The identification of Listeria species

International Journal of Food Microbiology 38 (1997) 77–81

Short Communication

The identification of Listeria species J. McLauchlin* Food Hygiene Laboratory, Public Health Laboratory Service Central Public Health Laboratory, 61 Colindale Ave, London NW9 5 HT, UK Received 12 May 1997; received in revised form 16 July 1997; accepted 12 August 1997

Abstract The purpose of this study was to compare methods for the identification of Listeria species. Three hundred and fifty cultures representing the six species of Listeria were tested using conventional sugar fermentation and haemolytic reactions, as well as the hydrolysis of the DL-alanine b-naphthylamide (DLABN), and the API Listeria identification test kit. Using conventional tests, 99% of cultures were correctly identified: four L. monocytogenes were misidentified as L. innocua. The DLABN hydrolysis test distinguished L. monocytogenes from the remainder of the genus for 98% of the cultures: 6 out of 14 L. ivanovii isolates gave atypical results. There was correct identification for 97% of the cultures using the API Listeria test kit and no misidentifications were obtained: nine cultures (six L. monocytogenes and three L. innocua) gave equivocal profiles which were not ascribed to any species.  1997 Elsevier Science B.V. Keywords: Listeria spp.; Listeria monocytogenes; Identification tests

1. Introduction. Listeriosis was first described as a spontaneous epidemic of infection amongst laboratory animals in Cambridge during the 1920s (Murray et al., 1926). However, human listeriosis received relatively little attention until the 1980s and 1990s when an increase in the numbers of reported cases in several countries, together with the description of foodborne transmission for both sporadic and epidemic disease, was reported (Farber and Peterkin, 1991; McLauchlin, 1996). *Corresponding author. Tel.: 1 44 181 2004400; fax: 1 44 181 2008264; e-mail: [email protected]

The genus Listeria comprises six species, i.e. L. monocytogenes, L. innocua, L. ivanovii, L. welshimeri, L. seeligeri, and L. grayi. In humans, L. monocytogenes is the major pathogen, although very rare cases of infection due to L. ivanovii and L. seeligeri have been described (McLauchlin, 1997). The presence of any Listeria species in food may be an indicator of poor hygiene. However, since L. monocytogenes is the major human pathogen, there is widespread agreement that the goal should be to exclude this organism from the food chain wherever possible, and to maintain conditions which will inhibit its multiplication in foods in which this bacterium can grow. Food Regulatory Agencies in a number of countries have adopted standards for the

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J. McLauchlin / International Journal of Food Microbiology 38 (1997) 77 – 81

absence of L. monocytogenes in specific food products (Shank et al., 1996; Skinner, 1996). In the interests of public safety and for considerations of public health microbiology, all L. monocytogenes should be regarded as potentially pathogenic (McLauchlin, 1997), and similar views have been stated by others (Hof and Rocourt, 1992; Brosch et al., 1993). Hence, it is important to be able to correctly identify the different Listeria species, or at least differentiate L. monocytogenes from the remainder of the genus. Conventional methods for the identification of Listeria species have relied on the results of fermentation of sugars and haemolytic reactions (Seeliger ´ and Jones, 1986), and the API Listeria (BioMerieux) identification kit based on some of these tests is commercially available. Problems have been described in the interpretation of haemolytic reactions, particularly the CAMP test (Schuchat et al., 1991; Vazquez-Boland et al., 1992; McKellar, 1994; Fernandez-Garayzabal et al., 1996), and although generally working well, the API Listeria test kit has been reported to produce occasional discrepant results (Bille et al., 1992). ¨ ¨ Kampfer and colleagues (Kampfer et al., 1991; ¨ Kampfer, 1992) reported that all species of Listeria with the exception of L. monocytogenes produce amino acid peptidase activity on alanine substituted substrates. It was further reported that a modification of this reaction for the identification of Listeria could be successfully carried out within 5 h using DLalanine b-naphthylamide (DLABN) as the substrate (Clark and McLauchlin, 1997). The purpose of this paper is to describe the conventional identification techniques for the genus Listeria and compare these to the API Listeria identification test kit together with the hydrolysis of the DLABN substrate.

2. Materials and methods

2.1. Cultures of Listeria All type strains of Listeria species were obtained from the National Collection of Type Cultures (NCTC, Public Health Laboratory Service, Central Public Health Laboratory, London, England) and comprised: L. monocytogenes (NCTC 10357); L.

innocua (NCTC 11288); L. ivanovii (NCTC 11846); L. welshimeri (NCTC 11857); L. seeligeri (NCTC 11856); and L. grayi (NCTC 10856). L. monocytogenes (NCTC 11994), L. ivanovii (NCTC 11007), and L. grayi subsp. murrayi (NCTC 10812), were also included plus 341 wild-type cultures of the six Listeria species listed together with their origins in Table 1. To avoid duplication of wild-type strains, a single culture only was included from either an individual food or clinical case of listeriosis. There was no selection of the wild-type cultures for atypical phenotypic characteristics.

2.2. Conventional identification tests Tests were carried out similarly to that described ¨ previously (Seeliger and Hohne, 1979; Seeliger and Jones, 1986; Rocourt et al., 1983). All cultures were grown on Tryptose Phosphate Agar (Difco Laboratories, West Molesey, Surrey, England) and layered 5% horse blood agar (Nutrient Agar No.1 base; Oxoid, Basingstoke, England) incubated overnight at 378C. Colonies were examined for characteristic morphology, the presence of haemolysis and catalase production, and were examined microscopically by the Gram’s staining technique. The following identification tests were also carried out: (a) Motility test was done using a hanging drop technique with the growth from a Brain Heart Infusion Broth (Oxoid) incubated for 4–18 h at room temperature. A characteristic ‘tumbling’ pattern of motility was noted. (b) CAMP test was carried out on layered 5% Table 1 Number and origin of wild-type Listeria cultures tested Species

Numbers of cultures Total

L. monocytogenes L. innocua L. ivanovii L. welshimeri L. seeligeri L. grayi Total

179 118 12 8 22 2 341

Origin H

A

F and E

92 0 0 0 0 0 92

48 0 6 0 0 0 54

39 118 6 8 22 2 195

H, human clinical (associated with disease). A, animal clinical (associated with disease). F and E, food or the environment.

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(v:v) sheep blood (Oxoid; product code SR 53) agar using a Nutrient Agar No. 1 base (Oxoid). The layer of sheep blood agar was 1–2 mm in depth and equivalent to 2–3 ml over the surface of a nutrient agar base in an 8 cm diameter petri dish. Cultures under test were inoculated onto the agar surface in lines which were perpendicular to either a culture of Staphylococcus aureus (NCTC 1803) or Rhodococcus equi (NCTC 1621), so that the bacterial growth was, at its closest, about 1–2 mm apart. Inoculated CAMP plates were incubated for 18 h at 378C and observed for the enhancement of haemolysis at the closest point between the cultures. (c) sugar utilization was carried out in peptone water broth bases plus phenol red (28 mg / l), and adjusted to pH 7.6 prior to autoclaving. Sugars ( Dmannitol, L-rhamnose, D-xylose, D-glucose, D-salicin, and a-methyl D-mannoside; Sigma-Aldrich, Poole, Dorset, England) were filter sterilized and added to the broths after autoclaving to give a final concentration of 1% (w:v) except for a-methyl D-mannoside which was added as above to give a final concentration of 0.5% (w:v). Broths were inoculated and incubated for up to 7 days at 378C and acidification recorded. ´ 2.3. API Listeria identification kit ( BioMerieux , Basingstoke, Hants, England) All cultures were tested and identified using the API Listeria identification kit which comprises a gallery of 10 microtubes containing dehydrated substrates for enzymatic or sugar fermentation tests. The API Listeria test kit includes an amino acid peptidase substrate (DIM reaction) which is hydrolysed by all Listeria species with the exception of L. monocytogenes. Kits were used in accordance with the manufacturers’ instructions.

2.4. Hydrolysis of ( DLABN)

DL-alanine

b -naphthylamide

This test was carried out as described previously (Clark and McLauchlin, 1997). Listeria were harvested into saline (approximately 10 8 cfu / ml) from the growth obtained after overnight incubation at 378C on blood agar. A portion (25 ml) of the bacterial suspension was added to wells in a U-

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bottomed microtitre plate (Sterilin, Stone, Staffordshire, England), and to this was added 25 ml of DLABN substrate (Sigma-Aldrich, product code A2503; 20 mM dissolved in 0.05 M Tris HCl pH 7.0). The plate was sealed with an adhesive cover, incubated at 378C for 4 h, after which 50 ml of fast violet solution was added (Sigma-Aldrich, code number 85-1, one 12 mg capsule dissolved in 4.8 ml of distilled water just prior to use), and incubated in the dark at room temperature for a further 15 min. The presence of an orange precipitate was recorded as a positive result.

3. Results and discussion All Listeria cultures tested showed typical 1–2 mm diameter, non-pigmented colonies with a crystalline / ground glass appearance on blood agar and gave a characteristic caramel / sour buttery smell. All cultures fermented D-glucose and D-salicin, were gram-positive coccobacilli, catalase test positive, and (with the exception of the L. monocytogenes type strain, NCTC 10357) showed the characteristic tumbling motility at room temperature. With the exception of the type strain of L. monocytogenes (NCTC 10357) and an additional three cultures isolated from food, all L. monocytogenes were haemolytic and CAMP test positive with S. aureus (Table 2). The remaining Listeria species produced typical sugar fermentation patterns and haemolytic reactions (Table 2). Using conventional haemolytic and sugar fermentation tests therefore, 346 (99%) of the 350 cultures were correctly identified. The four non-haemolytic L. monocytogenes were misidentified as L. innocua. The identity of these cultures is further discussed. All cultures of L. monocytogenes and L. innocua fermented L-rhamnose and a-methyl D-mannoside within 24 h. An extended incubation period (up to 7 days) was sometimes necessary for some of the other cultures, particularly the fermentation of D-xylose by L. ivanovii. With the exception of the 181 L. monocytogenes cultures tested, the DLABN substrate was hydrolysed by 163 (96%) of the remaining 169 cultures (Table 2). Using the DLABN test, L. monocytogenes was distinguished from the remainder of the genus in 344 (98%) of the 350 cultures described here. Six of

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Table 2 Results of phenotypic characterization and API Listeria identification test Species

Total

Percentage of cultures giving positive test Hemolysis

L. monocytogenes 181 L. innocua 119 L. ivanovii 14 L. welshimeri 9 L. seeligeri 23 L. grayi 4

98% 0% 100% 0% 100% 0%

API profile

CAMP test

Sugar utilization

Hydrolysis

Sa

Re

M

R

X

aM

of DLABN

98% 0% 0% 0% 100% 0%

0% 0% 100% 0% 0% 0%

0% 0% 0% 0% 0% 100%

100% 100% 0% 0% 0% 0%

0% 0% 100% 100% 100% 0%

100% 100% 0% 0% 100% 100%

0% 100% a 57% b 100% 100% 100%

(correct identification)

97% 97% 100% 100% 100% 100%

M, D-mannitol; R, L-rhamnose; X, D-xylose; aM, a-methyl D-mannoside; DLABN, DL-alanine b-naphthylamide. Sa, Staphylococcus aureus; Re, Rhodococcus equi. a Two cultures gave weak reaction, but became strongly positive after 18 h of incubation. b Six cultures negative, but all became positive after 18 h of incubation.

the 14 L. ivanovii cultures tested gave negative results, however, all of these gave positive results when the incubation period with the DLABN substrate was increased to 18 h. Two cultures of L. innocua which initially gave weak reactions also gave strongly positive reactions after the 18 h incubation. L. monocytogenes cultures which were similarly incubated for 18 h with the DLABN substrate remained negative. The API Listeria test correctly identified 341 (97%) of the 350 cultures tested (Table 2) with no misidentifications with this system. Equivocal profiles, however, not ascribed to any species, were obtained with six cultures of L. monocytogenes and three L. innocua (all of which were isolated from foods). Reactions responsible for these equivocal profiles were: xylose fermentation for the three L. innocua and four L. monocytogenes cultures; tagatose fermentation for one L. monocytogenes; xylose and ribose fermentation for one L. monocytogenes. None of these cultures fermented xylose using the conventional ‘in-house’ fermentation tests using peptone water sugar broths (Table 2). Commercially available test kits and ‘in house’ systems for the identification or detection of L. monocytogenes are available, and these are based on, ´ for example, immunoassays (VIDAS, BioMerieux), DNA hybridization (Gene-Trak, Gene-Trak Systems) and the polymerase chain reaction (TaqMan, Perkin Elmer). In addition, identification of this bacterium can be achieved by the use of DNA probes, the polymerase chain reaction or by nucleic acid finger-

printing. However, despite the availability of alternative identification techniques, conventional methods based on sugar fermentation and haemolytic reactions are most commonly used. Difficulties have been reported in the detection of haemolysis and with the CAMP reaction. It is the author’s experience that there is variation in the degree of haemolysis between cultures of L. monocytogenes, and the use of layered blood agar plates facilitates the recognition of weakly haemolytic cultures. The four non-haemolytic L. monocytogenes cultures (including the type strain of this species) were misidentified as L. innocua, and these have all been confirmed as L. monocytogenes by detection of prfa and hly gene sequences, together with some (two cultures only) rRNA sequence data (McLauchlin et al., results in preparation). However, these cultures are rare, and for most cultures of Listeria recovered from foods, which are usually either L. monocytogenes or L. innocua, (Farber and Peterkin, 1991) satisfactory identification using conventional tests can usually be achieved within 18–24 h of obtaining a pure culture on non-selective solid media. The CAMP test has also presented difficulties for some workers, and again the use of layered plates (in this instance using a thin overlay of agar containing sheep blood) is recommended. The source of blood may be of importance, and some manufacturers’ products may be improved by washing the erythrocytes in physiological solutions. However, satisfactory CAMP plates have been produced from all of a

J. McLauchlin / International Journal of Food Microbiology 38 (1997) 77 – 81

large numbers of batches of the Oxoid product described here. The choice of S. aureus and R. equi is also important since some strains completely haemolyse the erythrocytes proximal to their bacterial growth. The choice of a nutrient agar basal medium is also of importance since richer media may produce excessive growth of S. aureus and R. equi, which can obscure the enhancement reactions because of excessive haemolysis of the erythrocytes. Fernandez-Garayzabal et al. (1996) described some enhancement of haemolysis between R. equi and L. monocytogenes cultures, although these reactions differed in appearance to that seen with L. ivanovii. These reactions are occasionally observed, particularly with older batches of sheep erythrocytes (unpubl. data). However, in this laboratory these reactions have not been reproduced consistently and differ from the ‘arrow head’ appearance obtained with L. ivanovii, they have not been recorded here. The observations reported here that the DLABN test can differentiate L. monocytogenes from the remainder of the genus confirms previous observations (Clark and McLauchlin, 1997). This test has the added advantage of correctly identifying nonhaemolytic L. monocytogenes. The API Listeria test kit is easy to use, relatively rapid, shelf stable and useful for both specialist and non-specialist laboratories. The evaluation reported here is in agreement with that described elsewhere (Bille et al., 1992) and shows that the kit works well for the majority of cultures. Although the final choice of methods rests with the available resources and expertise, the API Listeria test kit should be seriously considered for ease of use and reliability for all laboratories involved with the identification of Listeria from food and the environment.

Acknowledgements The excellent assistance of Mr. R. Iafrate is acknowledged.

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