Chapter 5 Culture media and methods for the isolation of Listeria monocytogenes

Culture Media for Food Microbiology, J.E.L. Corry et al. (Eds.) 9 1995 Elsevier Science B.V. All rights reserved

63

Chapter 5 Culture media and methods for the isolation of Listeria monocytogenes G . D . W . C u r t i s a, W . H . Lee b a

Bacteriology Department John Radcliffe Hospital, Oxford OX3 9DU, UK b USDA-FSIS, Beltsville, MD 20705, USA

Abstract

The recovery of low numbers of Listeria monocytogenes from foods and environmental samples requires the use of enrichment cultures followed by selective plating and, where injured organisms are likely to be present, a pre-enrichment step. The development of selective and enrichment media for L. monocytogenes is traced and currently used media are discussed. Comparisons of media and methods for the culture of L. monocytogenes are reported but no single method can be recommended for all situations. Guidance is given on the choice of media and methods which is governed by the type of sample, number and nature of competing flora and cost.

During the past 10 years, large outbreaks of human listeriosis have been traced to contaminated coleslaw made from raw cabbage in Canada (Schlech et al., 1983), Mexican style soft cheese in the US (Linnan et al., 1988), soft cheese in Switzerland (Bille, 1990) and liver pat6 in the UK (McLauchlin et al., 1991) and in Western Australia (Kittson, 1992). A very large human listeriosis outbreak occurred in France in 1992, the source of which has been identified as pork tongues in aspic (Goulet et al., 1993). Sporadic or single unrelated cases of human listeriosis are also common in many countries. Sporadic listeriosis in the US was associated with Listeria monocytogenes contaminated (opened) packages of readyto-eat food from retail outlets in the domestic refrigerators of listeriosis patients by the US Centers for Disease Control ( C D C ) ( P i n n e r et al., 1992). Schuchat et al.

64 (1992) showed that consumption of soft cheese and food from a delicatessen counter were each significantly associated with an increased risk of listeriosis and Schwartz et al. (1992)showed an association between consumption of uncooked hot dogs or undercooked chicken and sporadic cases of listeriosis. The CDC (Schuchat et al., 1992) estimated that there are about 1,850 cases of sporadic human listeriosis in the US with 425 deaths annually. The whole subject of L. monocytogenes as a food borne pathogen has been well reviewed by Farber and Peterkin (1991). Because human listeriosis is such a serious and often fatal disease, many countries have imposed a zero tolerance for L. monocytogenes in cooked ready-to-eat foods. Sampling of foods and the food plant environment for L. monocytogenes has become a major preoccupation worldwide. This in turn has lead to rapid improvements of L. monocytogenes recovery methods. Testing for L. monocytogenes from such diverse sources as farms, food plants, foods and kitchens requires different selective media and sampling procedures. This paper will attempt to review the current and some promising new recovery methods.

1. Listeria enrichment broths (LEB) A liquid enrichment step is required for the recovery of low numbers ( < 20/g) of L. monocytogenes from foods and food plant environmental samples. For many years the only method available was the cold enrichment technique first advocated by Gray et al. (1948) in which sample material was inoculated into a nutrient broth without selective agents and held at 4~ for long periods. The method was primarily used for isolation from infected animal or human tissue but its usefulness in the examination of silage (Gray, 1960) and milk (Larsen, 1966) was also demonstrated. Its chief drawback was the need to make subcultures from the broth at intervals up to several months or even a year. Early attempts at producing a selective broth which would allow the isolation of listeria within days of inoculation resulted in tryptose phosphate broth with polymyxin (Bojsen-Mr 1972) and Levinthal broth with trypaflavine and nalidixic acid (Ralovich et al., 1972). Watkins and Sleath (1981) combined a period of non-selective cold enrichment with subsequent selective enrichment in a broth containing potassium thiocyanate and nalidixic acid as selective agents. Although originally designed to recover listeria from sewage this system has been successfully used by Lewis and Corry (1991) for foods. Increased interest in L. monocytogenes as a food borne pathogen led to the development of many selective enrichment broths, the chief features of which are shown in Table 1. With the exception of L-PALCAMY (van Netten et al., 1989) all these selective broths rely on nalidixic acid to inhibit Gram-negative organisms and acriflavine to provide additional selectivity especially with regard to Gram-positive organisms. L-PALCAMY incorporates only 5 mg/1 acriflavine and substitutes polymyxin, ceftazidime and lithium chloride for nalidixic acid. Lithium chloride is also used in Fraser's broth (Fraser and Sperber, 1988) whilst the FDA broth of Lovett et al. (1987) includes cycloheximide to inhibit yeasts and moulds.

Table 1 Listeria enrichment broths (LEBs): selective agents and other additions

Buffer

Other

References

0 cycloheximide 0 cycloheximide cysteine, N 2 cycloheximide 0

Greenwood et al. (1991) IDF 143:1990 Ralovich et al. (1972) Lovett et al. (1987) Knabel, unpublished Anon. (1991b) Walker et al. (1990)

5

phosphate phosphate none phosphate phosphate phosphate phosphate MOPS none

van Netten et al. (1989)

20 0

20 5

phosphate phosphate

ceftazidime, polymyxin, LiCl, egg yolk 0 MgSO4, pyruvate

25

20

20

phosphate

LiCl

Fraser & Sperber (1988)

15 25

40 20

5 20

phosphate phosphate

cycloheximide 0

Lovett et al. (1987) McClain & Lee (1988)

12 f/15 g /

20 f / 40 g

5

phosphate MOPS

cycloheximide g glucose, yeast extract, MgSO4, FeSO 4 pyruvate

Busch & Donnelly (1992)

Acriflavine (mg/1)

Nalidixic acid (mg/l)

Primary LEBs Buffered peptone water (PHLS) IDF Levinthal a Lovett (FDA) b m-Lovett K m-Lovett OX m-Lovett MOPS

0 10 15 15 10 15 15

0 40 40 40 40 40 40

5 5 5 5 5 5 5

L-PALCAMY c

5

0

12 0

UVM I d Universal Secondary LEBs Fraser e Lovett (PHLS method) UVM I I d

Combined Listeria repair broth (LRB)

NaCl (g/l)

Donnelly & Baigent (1986) Bailey & Cox (1992)

Monographs relating to these media are to be found in this volume. f-g These additions are made after 5 h incubation of the non-selective basal repair broth (f USDA-FSIS concentrations) (g F D A concentrations)

a-e

66 The original formulation of Lovett et al. (1987) was designed for isolation of L. monocytogenes from milk and the University of Vermont (UVM) broths of Donnelly and Baigent (1986) were developed by the USDA for use with meats (McClain and Lee, 1988). In view of the fact that the sample often constitutes 10% of the incubation mixture, it may prove useful to keep these applications in mind when choosing a selective broth. Samples likely to be heavily contaminated with interfering organisms may best be enriched in the more selective L-PALCAMY or even the thiocyanate nalidixic acid broth of Watkins and Sleath (1981). However, highly selective media should not be used unnecessarily as the concentrations of inhibitors may slow or prevent the growth of sublethally-damaged or of exceptionally sensitive strains of Listeria spp. Care should also be taken to adhere to the recommended incubation temperature as many listeria strains show an increased susceptibility to antibiotics when incubated at 37 rather than 30~ (Curtis et al., 1989b). The various modifications of Lovett's broth are the results of different attempts to prevent a rapid drop in pH which occurs particularly after the inoculation of soft cheese and inhibits growth of listeria (Anon., 1991b; Walker et al., 1990). This does not happen with the UVM broths which are more strongly buffered. The unpublished modification of Knabel is designed to create a degree of anaerobiosis to enhance the recovery of injured cells (see below). Two enrichment broths, Fraser's and L-PALCAMY, include an indicator system consisting of aesculin and ferric iron. Organisms which hydrolyse aesculin produce a blackening of the broth due to the formation of iron phenolic compounds derived from the aglucon. Broths without blackening are considered negative for listeriae and require no further examination. Up to 38% false positive results have been recorded with Fraser's broth but no false negative results were found provided incubation was continued for 48 h (Warburton et al., 1991a).

2. Recovery of injured Listeria monocytogenes The selective agents in Lovett, Fraser, UVM and L-PALCAMY (Table 1) inhibit the growth and recovery of injured Listeria spp. from environmental and food samples (Busch and Donnelly, 1992). To overcome this problem, the UK Public Health Laboratory Service (Greenwood et al., 1991; Morris and Ribeiro, 1991) used the totally non-selective buffered peptone water incubated at 30~ for 24 h as the primary enrichment broth and the Lovett LEB incubated for 24 and 48 h for the secondary enrichment. Another nonselective universal pre-enrichment broth is that described for the recovery of both Listeria and Salmonella spp. from foods by Bailey and Cox (1992). Comparisons of these non-selective broths with other enrichment systems need to be made to assess their performance in the recovery of injured and uninjured Listeria from various naturally contaminated foods, particularly with regard to the possibility of overgrowth by competing organisms during the later stages of incubation.

67 Busch and Donnelly (1992) introduced a new concept, that of the dual purpose non-selective/selective broth. A non-selective 3-(N-morpholino)propanesulphonic acid (MOPS) buffered enrichment broth base (LRB)with magnesium sulphate and sodium pyruvate added to improve recovery of injured cells, was inoculated with the sample. After allowing 5 h for the injured Listeria to recover, the selective agents acriflavine and nalidixic acid were then added to the LRB base and incubation continued in the usual way. A similar technique may conveniently be used with some commercial presentations of other LEBs where the basal broth is prepared from a dehydrate and the selective supplements are supplied in vials for reconstitution and addition to the broth after sterilization. Delaying this addition for some hours after inoculation of the basal broth will allow resuscitation of injured bacteria. Knabel et al. (1990) reported that strict anaerobic incubation aided in the recovery of severely injured L. monocytogenes. In 1992, Knabel (personal communication) simplified this anaerobic recovery procedure by using a modified Lovett LEB (m-Lovett K), with 10 mg/1 of acriflavine and 0.5 g/1 filter sterilized cysteine added, then flushing the flasks with nitrogen to remove atmospheric oxygen. This was used as the primary enrichment step for the recovery of severely injured L. monocytogenes which had been heated to 62.8~ for 5 min and inoculated into raw milk. After 48 h incubation at 30~ the primary enrichment broth was transferred to Fraser's LEB. Knabel (personal communication) claimed that severely injured L. monocytogenes did not recover in the aerobic LRB of Busch and Donnelly (1992) or in nitrogen-flushed UVM I LEB with 0.5 g/1 filter sterilized cysteine added. It is possible that oxygen is toxic to the growth of severely injured listeriae, but not to the growth of moderately injured listeriae heated to 56~ for 50 min (Busch and Donnelly, 1992). Smith (1990) reported that injured L. monocytogenes did not recover in a non-selective medium at 5 or 12~ but Meyer and Donnelly (1992) stated that injured listeria can recover at 5~ in milk. Thus the media and conditions for the recovery of injured Listeria spp. remain uncertain and need further study and substantiation. The ability to isolate injured L. monocytogenes from foods and environments is important because if injured Listeria spp. are not detected, the bacteria may slowly recover and colonize foods and food plant environments.

3. Listeria selective agars (LSA)

The first significant step in producing a selective agar for listeria was that of Gray et al. (1950)who used potassium tellurite to inhibit Gram-negative organisms and examined the growth under a dissecting microscope. In 1960, McBride and Girard added lithium chloride, glycine and blood to phenyl ethanol agar to produce a medium which, until relatively recently, was the only readily available commercially produced selective agar for listeria isolation. Its chief drawback was its inability to inhibit enterococci. Ralovich et al. (1971) published the first truly selective agar using trypaflavine and nalidixic acid in a serum agar base and

68 Henry's illumination method to identify listeria colonies. Lee and McClain (1986) modified McBride's agar by substituting glycine anhydride for glycine, increasing the lithium chloride content and adding moxalactam. Omitting blood allowed Henry's method to be used for identification of listeria colonies. The discovery that listeriae tolerate up to 15 g/1 of lithium chloride, a concentration which inhibits almost all enterococci, led to many of the currently used formulations, most of which also incorporate the aesculin-ferric iron indicator system thus obviating the need for special illumination techniques to identify presumptive listeria colonies. Since the advent of Oxford agar (Curtis et al., 1989a) the trend has generally been towards a simplification of media, more reliance being placed on the use of second and third generation cephalosporin antibiotics and less on acriflavine, whilst polymyxins are still used for inhibiting most Gram-negative bacteria. The simplest LSA is the modified lithium chloride-ceftazidime agar (LCAM) developed by Lachica (1990 and personal communication). The selective agents in the commonly used agars are shown in Table 2. A problem in the use of cephalosporins in selective media for listeria was noted by Curtis et al. (1989b). They showed that the three commonly used cephalosporins, cefotetan, ceftazidime and moxalactam were more inhibitory to some strains of L. seeligeri when incubation was carried out at 37~ than at 30~ Two out of 14 strains of L. monocytogenes also showed this effect with ceftazidime but not with cefotetan or moxalactam. A similar effect has been observed with L. monocytogenes ATCC 19111, which is inhibited on agar at 35~ with ceftazidime but grows well at 30~ (Lee, unpublished results). Ceftazidime at 20 mg/1 renders media such as PALCAM (van Netten et al., 1989) and modified Oxford (MC) agar (Lee, unpublished results)very selective and this may account for the occasional failure to recover strains of L. monocytogenes in comparative trials (Warburton et al., 1991b; Kovacs and Ralovich, 1991; Westoo and Peterz, 1992). These workers found Oxford agar best for isolation of Listeria spp. from enrichment broths followed by LPM, PALCAM and modified Oxford agar. A feature of PALCAM agar is the incorporation of a second indicator system, mannitol and phenol red, in addition to the aesculin-ferric iron complex. Microaerobic incubation is recommended by the authors for this agar. Dominguez-Rodriguez LSAMm agar includes potassium tellurite to enhance haemolysis by L. monocytogenes colonies in a secondary blood containing overlay (Blanco et al., 1989; Fernandez-Garayzabel et al., 1992). Whilst undoubtedly useful in differentiating haemolytic from non-haemolytic colonies the technique is time consuming and adds a further day's incubation to the process time. This is avoided in the enhanced haemolysis agar (EHA) of Cox et al. (1991a,b) but at the considerable expense of adding sheep blood, sphingomyelinase and 4-methyl umbelliferyl-/3-D-glucoside to a basal medium with PALCAM supplements. It is reported (Cox et al., 1991b) that this EHA has recovered significantly more Listeria spp. and L. monocytogenes from food samples than Oxford agar. Modified Vogel Johnson agar (MVJ) has been further modified (Smith and Buchanan, 1990) by the addition of Tween 80 and egg yolk to promote the recovery of injured listeriae. However, fats, Tween 80 and especially Tween 20 can be inhibitory to growth of Listeria spp. if they are hydrolysed to fatty acids by the

Table 2 Selective agents used in Listeria selective agars Tellurite (mg/1)

LiC1 (g/1)

Polymyxin (P) or Colistin (C) (mg/1)

Acriflavine (mg/1)

EHA LCA(M) LPM a

10 5 5

P10 0 0

5 0 0

0 0 0

LSAMm b MC e MVJ(M)

15 12 5

P10 C10 0

5 0 0

200 0 105

Oxford c

15

C20

5

0

PALCAM d

10

P10

5

0

a-e Monographs relating to these media are to be found in this volume.

Other (mg/1)

References

Ceftazidime 30 Ceftazidime 50 Moxalactam 20, Glycine anhydride 10 Phenylethanol 2500 Ceftazidime 20 Ceftazidime 20 Bacitracin 20 Nalidixic acid 50 Moxalactam 5 Glycine 10 Fosfomycin 10 Cefotetan 2 Cycloheximide 400 Ceftazidime 30

Cox et al. (1991b) Lachica (1990 & unpublished) Lee & McClain (1986)

Blanco et al. (1989) Lee, unpublished Smith & Buchanan (1990)

Curtis et al. (1989a)

van Netten et al. (1989)

70 microorganisms in the foods (Wang and Johnson, 1992). Seeliger and Jones (1986) reported that the growth of Listeria spp. was inhibited on the lactobacillus se|ective (MRS) medium of de Man et al. (1960), which contains 1 g/1 Tween and 5 g / l sodium acetate as the principle selective agents. The addition of 3 g/1 of sodium diacetate to processed meat products also controls the growth of Listeria spp. in processed meats (Schmidt and Leistner, 1991). Thus in medium development, there should be an awareness of compounds which may be used to inhibit the growth of listeriae in foods.

4. Procedures for the isolation of L. monocytogenes

Current procedures cannot recover very low levels (less than 0.3/g) of L. monocytogenes from foods (Hayes et al., 1991; Norrung et al., 1991; Warburton et al., 1991b) and the ability of present methods to achieve complete recovery of all injured cells is in doubt. For the best possible recovery a three stage procedure is recommended (i) pre-enrichment or resuscitation (ii) selective enrichment and (iii) selective plating. Media for each of these stages have been discussed above and consideration must now be given to the various combinations of broths and agars which have been assessed. Two methods were developed in the US, the FDA method (Lovett, 1987; Lovett and Hitchens, 1988) and the USDA method (McClain and Lee, 1988). Both have been subject to a number of modifications and details of the original methods together with some modifications are given in tabular form by Warburton et al. (1991a). A group of experts from the International Dairy Federation (IDF), the International Standards Organisation and the Association of Official Analytical Chemists have also produced a provisional standard method (IDF, 143:1990). This recommends the use of a modification of Lovett's broth with reduced acriflavine for enrichment, followed by plating onto Oxford agar. The US CDC (Hayes et al., 1992) compared the recoveries of L. monocytogenes from naturally contaminated foods by the revised FDA procedure using Lovett LEB, with LPM and Oxford agars, the revised USDA procedure using UVMI and Fraser LEBs with modified Oxford (MC) agar and the Netherlands procedure using L-PALCAMY LEB with PALCAM agar. They found these procedures to be almost equally effective, but the recovery of L. monocytogenes by a combination of any two methods was significantly better than by one method used alone (P < 0.02). Westoo and Peterz (1992) found no difference in the recovery of inoculated L. monocytogenes when comparing Lovett's, UVMI and UVMII broths. Rollier et al. (1991) found that the use of UVMI and UVMII broths with plating on Oxford agar was more effective than using Lovett LEB and L-PALCAMY enrichment when examining fermented sausages. They found no difference between Oxford and PALCAM agars for plating. An examination of commercial minced meat was carried out by Kokubo et al. (1990) in Japan using Lovett's (FDA) broth and trypaflavine nalidixic acid broth with Oxford, LPM, McBride and trypaflavine nalidixic acid agars. The best results

71 were obtained with Lovett's broth incubated for 7 days and subcultured onto Oxford agar. Subculture onto LPM was almost as successful. Very extensive collaborative tests were carried out by Warburton et al. (1991a,b). Using both the original and revised F D A and U S D A methods, they found that no single procedure recovered all of the L. monocytogenes in naturally contaminated samples. They recommended for all samples the use of the modified U S D A method with subculture of UVMI broth to Fraser's broth and plating media at 24 and 48 h. They also recommended the use of Oxford and at least one other agar, either LPM or M O X (Warburton et al., 1991b). The use of several plating media was shown to give substantially improved isolation rates (up to 46%) in the recovery of L. monocytogenes (Warburton et al., 1991a).

5. Enumeration of L. monocytogenes in foods by direct plating Several countries in Europe are considering setting limits for L. monocytogenes in certain categories of foods at the retail level (Anon., 1991a). Since many refrigerated ready-to-eat foods present no growth barrier to L. monocytogenes, it can grow to high numbers. In an extensive search for the possible causes of sporadic human listeriosis in the US, the CDC (Pinner et al., 1992) found strong positive correlation of human listeriosis to the foods in which high numbers of L. monocytogenes could be recovered by direct plating on LPM agar. Also, in the UK, pat6 with high counts of L. monocytogenes enumerated using Oxford agar was epidemiologically linked to a significant number of cases of listeriosis (McLauchlin et al., 1991; Morris and Ribeiro, 1991). High numbers of L. monocytogenes can easily be detected by plating the 1 in 10 food suspension in primary enrichment broth, immediately after preparation at 0 h onto LPM, Oxford, MC or PALCAM agars. This method assumes that L. monocytogenes growing in food samples is not injured and is able to form colonies on the selective plating media. However, in a few instances, high numbers of L. monocytogenes have been recovered by the MPN enrichment method but not by direct plating on LPM agar (Hayes et al., 1991, Pinner et al., 1992). Oxford agar has been used extensively by PHLS laboratories (Greenwood et al., 1991; McLauchlin et al., 1991; Morris and Ribeiro, 1991) for the direct enumeration of high numbers of L. monocytogenes in retail foods in the UK. However, the presence of large numbers of some competing bacteria can sometimes overwhelm the selective agents in this medium. Ringle et al. (1991) preferred to use the more selective PALCAM to Oxford agar for direct plating of soft cheeses. Vazquez-Boland et al. (1992) found that the more selective LSAMm and PALCAM agars were suitable for direct plating of silage samples for Listeria spp., but that Oxford agar was overwhelmed by the competing bacteria. Warburton et al. (1991b) drew attention to considerable (1-3 log)variation in recovery of certain strains on selective plating media and Tiwari and Aldenrath (1990) reported a strain which they were able to grow only on Oxford agar. Direct counting of listeriae in foods is therefore dependent on the nature and numbers of interfering organisms and on the ability of the chosen plating media to recover

72 quantitatively all of the strains sought. It should be n o t e d also that n o n e of the m e d i a in c o m m o n use at the m o m e n t will distinguish L. m o n o c y t o g e n e s from o t h e r listeriae. T h e growth of L. m o n o c y t o g e n e s in naturally c o n t a m i n a t e d food is b o t h u n e v e n a n d erratic. M i c h a r d et al. (1989) r e p o r t e d 100-fold higher n u m b e r s of L. m o n o c y togenes on the surface of soft cheeses t h a n in the centre, and a similar distribution of Listeria spp. has b e e n n o t e d in retail v a c u u m p a c k a g e d p r o c e s s e d m e a t s (Lee, u n p u b l i s h e d results). Thus counts of Listeria spp. in such foods may d e p e n d on which parts of the sample are examined. W o r k by G r e e n w o o d et al. (1991) on soft c h e e s e s a n d Schmidt and Kaya (1990) and G r a u and V a n d e r l i n d e (1992) on p r o c e s s e d m e a t s f o u n d counts in excess of 1 0 0 0 / g of L. m o n o c y t o g e n e s in 8.4, 2.3 a n d 4% of samples, respectively, d e m o n s t r a t i n g the n e e d to e x a m i n e large n u m bers of samples in o r d e r to d e t e c t sporadic c o n t a m i n a t i o n . In this respect the use of the swabbing t e c h n i q u e of L e e (1989) will allow investigation of large n u m b e r s of, for example, out of date v a c u u m packs of meat, d e t e c t i n g counts in excess of 1 0 0 / g with comparatively little effort or expense. In summary, c u r r e n t isolation m e d i a allow the recovery of L. m o n o c y t o g e n e s from a variety of foods and food plant e n v i r o n m e n t s with relative ease. Yet, to date, identification and recovery of c o n t a m i n a t e d foods responsible for h u m a n listeriosis o u t b r e a k s r e m a i n s a slow and difficult process (Anon., 1992; M c L a u c h l i n et al., 1991).

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