Characterization of Listeria monocytogenes isolates by Southern blot hybridization

Veterinary Microbiology, 24 ( 1990 ) 341-353 Elsevier Science Publishers B.V., Amsterdam

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Characterization of Listeria rnonocytogenes isolates by Southern blot hybridization Irene V. Wesleya, Ronald D. Wesleya, Judy Heisick b, Fannie Harrell b and Dean Wagner b aphysiopathology Research Unit, National Animal Disease Center, USDA-Agricultural Research Service, Ames, IA, U.S.A. bDepartment of Health and Human Services, Public Health Service, Food and Drug Administration, Minneapolis, MN, U.S.A.

ABSTRACT Wesley, I.V., Wesley, R.D., Heisick, J., Harrell, F. and Wagner, D., 1990. Characterization of Listeria monocytogenes isolates by Southern blot hybridization. Vet. Microbiol., 24:341-353. A synthetic deoxyribonucleotideprobe for virulent Listeria monocytogenes, designated ADO7, was evaluated for its ability to identify restriction fragments of L. monocytogenes with nucleic acid sequences homologous with the fl-hemolysin gene by Southern blot hybridization of clinical and food isolates. The synthetic probe hybridized with three restriction fragments (approximately 1.1, 0.86, and 0.76 kb) of the serotype 1/2A isolates. Southern blot hybridization of the serogroup 4B isolates indicated that the nucleic acid sequences homologous with the p-hemolysin gene probe were limited to a single restriction fragment of approximately 1 kb.

INTRODUCTION

Listeria monocytogenes is a Gram-positive, motile, cryophilic, facultative intracellular bacterium (Murray et al., 1926) which has recently emerged as an important food-borne pathogen (Kvenberg, 1988). Human epidemics have been traced to ingestion of contaminated milk (Seeliger, 1961; Fleming, 1985 ), cheese (James et al., 1985; Malinverni et al., 1985; Linnan et al., 1988 ), coleslaw (Schlech et al., 1983 ), vegetables (Ho et al., 1986 ), poultry and meat products (Schwartz et al., 1988 ). Twelve serotypes are recognized for L. monocytogenes, although most cases of listeriosis are caused by either serovar 1/2A or 4B. Because serotyping is of little help in epidemiological studies (McLaughlin, 1987), alternative methods of discriminating among strains involved in epidrmics and in crossinfection are needed. The fl-hemolysin is secreted only by virulent L. monocytogenes (Gaillard et al., 1986) and is required for intracellular survival (Kuhn et al., 1988 ). A 0378-1135/90/$03.50

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Hind III-Hinc II DNA fragment of 500 base pairs from the//-hemolysin gene of Listeria strain 10403S has been cloned in pUC8 (Datta et al., 1987, 1988 ). A 20-nucleotide base probe, designated ADO7, specific for the fl-hemolysin gene of L. monocytogenes has been synthesized from sequence data obtained from subclones of this fragment in M13 (Datta et al., 1988). The oligonucleotide probe has been shown to be highly specific for L. monocytogenes and does not cross-react with other species of the genus, including hemolytic strains of L. seeligeri and L. ivanovi. In addition, no cross-reactivity has been reported with members of the genus Streptococcus, Staphylococcus, Proteus, or Vibrio (Datta et al., 1988 ). The purpose of this study was to identify the restriction fragment (s) with nucleic acid sequences homologous with the fl-hemolysin gene in order to detect restriction fragment length polymorphism in serogroups 1/2A and 4B. MATERIALS AND METHODS

Isolates Reference strains representing 11 of the 12 recognized serotypes (kindly provided by Dr. R. Weaver, Center for Disease Control, Atlanta, GA) are listed in Table 1. The origin, serotype and surface antigens (factors) of the field isolates examined in this study are given in Tables 2 and 3. All isolates were characterized with respect to Gram stain, motility, hemolysis, and activity in rhamnose and xylose. Field isolates were antigenically characterized with respect to serotype and surface factors as described (Lovett, 1987 ), exTABLE 1 Serotype reference strains ofListeria monocytogenes examined Laboratory

CDC No.

Serovar

G845 G848 G1431 G1127 G1345 G1407 KCI710 (from Donker-Voet G1310 KC 1705 (from Donker-Voet KC1706 (from Donker-Voet KC1717 (from Donker-Voet

88013731 88013665 88027646 88022440 88027355 88027622

I/2A 1/2B 1/2C 3A 3B 3C

88024944

4A 4B

No. 5214 )

No. 10)

4C

No. 21 )

4D

No. 14)

4E

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cept that live bacteria were used. Monospecific polyclonal sera which define somatic antigens or factors of serogroups 1/2A and 4B (R. Bennett, personal communication) were used in this current study. All serotype 4B isolates expressed the surface antigen, factor 6; serotype 1/2A isolates uniformly exhibited factor 1. All isolates described herein are identified by their National Animal Disease Center accession n u m b e r shown in parenthesis. The first set of isolates studied included 13 field isolates (2063-2074, 2104) with no known epidemiological association. The second set of isolates included field strains identified as serotype 1/2A. The third set of isolates included field isolates identified as serotype 4B. Twelve isolates recovered during the 1985 listeriosis outbreak in California which were identified as serotype 4B were also examined. These included five clinical isolates from Los Angeles (2173-2177) and an isolate (2149) from an elderly diabetic who had purchased the cheese in Arizona. Six isolates obtained from Mexican-style soft cheese bearing package code dates corresponding to the epidemic were also evaluated. TABLE2 Serotype 1/2A isolates o f L i s t e r i a monocytogenes examined NADC Number

Source

G845-888013731 2063-AK 1/2a a 2064-AT46 a 2066-H-P238 a 2067-H-342 a 2068-H-1022 ~ 2071-A8694 a 2072-A8801 a 2074-H-25380 a 2075-DV- I b 2076-DV-2 b 2078-DV-4 b 2080-DV-6 b 2082-DV-8 b 2083-DV-9 b 2084-DV- 10~ 2085-DV- 11 b 2100-V7 2102-840300184-F5416 2103-84026817-F5380 2104-84021544-F5260 2107-87-479-752 2110-86-448-196

Reference strain Cow; uterine swab Sheep; septicemia Human; abortion Human; CSF Human; CSF Cow; milk Cow; milk Human; CSF Human; CSF Human; CSF Human; CSF Human; CSF Human; blood Human; blood Human; blood Human; blood Bulk milk Human; blood Human; CSF Human; paracentesis fluid Ice cream Floor drain

aDr. M. Svabic-Vlahovic, Medical Faculty, Belgrade, Yugoslavia. bDr. Mary Scheier, Veterans Administration Hospital Denver, CO, U.S.A.

I.V. WESLEYET AL,

344 TABLE 3 Serotype 4B field isolates of Listeria monocytogenes examined Isolate Number

Source

G 1310-88024944 2047-RM I 2048-RM II 2065-HB220 a 2069-H 1195 a 2070-A8037 a 2077-DV-3 b 2079-DV-5 b 2081-DV-7 b 2098-Scott A 2099-Murray B 2101-V37 2105-84021167-F5234 2106-87-352-900 2108-86-445-642 c 2112-87-340-892 2114-87-340-897 2115-85-354-9185E 2117-85-354-91818E 2118-85-354-91820D 2121-85-354-91838A 2122-85-354-91821C 2123-85-354-91815B 2125-CIP7838-ATCC 19115 d 2149-DA-3 e 2173-F7149 f 2174-F7150 f 2175-F7157 f 2176-F7206 f 2177-F7207 f

Reference strain Raw milk Raw milk Human; vaginal swab Human; vaginal swab Cow; milk Human; CSF Human; CSF Human; blood Human; CSF Human Raw milk Human; blood Ice cream Floor drain Ice cream Ice cream Mexican-style soft cheese Mexican-style soft cheese Mexican-style soft cheese Mexican-style soft cheese Mexican-style soft cheese Mexican-style soft cheese Human; CSF Human Human Human Human Human Human

aDr. M. Svabic-Vlahovic, Medican Faculty, Belgrade, Yugoslavia. bDr. Mary Scheier, Veterans Administration Hospital Denver, CO, U.S.A. CIdentified as L. innocua. dDr. J. Rocourt, Institut Pasteur, Paris, France. eDr. Joseph Lovett, Food and Drug Administration, Cincinnati, OH, U.S.A. fDr. Robert Weaver, Center for Disease Control, Atlanta, GA, U.S.A.

Extraction of chromosomal DNA All isolates were plated onto BHIA containing 5% defibrinated bovine and incubated at 37°C for 24 h. Bacterial lawns were harvested in PBS (0.01 M, pH 7.2), pelleted (8000 ×g, 30 m i n ) and resuspended in 0.4 ml of 25% sucrose in TE buffer ( 1 m M ethylenediaminetetraacetic acid (EDTA), 10 m M Tris, pH 7.2 ), and frozen ( - 20°C ) until the time of DNA preparation. DNA was extracted as follows. The bacterial suspensions ( 300 #1 ) were transferred

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to 13 × 51 ultracentrifuge tubes (Beckman Instruments, Palo Alto, CA), and lysozyme (Sigma Chemical Co., St. Louis, MO) was added ( 130 #1 at 50 mg/ ml). After incubation (37°C, 1 h), 20/tl of proteinase K (Sigma Chemical Co. ) at 50 mg/ml and 130/tl of 0.5 M EDTA (pH 8.0) were added and mixed gently. Cells were lysed by the addition of 130 pl of sarkosyl (25%; sodium salt of N-lauryl-sarcosine, Sigma Chemical Co.) and incubated overnight at 65 ° C. DNA was recovered by equilibrium centrifugation (416 000 × g, 4 h, 15°C) of the lysate in CsC1 ( 1.25 g/ml of 50 mM Tris, 5 mM EDTA, 5 mM NaC1) in a VTi 65.2 rotor (Beckman Instruments, Palo Alto, CA). The viscous DNA band was harvested from the side of the tube through a 16-gauge needle and dialysed extensively against TE buffer (pH 8.0). The final DNA concentration was determined spectrophotometrically in an Ultra-Spec II (Model 4050, LKB Instruments, Gaithersburg, MD) with an OD26o of 1= 50 #g DNA/ml (Maniatis et al., 1982).

Synthetic oligonucleotide probe for L. monocytogenes The synthetic oligonucleotide probe, designated ADO7, was used in this study. The origin, nucleotide sequence, specificity for L. monocytogenes, and method of 5' terminus labeling of ADO7 have been described (Datta et al., 1988).

Slot blot analysis Two micrograms of chromosomal DNA extracted from each of the serotype reference strains was applied to a nylon membrane (Gene Screen, NEN Dupont, Boston, MA) using a slot blot microfiltration apparatus (Bio-Rad, Richmond, CA). Membranes were denatured, neutralized and DNA crosslinked as described by the vendor and refrigerated (4 ° C ) until hybridization.

Restriction endonuclease digestion of DNA Two micrograms of purified DNA was digested (37 ° C, 3-4 h) with Hha I (Bethesda Research Laboratories, Gaithersburg, MD) in a 20-/~1 reaction mixture in buffer supplied by the manufacturer. DNA fragments were separated on 0.8% agarose gels ( 60 V, 16 h) in a horizontal gel bed ( 120 × 25 cm) with Tris-borate EDTA (89 mM Tris borate, 89 mM boric acid, 2 mM EDTA) as the running buffer. At the completion of electrophoresis, gels were stained ( 1 h) with ethidium bromide (0.125 pg/ml), visualized with shortwave UV light and photographed using a Kodak 23A red filter (Foto UV 300/mp-4 DNA Photographic Transilluminator System, Fotodyne, Inc., New Berlin, WI).

Southern transfer Restriction fragments were transferred from agarose gels onto nylon membranes (Gene Screen, NEN Research Products, Boston, MA) as described

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(Southern, 1975 ). UV crosslinking was used to covalently bind the DNA to the membrane filters (Church and Gilbert, 1984). Membranes were refrigerated at 4 °C until hybridization.

Hybridization Prehybridization was carried out for 3 h at 37°C in 6 × S S C (SSC is 0.15 M NaCI, 15 m M Na citrate, pH 7.0), 5 x D e n h a r t ' s solution (0.1% Ficoll, 0.1% polyvinyl pyrrolidone, 0.1% bovine serum albumin), 0.5% SDS, and 100/tg/ml ofsonicated denatured salmon sperm DNA (Maniatis et al., 1982 ). Hybridization was carried out at 37°C for 18 h in fresh prehybridization solution containing 1-2 X 106 cpm/filter of the end-labelled 32p synthetic oligomer. After incubation, filters were washed briefly at room temperature with 2 X SSC, followed by two washes of 6 X SSC at 50°C for 1 h each with gentle shaking. Dried filters were exposed to Kodak X-OMAT AR film with two intensifying screens for 2-4 days at - 80 oC. Films were developed using the Kodak X-Omat M35 Processor (Eastman Kodak, Rochester, NY). RESULTS

Slot blot hybridization The ability of the synthetic oligonucleotide to react with chromosomal DNA prepared from each of the 11 serotype reference strains was evaluated in a 1

2

3

4

5

6

B

E

Fig. 1. DNA-DNA hybridization results obtained with L. monocytogenes strains in a slot blot format using the synthetic probe AD07. Positive results were obtained for L. monocytogenes (slots A1, B1 ) and its serotypes 1/2A (C1, DI ), 1/2B (C2, D2), 1/2C (C3, D3), 3A (C4, D4), 3B (C5, D5), 3C (C6, D6), 4B (E2, F2), 4D (E4, F4), and 4E (E5, F5). Negative results were obtained with L. innocua (A2, B2), L. ivanovi (A3, B3), L. seeligeri (A4, B4), L. welshimeri (A5, B5) and L. monocytogenes serotypes 4A (El, F1 ), and 4C (E3, F3). Empty slots: A6, B6, E6, F6.

347 14

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2.32 2,03

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Fig. 2. (A) Hha I restriction enzyme digests of DNA from clinical isolates. L. innocua (lane 1), 2065 (lane 2), 2104 (lane 3), 2068 (lane 4), 2066 (lane 5), 2074 (lane 6), 2071 (lane 7 ), 2063 (lane 8), 2064 (lane 9), 2067 (lane 10), 2070 (lane 11 ), 2069 (lane 12), 2072 (lane 13), 2073 (lane 14). Hind III digest of bacteriophage lambda DNA is included as size marker. Sizes are indicated in kilobases (kb). (B) Southern blot of this gel and localization offl-hemolysin gene within three low molecular weight restriction fragments (arrows).

slot blot f o r m a t (Fig. 1 ). O f the 1 1 reference serotype strains o f L. m o n o c y togenes which we e x a m i n e d , n i n e c o n t a i n e d nucleic acid sequences h o m o l o gous with the synthetic oligonucleotide. These i n c l u d e d serotypes 1/2A, 1/ 2B, 1/ 1C, 3A, 3B, 3C, 4B, 4D, a n d 4E. R e f e r e n c e strains o f serotypes 4A a n d 4C d i d n o t h y b r i d i z e w i t h the synthetic oligonucleotide A D O 7 .

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9.42 6.55

4.35

2.32

2.03

.564 b

I

2

3 4

5

6 7 8

9 I0 II 12 13 14 15 16 17 18 19

Fig. 3. (A) Hha I restriction enzyme digest of DNA from serotype 1/2A isolates. Serotype 1/ 2A reference strain (lane 1) and field isolates 2063 (lane 2), 2064 (lane 3), 2100 (lane 4), 2102 (lane 5), 2103 (lane 6), 2104 (lane 7), 2075 (lane 8), 2076 (lane 9), 2078 (lane 10), 2080 (lane 11 ), 2083 (lane 12), 2084 (lane 13), 2082 (lane 14), 2085 (lane 15), 2066 (lane 16 ), 2067 (lane 17 ), 2107 (lane 18 ), 2110 (lane 19), Hind III digest of bacteriophage lambda DNA is included as size marker. Sizes are indicated in kilobases (kb). (B) Southern blot of this gel and localization of fl-hemolysin gene within three restriction fragments of approximately 1.1,0.86 and 0.76 kb (arrows).

Examination ofL. monocytogenes field isolates Twelve isolates (9 o f serotype 1 / 2 A and 3 o f serotype 4B) cultured from animal and h u m a n cases ( 2 0 6 3 - 2 0 7 4 , 2104) were evaluated. Following Hha I digestion, six different restriction, patterns were evident in the isolates identified as serotype 1/2A (Fig. 2A). Each of the three isolates (2065, 2069, 2070) identified as serotype 4B exhibited a unique restriction pattern (Fig. 2A). Following Southern transfer, the fl-hemolysin probe hybridized with three low molecular weight restriction fragments of the isolates identified as serotype 1/2A. However, for the three isolates o f serotype 4B, nucleic acid sequences homologous with the fl-hemolysin probe were detected in a single

349 15 14 15 16 IT 18 19

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12

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Fig. 4. (A) Hha I restriction enzyme digest of DNA from serotype 4B (factor 6) isolates. Serotype 4B reference strain (lane 1), and field isolates 2115 (lane 2), 2069 (lane 3), 2101 (lane 4), 2077 (lane 5), 2079 (lane 6), 2108 (lane 7), 2081 (lane 8), 2065 (lane 9), 2125 (lane 10), 2098 (lane 11), 2099 (lane 12), 2105 (lane 13), 2106 (lane 14), 2112 (lane 15), 2114 (lane 16), 2047 (lane 17), 2048 (lane 18), 2070 (lane 19). Hind III digest of bacteriophage lambda DNA is included as size marker. Sizes are indicated in kilobases (kb). (B) Southern blot of this gel and localization offl-hemolysin gene within a single restriction fragment of approximately 1 kb.

restriction fragment of approximately 1 kb. Isolate 2073 exhibited a restriction enzyme pattern typical of L. innocua, failed to hybridize with the fl-hemolysin probe, and was subsequently identified as L. innocua. A larger number of isolates representative of serotypes 1/2A and 4B was evaluated. Nineteen isolates which were recovered from foods and clinical specimens were serologically identified as serotype 1/2A. Restriction enzyme

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4 5 6 78 9101112

9.42 6.56

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Fig. 5. (A) Hha I restriction enzyme digest of DNA from clinical (lanes 1-6 ) and cheese (lanes 7-13) isolates associated with the 1985 listeriosis epidemic. Human isolates 2173 (lane 1), 2174 (lane 2), 2175 (lane 3), 2176 (lane 4), 2177 (lane 5), 2149 (lane 6), and cheese isolates 2115 (lane 7), 2121 (lane 8), 2123 (lane 9), 2117 (lane 10), 2118 (lane 11), 2122 (lane 12). Hind II1 digest of bacteriophage lambda DNA is included as size marker. Sizes are indicated in kilobases (kb). (B) Southern blot of 5A and localization of hemolysin gene within a single restriction fragment of approximately 1 kb. analysis o f these strains y i e l d e d 15 d i f f e r e n t r e s t r i c t i o n p a t t e r n s after H h a I digestion (Fig. 3 A ) . T h r e e h u m a n isolates o r i g i n a t i n g f r o m s p o r a d i c unrelated cases ( 2 0 8 0 , 2083, 2 0 8 4 ) e x h i b i t e d similar r e s t r i c t i o n e n z y m e patterns. S o u t h e r n blot h y b r i d i z a t i o n i d e n t i f i e d t h r e e r e s t r i c t i o n f r a g m e n t s o f a p p r o x i m a t e l y 1.1, 0.86, a n d 0.76 kb with s e q u e n c e h o m o l o g y to the fl-hemolysin gene (Fig. 3 B ) .

CHARACTERIZATION OF LISTERIA MONOCYTOGENES

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Nineteen isolates obtained from foods, animal and human sources were antigenically identified as serotype 4B. Restriction enzyme analysis of these strains yielded seven different restriction enzyme patterns after Hha I endonuclease digestion (Fig. 4A). Nine isolates exhibited identical DNA fingerprint. These included the two human isolates (2098, 2099) associated with the 1983 epidemic in Massachusetts in which milk was the suspected vehicle of transmission (Fleming, 1985 ), a clinical isolate from the Northeast unrelated to that outbreak (2105 ), three ice cream isolates originating in the Midwest (2106, 2112, 2114), and three isolates recovered from bulk milk tanks (2047, 2048, 2070). Southern blot hybridization of these isolates localized nucleic acid sequences homologous with the//-hemolysin gene within a single restriction fragment of approximately 1 kb (Fig. 4B). Isolate 2108 exhibited a restriction enzyme pattern typical of L. innocua, and failed to hybridize with the//-hemolysin probe. Twelve isolates associated with the 1985 listeriosis outbreak in California were examined. The six clinical samples and six cheese samples were of serotype 4B. The 12 isolates exhibited identical DNA patterns after Hha I digestion (Fig. 5A). Nucleic acid sequences homologous with the fl-hemolysin gene probe were present within a single restriction fragment of approximately 1 kb (Fig. 5B). DISCUSSION

//-hemolysin, secreted by virulent Listeria monocytogenes (Gaillard et al., 1986), is required for intracellular survival (Kuhn et al., 1988) and, therefore, is the virulence marker for the species. The//-hemolysin probe was tested in a slot blot format with chromosomal DNA prepared for strains of the 11 reference serotypes of L. monocytogenes. Hybridization occurred with serotypes 1/2A, 1/2B, 1/2C, 3A, 3B, 3C, 4B, 4D and 4E. No hybridization was observed with the reference strains of serotypes 4A and 4C, which represent less than 4% of the L. monocytogenes isolates (McLaughlin, 1987 ). Recently, studies using the 1.1 kb gene probe encoding a delayed hypersensitivity factor (DTH) of virulent L. monocytogenes also failed to hybridize with strains of serogroup 4A (Notermans et al., 1989). It was the goal of this study to evaluate the extent of genetic diversity via restriction fragment length polymorphism in field strains of the serotypes 1/ 2A and 4B, which are the serogroups encompassing nearly 86% of the L. monocytogenes isolates. Field strains were analyzed by Southern blot hybridization with the oligonucleotide probe specific for the//-hemolysin gene. After Hha I digestion, 15 different restriction enzyme patterns occurred in the 20 representatives of serotype 1/2A. Despite the multiple restriction enzyme patterns, the//-hemolysin probe hybridized with three low molecular weight restriction fragments of approximately 1.1, 0.86, and 0.76 kb.

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The major listeriosis food-borne outbreaks have been caused by serotype 4B. Although at least seven restriction enzyme patterns were evident in the 30 isolates of serogroup 4B that we examined, including strains associated with the major food-borne outbreaks, the synthetic oligonucleotide hybridized with a single restriction fragment of approximately 1 kb. To date, we have evaluated approximately 100 strains ofserotype 4B and have found that the fl-hemolysin probe invariable associates with a 1 kb restriction fragment. In this study, we observed multiple restriction enzyme patterns after Hha I digestion in field strains of serotype 1/2A and 4B. However, isolates recovered from a single epidemic exhibited the identical pattern which was characteristic of that outbreak. For example, the 12 isolates (clinical and cheese) associated with the California outbreak exhibited the identical restriction enzyme pattern which differed from that seen in strains originating from the Massachusetts and Halifax outbreaks (manuscript in preparation). In addition, we observed that the restriction enzyme pattern ofL. monocytogenes was distinct from that of L. innocua. During this study, we evaluated two isolates (2073, 2108 ) which failed to react with the fl-hemolysin probe in slot blot format and in Southern hybridization assays, exhibited restriction enzyme patterns characteristic of L. innocua, and which were confirmed biochemically as L. innocua. We have subsequently examined four additional isolates incriminated in human to animal transmission which failed to react with the fl-hemolysin probe, displayed the restriction enzyme pattern typical ofL. innocua, and were identified biochemically as L. innocua. In conclusion, the observed restriction fragment length polymorphism exhibited by serogroup 1/2A isolates suggest that this is a more heterogeneous cluster than the 4B group. In addition, these data indicate the value of restriction enzyme analysis as an important adjunct in verifying species identification and in characterizing epidemic strains. ACKNOWLEDGMENTS

The unfailing cooperation of the Visual Information staff in preparing illustrations, the editorial assistance of Ms. Annette Bates, and the technical assistance of Ms. Laura Rohrbough and Ms. Audra Kreykes are deeply appreciated.

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tide probes for detection of Listeria monocytogenes. Appl. Environ. Microbiol., 54: 29332937. Fleming, D.W., Cochi, S.L., MacDonald, K.L., Brondum, J., Hayes, P.S., Plikaytis, B.D., Holmes, M.B., Audurier, A., Broome, C.V. and Reingold, A.L., 1985. Pasteurized milk as a vehicle of infection in an outbreak of listeriosis. New Engl. J. Med., 312: 404-407. Gaillard, J.L., Berche, P. and Sansonetti, P., 1986. Transposon mutagenesis as a tool to study the role of hemolysin in the virulence ofListeria monocytogenes. Infect. Immun., 52: 50-55. Ho, J.L., Shands, K.N., Friedland, G., Eckind, P. and Fraser, D.W., 1986. An outbreak of type 4B Listeria monocytogenes infection involving patients from eight Boston hospitals. Arch. Intern. Med., 146: 520-524. James, S.M., Fannin, S.L., Agee, B.E., Hall, B., Parker, E., Vogt, J., Run, G., Williams, J., Lieb, L., Salminen, C., Prendergast, T., Werner, S.B. and Chin, J., 1985. Listeriosis outbreak associated with Mexican-style cheese in California. Morbid. Mortal. Weekly Rep., 34: 357359. Kuhn, M., Kathariou, S. and Goebel, W., 1988. Hemolysin supports survival but not entry of the intracellular bacterium Listeria monocytogenes. Infect. Immun., 56: 79-82. Kvenberg, J.E., 1988. Outbreaks of listeriosis/Listeria-contaminated foods. Microbiol. Sci., 5: 355-358. Linnan, M.J., Mascola, L., Lou, X.D., Goulet, V., May, S., Salminen, C., Hird, D.W., Yonekura, M.L., Hayes, P., Weaver, R., Audurier, A., Plikaytis, B.D., Fannin, S.L., Kleks, A. and Broome, C.V., 1988. Epidemic listeriosis associated with Mexican-style cheese. New Eng. J. Med., 319: 823-828. Lovett, J., 1987. Listeria isolation. In: FDA Bacteriological Analytical Manual, Suppl. 6 edn. Association of Analytical Chemists, Arlington, VA. Malinverni, R., Bille, J., Perret, C., Regli, F., Tanner, F. and Glauser, M. P., 1985. Listeriose epidemique. Schweiz. Med. Wochenschr., 115: 2-10. Maniatis, T., Fritsch, E.F. and Sambrook, J., 1982. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, pp. 387-389. McLauchlin, J., 1987. Listeria monocytogenes, recent advances in the taxonomy and epidemiology of listeriosis in humans. J. Appl. Bacteriol., 63:1-11. Murray, E.G.D., Webb, R.A. and Swann, M.B.R., 1926. A disease of rabbits characterized by large mononuclear leukocytosis, caused by a hitherto undescribed bacillus, Bacterium monocytogenes (n.sp.). J. Pathol. Bacteriol., 29: 407-439. Notermans, S., Chakraborty, T., Leimeister-Wachter, M., Dufrenne, J., Heuvelman, K.J., Maas, H., Jansen, W., Wernars, K. and Guinee, P., 1989. Specific gene probe for detection ofbiotyped and serotypes Listeria strains. Appl. Environ. Microbiol., 55: 902-906. Schwartz, B., Ciesielski, C.A., Broome, C.V., Gaventa, S., Brown, G.R., Gellen, B.G., Hightower, A.W. and Mascola, L., 1988. Association of sporadic listeriosis with consumption of uncooked hot dogs and undercooked chicken. Lancet, i: 779-782. Schlech, W.F., Lavigne, P.M., Bortolussi, R.A., Allen, A.C., Haldane, E.V., Wort, A.J., Hightower, A.W., Johnson, S.E., King, S.H., Nicholls, E.S. and Broome, C.V., 1983. Epidemic listeriosis: Evidence for transmission by food. New Eng. J. Med., 309: 203-206. Seeliger, H.P.R., 1961. Listeriosis, 2 edn. New York, Macmillan Publishing Company, 121 pp. Southern, E., 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol., 98:503-517.