International Journal of Food Mwrobwlogv, 12 (1991) 281-286

281

© 1991 Elsevier Science Publishers B.V. 0168-1605/91/$03.50 FOOD 00387

Short communication

Comparison of a cold enrichment and the F D A method for isolating Listeria rnonocytogenes and other Listeria spp. from ready-to-eat food on retail sale in the U.K. Stephanie .l. Lewis and Janet E.L. Corry * Food Soence Division, Mimstry of Agriculture Fisheries and Foo~ Colney Lane. Norwwh, U.K.

(Received 12 July 1990, accepted 30 October 1990)

A cold ennchment and the modified FDA selective enrichment method were compared for their ability to detect l.asterza rnono~togenes and other l.aster~a species from various ready-to-eat foods on sale m the UK. Of 57 food samples examined using cold enrichment, five yielded L, monocytogenes, and two L. mnocua. The FDA enrichment method yielded three samples positive for L. monocytogenes only. Foods exanuned mchided soft cheeses, fermented meat sausages, pates and salads.

Key words" LJsterta monocytogenes; Listeria mnocua; Listeria spp.; Ready-to-eat food; Isolation method; Cold enrichment

Introduction U n t i l the recent w i d e s p r e a d interest in isolating Listeria species f r o m f o o d s a n d the food e n v i r o n m e n t , m o s t i s o l a t i o n m e t h o d s i n v o l v e d a l e n g t h y cold e n r i c h m e n t . C o n s i d e r a t i o n s o f speed a n d p r a c t i c a l i t y a p p e a r to have i n f l u e n c e d the d e v e l o p m e n t o f a h e r n a t i v e m e t h o d s o f i s o l a t i o n using selective e n r i c h m e n t at h i g h e r t e m p e r a tures. T h e s t u d y d e s c r i b e d in this r e p o r t c o m p a r e d a c o l d (4 o C) e n r i c h m e n t m e t h o d ( W a t k i n s a n d Sleath, 1981) with a w i d e l y used, slightly m o d i f i e d U n i t e d S t a t e s F o o d a n d D r u g A d m i n i s t r a t i o n ( F D A ) 30 ° C selective e n r i c h m e n t m e t h o d for the isolation of L. m o n o c y t o g e n e s a n d o t h e r Listeria species o c c u r r i n g n a t u r a l l y in r e a d y - t o eat food.

Correspondence (Present) address: S.J. Lewis, RHM Research and Engineering, Lord Rank Research Centre. Lincoln Road, High Wycombe, Buckinghamshire HP2 3QR, U.K. • Present address: J Sainsbury pie, Stamford House, Stamford Street, London SEI 9LL, U.K.

282 Materials and

Methods

Methods of tsolation T w o m e t h o d s o f i s o l a t i o n w e r e u s e d i n p a r a l l e l a s w e l l as a d i r e c t p l a t i n g m e t h o d . These were a slightly modified FDA method (FDAM) and a cold enrichment t e c h n i q u e ( C E ) w h i c h u t i l i s e d a n o n - s e l e c t i v e c o l d p r e - e n r i c h m e n t (4 ° C ) f o l l o w e d by the enrichment broth of Watkins and Sleath (1981) which contains sodium t h i o s u l p h a t e a n d a c r i f l a v i n e . T h e s e m e t h o d s a r e s h o w n s c h e m a t i c a l l y in Fig. 1. M o d i f i c a t i o n s to t h e F D A m e t h o d w e r e i n t h e s a m p l e size, w h e r e 10 g i n s t e a d o f 25 g w a s u s e d , a n d i n t h e o m i s s i o n o f d i l u t i o n i n 0.5% p o t a s s i u m h y d r o x i d e p r i o r to plating out. Dilution in potassium hydroxide was investigated (results not shown) 10 g f o o d + 9 0 m l FDAEB

Streaked onto

a

, 24 h & 7 days

, MMA

i n c u b a t e d a t 30 ° C

10 g f o o d

b incubated

a t 37 ° C f o r 48 h

Incubated at 4°C

+ 9 0 m l N B 2 ........... • a n d s a m p l e d w e e k l y

0.5 ml + 5 ml ...........

C E B d a t 30 ° C for 24 h

w

A p p r o p r i a t e d i l u t i o n .......... ~ H e n r y t e c h n i q u e • spread on TA f incubated a t 37 ° C f o r 24 h /!/

~, 0.1 m l

~~s s s ~~~~s ~~s ~~~~~~ ~~~s ~~~ Direct-plated

Up to 5 colonies purified on TAf

~~

onto MMA" Confirmatory

F~g. 1. Dmgram showing isolation of hstenae by the modified FDA method (FDAM; - - ) , enrichment (CE; . . . . . . ), and by the direct-plating method ( - - - --). "

b c a c f

tests

by cold

F D A E B FDA enrichment broth (tryptose soya broth (Difco) containing 6 g/I yeast extract (Oxoid), 15 mg/I acriflavm. HCI (Sigma), 40 mg/I nalidixic acid (sodmm salt: Sigma), and 50 mg/I cyclobeximlde (Stgma)). MMA: modified McBride agar (McBride agar (Oxoid) with 200 mg/I cyclohexarmde (Sigma)). NB2: nument broth number 2 (Oxoid). CEB: cold ennchment broth (NB2 containing 37.5 g/I potassium thaocyanate and 100 mg/I nalidlxic acid (Sigma)). Henry techmque: according to Henry (1933). T A : tryptose agar (Difeo).

283

and found to have no beneficial effect on the isolation rate of listeriae. For direct-plating, duplicate 0.1 ml-volumes of the one in ten suspension of food in nutrient broth were spread on modified McBride agar (see Fig. 1).

Identification methods The isolates were identified biochemically and morphologically using criteria described by McLaughlin et al. (1986). Isolates considered to be L. monocytogenes were sent to the Division of Microbiological Reagents and Quality Control. Central Public Health Laboratory, London, for confirmation of identity and serotype.

Foods sampled Fifty-seven food samples listed in Table I were purchased from retail outlets in the London area from November 1987 to May 1988. No special precautions were taken to avoid cross-contamination at the counter during weighing and packaging. Each sample was however individually packed and no cross-contamination would have occurred during or after transport back to the laboratory.

TABLE I Species and serotype of isolates from food found to contain listenae Food

pH

Spies of Listena

Serotype

Method of isolation a (week of enrichment) FDAM

Dairy Soft cheese from raw cows milk

5.2

Soft cheese from

6.0

raw cows m i l k

Soft cheese from raw cows milk Soft cheese from raw cows milk Blue Stilton from raw cows milk Meat Ardenne pate

monocytogenes mnocua monocytogenes monocytogenes

4b 1/2 1/2

mnocua

5.9 6.2

monocytogene$ monocytogene$ monocytogenes

5.9

innoeua

5.2

monocywgenes

4b 1/2 1/2

1

innocua

Fermented raw meat sausage (Gyula) Salad (drlmed) Potato and vegetable salad

4.7

monocytogenes

4.9

mnocua

4b

* FDAM, Modified FDA method; CE, cold enrichment.

CE(0)

CE(1)

CE(2)

CE(3)

CE(4)

284 The samples investigated comprised 25 dairy samples, i.e. five yoghurts and 20 cheeses; 21 meat samples including fermented sausage, patds, dried beef and ham, roast beef and cooked chicken products; and 11 salads including raw mushrooms, salads with and salads without dressing. Of the 20 cheeses tested, one was made from goats' milk, two from sheeps' and 17 from cows' milk. Eight of the cows' milk cheeses were stated to have been made using unpasteurised milk, three from pasteurised milk and no information with regard to pasteurisation was supplied for the remainder. The pH of each food was measured, by taking a representative sample of approximately 10 g homogenised in an equal quantity of distilled water, and is shown in Table I for those foods which were found to contain listeriae.

Results and Discussion

The results are shown in Table I. Eight out of 57 samples were found to contain listeriae. Only L. monocytogenes and L innocua were detected. Table I shows the method of isolation and the week of enrichment at which listeriae were isolated from CE. The CE method yielded seven positive samples, the F D A M method three, and none were detected by direct plating. Considering L. monocytogenes, the CE method identified five of the six positive samples and the F D A M method only three (Table I). Positive results by both methods on the same sample were obtained only twice. In both cases different serotypes of L. monocytogenes were isolated. The F D A M method never isolated more than one strain of Listeria from one sample, but the cold enrichment method yielded both L monocytogenes and L. innocua on three occasions. Five of the eight positive samples were soft cheeses made from raw cows' milk. Other types of food found positive were single samples of pat& raw fermented sausage and a dressed potato salad (Table I). With two of the cheeses, isolation was made only after 2 or more weeks' cold enrichment, indicating that the organisms were present in very small numbers. As no listeriae were found by direct-plating, the numbers in all samples were probably low (less than 100 per g). This study and our study on raw chickens (Lewis and Corry, 1991) when 30 of 57 chickens were found positive using the CE method, and only three using the F D A M method, indicate that the cold enrichment method is superior to the F D A M method for isolating L. monocytogenes and L. innocua from foods. These results differ from those of Pini and Gilbert (1988a,b) who found that the FDA method was better than a cold enrichment technique for isolating listeriae from soft cheese. However, their cold enrichment differed from ours since it involved plating directly on to Modified McBrides Agar (MMA) from the cold enrichment, while our method included a secondary enrichment at 30 ° C. Their F D A technique also differed from our FDA technique in plating onto a blood agar as well as MMA. The consequence may have been that Pini and Gilbert's FDA technique was more efficient than ours, while our cold enrichment was better than theirs at detecting low numbers of organisms not detectable by the F D A M method. There appear to be few other

285 comparisons of cold enrichment methods for isolating listeriae, but Doyle and Schoeni (1986) found a cold enrichment better than the FDA method for isolating L. monocytogenes from artificially contaminated cheese. It appears that most workers have judged that sensitivity has to be sacrificed for speed in obtaining results. This may well be necessary when screening foods for positive release, but speed may be a lesser consideration for regulatory agencies carrying out surveys. However, there is no evidence that the low numbers of L. monocytogenes ( < 100 per g) detected during this survey pose a ha~urd to health. Since this survey was carried out there has been another report of listeriae in pat6 (Morris and Ribiero, 1989). Contamination probably occurs after cooking a n d / o r during handling and sale of the product. Listeriae are generally able to grow well in patr. The pH of the pat6 which contained the listeriae in this study was relatively low (5.2), possibly due to the metabolic activity of lactic acid bacteria, which often grow to high numbers in this product. The low pH would probably have prevented the listeriae from multiplying rapidly. There are reports that L. monocytogenes demonstrates a poor ability to survive and grow at a reduced pH; Gray and Killinger (1966) stated that it usually died at a pH lower than 5.6; Irwin (1968) reported pH 5.5 as the critical level below which death occurred in a grass silage medium; Conner et al. (1986) reported growth in unclarified cabbage juice at pH 4.8. In the present study listeriae were found in two products, potato salad and fermented meat sausage, with pH values of 4.9 and 4.7 respectively. George et al. (1988) reported growth of L. monocytogenes at pH 4.4 in a nutrient medium acidified with HC1, and Oguru (1988) reported growth at pH 4.5. Sorrells et al. (1989) observed growth of some strains o f / - monocytogenes at pH 4.6 in a nutrient medium containing lactic acid, and at pH 5.0 in the presence of acetic acid. It is expected that the pH tolerance of L. monocytogenes is influenced by the growth environment. It is therefore possible that the listeriae had grown in these foods, but more likely that they had merely survived from an initial contamination (i.e. from the raw meat used to make the salami sausage and the raw vegetable ingredients in the potato salad). Four of the five soft cheeses found to contain listeriae had pH levels < 5.9, and although only low numbers were detected, the pH was sufficiently high for them to multiply to high numbers.

Acknowledgements We would like to thank Dr. J. McLaughlin and Dr. A.G. Taylor, Division of Microbiological Reagents and Quality Control, Central Public Health Laboratory, for serotyping our strains.

References Conner, D.E.. Brackett, R.E. and Beuchat, L.R. (1986) Effect of temperature, sodium chloride and pH on growth of ~stena monocytogenes in cabbage juice. Appl. Environ. M:crobiol. 52. 59-63.

286 Doyle, M.P. and Schoeni, J.L. (1986) Selective enrichment procedure for isolation of Lzsler:a monocvtogenes from faecal and biological specunens. Appl. Environ. Mlcroblol. 51, 1127-1129. George. S., Lund, B.M. and Brocklehurst, T.F. (1988) The effect of pH and temperature on imtiatlon of growth of Lzsterla monocytogenes. Lett. Appl. Mlcrobiol. 6, 153-156. Gray, M.L. and IGllinger, A.H. (1966) ]_~sterta monocytogenes and listeric infections. Bacterlol. Rev. 30, 309-382. Henry, B.S. (1933) Dissociation m the genus Brucella. J. Infect. Dis. 30. 51-56. Irwin, A.D. (1968) The effect of pH on the muluplication of ].aster:a monocvtogenes m grass silage media. Vet. Rec. 82, 115-116. Lewis, S.J. and Corry, J.E.L. (1991) Survey of the incidence of Ltster:a monocvtogenes and other Ltsterta spp. in experimentally wradiated and in matched unirradiated raw chicken. Int. J. Food Mscroblol. 12, 257-262. McLaughhn, J., Audurier, A. and Taylor, A.G. (1986) The evaluation of a phage-typing system for ]_asterm monocytogenes for use in epidemiological studies. J. Med. Microbiol. 22, 357-365. Morris, I.J. and Ribeiro, C.D. (1989) Llster:a monocytogenes and pate. Lancet ti, 1285-1286. Oguru, P.O. (1988) Evaluation of Antimicrobial Factors Acting in Combination: Three Methods Compared Using Three Bacteria. MSc Dissertation. University of Bristol, Bristol. Pini, P.N. and Gilbert, R.J. (1988a) The occurrence in the U.K. of Ltster:a species in raw chickens and soft cheeses. Int. J. Food Microbiol. 6, 317-326. Pim, P.N. and Gilbert, R.J. (1988b) A comparison of two procedures for the isolation of Lzsterta monocytogenes from raw chickens and soft cheeses. Int. J. Food Microbiol. 7, 331-337. Sorrells, K.M., Enigl, D.C. and Hatfield, J.R. (1989) Effect of pH, acidulant, ume and temperature on the growth and survival of Listeria monocytogenes. J. Food Protect. 52. 571-573. Watkans, J. and Sleath, ICP. (1981) Isolation and enumeration of L~sterta monocytogenes from sewage sludge and river water. J. Appl. Bacteriol. 50, 1-9.