., Stephenson, P. and Hitchins, A. D. (1990) The effect of aerobic mesophilic microfloral levels on the isolation of inoculated Listeria monocytogenes strain LM 82 from selected foods. J. Food Safety 10, 267-275. van Netten, P., Perales, I., van de Moosdijk, A., Curtis, G. D. W. and Mossel, D. A. A. (1989) Liquid and solid selective differential media for the detection and enumeration of L. monocytogenes and other Listeria spp. Znt. J. Food Microbial. 8,299-316. Walker, S. J., Archer, P. and Appleyard, J. (1990) Comparison of the Listeria-Tek ELISA kit and cultural procedures for the detection of Listeria species in foods. Food Microbial. 7, 335-342. Warburton, D. W. and Farber, J. M. (1990a) Isolation of Listeria monocytogenes from all foods except meat. HPB Laboratory Procedure: MFLP-59. August 1990 update. In Volume 3. Compendium of analytical methods, pp. 1-18. Polyscience Publications Inc., Montreal, Quebec, Canada. Warburton, D. W. and Farber, J. M. (1990b) Isolation and identification of Listeria monocytogenes from meat. HPB Laboratory Procedure: MFLP-60. August 1990 update. In Volume 3. Compendium of analytical methods, pp. 1-15. Polyscience Publications Inc., Montreal, Quebec. Warburton, D. W., Farber, J. M., Armstrong, A., Caldeira, R., Hunt, T., Messier, S., Plante, R., Tiwari, N. P. and Vinet, J. (1991a) A comparative study of the ‘FDA’ and ‘USDA’ methods for the detection of Listeria monocytogenes in foods. Znt. J. Food Microbial. 13, 105-118. Warburton, D. W., Farber, J. M., Armstrong, A., Caldeira, R., Tiwari, N. P., Babiuk, T., Lacasse, P. and Read, S. (1991b) A Canadian comparative study of modified versions of the ‘FDA’ and ‘USDA’ methods for the detection of Listeria monocytogenes. J. Food Protect. 54, 669-676. Werner, B. S. and Lim, D. V. (1990) Growth of Listeria monocytogenes in different media. P-41. In Abstracts of the 90th Annual Meeting of the American Society for Microbiology, Anaheim, California, May 13-19, 1990, p. 285.