APMIS 101: 971-974, 1993 Printed in Denmark. All rights reserved

Copyright 0 A P M I S 1993

APUU8 ISSN 0903-4641

Subtyping of a frequent phagovar of Listeria mmcytugenes in Sweden by use of restriction

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endonuclease analysis

HENRIK ERICSSON,' MARIE-LOUISE DANIELSSON-THAM,' PER STALHANDSKE,' WILHELM'THAM' and JAN URSING'

'Department of Food Hygiene, Faculty of Veterinary Medicine, Swedish Agricultural University, Uppsala, and 'Department of Bacteriology, University of Lund, General Hospital, Malmo, Sweden

Ericsson, H., Danielsson-Tham, M.-L., Stilhandske, P., Tham, W. & Ursing, J. Subtyping of a frequent phagovar of Listeria monocytogenes in Sweden by use of restriction endonuclease analysis.

In Sweden, many Listeria monocytogenes strains belonging to serovar 4b and isolated during the last five years from different sources share the same phagovar - 2389:2425:3274:2671:47: 108:340. The object of the present study was to investigate if 31 L. monocytogenes serovar 4b strains belonging to this particular phagovar could be differentiated by use of a simple restriction endonuclease analysis (REA). Among the enzymes tested, Xho I was found to be the most useful, since this enzyme could divide the 3 1 strains into five groups. The profiles of all human clinical isolates were indistinguishable from each other, which indicates that these strains may represent a single clone. The food isolates and the strains of human origin did not share the same profile. This further characterization may be of epidemiological importance as this phagovar of L. monocytogenes has been associated with at least two outbreaks of human listeriosis in Europe.

Key words: Listeria monocytogenes; restriction endonuclease; subtyping; Xho I; Eco RI

H. Ericsson, Department of Food Hygiene, Faculty of Veterinary Medicine, Box 7009, SLU, S-750 07 Uppsala, Sweden.

APMIS 101: 971-974, 1993.

Typing of bacterial strains is important for identifying sources and tracing the transmission routes of food-borne infections. Division into serovars and phagovars is the system often used. However, for Listeria monocytogenes these methods have limitations. Gellin & Broome (10) found that 94% of 16 1 human L. monocytogenes strains divided up into only three serovars: 1 /2a, 1 /2b or 4b. They therefore considered serotyping to be of limited epidemiological use. McLauchlin (1 5) reported that 82% of serovar 4b and only 37% of serovar 1 /2a strains could

Received July 15, 1993. Accepted November 1, 1993

be characterized by bacteriophages. Almost 2500 strains of L. monocytogenes were phage typed by Audurier h Martin (1). Of serogroup 4 strains, 85% were phage typeable, but only 49% of the serogroup 1 strains.

Some authors have suggested restriction endonuclease analysis (REA) as a method to further characterize strains of L. monocytogenes (the papers reviewed here do not include works dealing with gene probes). The enzymes used were Eco RI (8, 16), Hue I11 (2), Hha I (1 1, 22), and Hind I11 (2, 20, 21). Typing of L. monocyto- genes by use of low-frequency-cleavage restric- tion endonucleases such as Apa I, Not I (4, 5 , 6, 7, 13), Smu I (4, 5 , 6, 13), and Asc I (13) has been applied. Pulsed-field gel electrophoresis is,

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however, necessary for separation and visualiza- tion of fragments generated by these enzymes.

In Sweden, many L. rnonocytogenes strains of serovar 4b isolated during the last five years from different sources share the same phagovar - 2389:2425:3274:2671:47:108:340 (9). The object of the present study was to investi- gate if the strains - indistinguishable by sero and phage typing - could be differentiated by use of a simple restriction endonuclease analy- sis (REA).

MATERIALS AND METHODS

Bacterial strains Thirty-one L. monocytogenes strains serovar 4b,

phagovar 2389:2425:3274:2671:47: 108:340 isolated from cheeses, deep litter, and from humans, sheep and one cow with clinical symptoms of listeriosis (Table 1) and one strain serovar 4b, phagovar 2389:3552: 1444: 13 17:3274:52: 108:3 12:340 isolated from soft cheese (SLU 149) were used. In addition, two reference strains (ATCC 191 11 serovar 1 /2a iso- lated from guinea pig and NCTC 11994 serovar 4b isolated from soft cheese) were included. All strains were subcultured onto blood agar and freeze-stored (- 70°C) in Brain Heart Infusion (BHI, Difco) broth with 20% (vol/vol) glycerol while awaiting further investigations.

Preparation of total DNA From each of the freeze-stored bacterial cultures

one loopful was streaked onto blood agar and incu- bated at 37°C for 24 h. Then two to three colonies of each strain were inoculated into 100 ml of BHI broth and incubated aerobically at 37°C for 24 h in an incubator shaker (225 revolutions per min, New Brunswick Scientific Co., Inc., New Brunswick, NJ, USA). Bacterial DNA was prepared essentially as described by Pitcher et al. (17). Briefly, 20 ml of the culture was centrifugated in a fiied angle rotor (15 min, 3100 x g, +4"C). The pellet was resuspended in 10 mlO.9% saline and treated with 1 ml lysozyme (50 mg/ml, Boehringer-Mannheim GmbH, Germany) at 37°C for 60 min. Bacterial cells were lysed with 5 ml 5 M guanidine isothiocyanate (Gibco BRL Life Technologies, Inc., Gaitherburg, MD, USA), vort- exed, and left at room temperature for 30 min. The lysate was cooled on ice, mixed with 2.5 ml ice-cold 7.5 M ammonium acetate, and left on ice for 45 min. Five ml chloroform-2-pentanol (24: 1; vol/vol) mixture was added. The tube was slowly inverted 5-6 times. The suspension was transferred to a corex tube and centrifugated in a fixed angle rotor (20 min, 10800 x g) . The supernatant was transferred to a sec-

ond tube and a 0.54 volume of ice-cold 2-propanol was added for selective precipitation of DNA. Subse- quently, the tube was centrifugated in a swing out rotor (1 min, 2100xg). The pellet was washed five times with ethanol (75%, vol/vol) and dried in vacuo. The pellet was then redissolved in 1 ml distilled water at +4"C. The concentration of DNA was determined by measuring the optical density (OD) at 260 nm and at 280 nm. A ratio (OD260/OD280) above 1.7 was considered acceptable.

DNA was prepared a second time with another method (16) from 18 of the strains. Among these strains was at least one representative from each of the groups formed using the former method of DNA preparation plus REA with Xho I described below (group I-V; see Results and Discussion).

Restriction endonuclease analysis (REA) and agarose gel electrophoresis

Restriction endonucleases Eco RI, Nco I, Cla I, Mlu I, Hind I11 and Xho I (Gibco BRL Life Techno- logies, Inc., Gaitherburg, MD, USA) were used as recommended by the manufacturer. Ten pg of DNA was cleaved with 20 units of enzyme and subsequently precipitated twice with 7.5 M ammonium acetate and 2.5 volumes of ethanol (99.8%, vol/vol). The pellet was washed twice with 2 ml ethanol (75%, vol/vol) and dried in vacuo. DNA was resuspended in TE buffer (10 mM Tris, 1 mM EDTA [PH 7.6]), heated at 65°C for 3 min, mixed with 3 p1 gel loading buffer (type IV, see ref. no. 19), and loaded onto an 0.9% agarose gel (SeaKem GTG Agarose, FMC Bioprod- ucts, Rockland, ME, USA), in TAE buffer (40 mM Tris acetate; 1 mM EDTA [PH 8.01). The gel was run for 21 h at 2 V/cm and at $4"C with buffer circula- tion; it was stained with ethidium bromide solution (final concentration 1.5 pg/ml) for 15 min, washed for 10 min in 1 mM magnesium sulphate, and photo- graphed over a 312 nm UV table.

Fingerprint groups were set up by examination of profiles generated on at least two independent gels.

RESULTS AND DISCUSSION

In the window analyzed (approximately 2-20 Kb), Xho I yielded a suitable cleavage pattern with good band resolution. Using this enzyme we have so far identified five different profiles, visually distinguished among 3 1 strains serovar 4b, phagovar 2389:2425:3274:267 1 :47: 108:340 (Table 1). The profiles are shown in Fig. 1. The other three strains (SLU 149, ATCC 191 11, NCTC 11994) cleaved with Xho I exhibited pro- files clearly different from the profiles of the 31 strains mentioned above (Fig. 1). The profiles of

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all human clinical isolates were indistinguishable from each other, which indicates that these strains may represent a single clone. This result is interesting from an epidemiological point of view, since this phagovar of L. rnonocytogenes has been associated with human outbreaks in Switzerland (3) and France (12, 14, 18).

The DNA restriction patterns of the human strains from the Swiss and the French outbreak were indistinguishable from patterns of isolates from soft cheese (16) and pig tongue in aspic (12), respectively. However, the restriction pro- files of the epidemic strains from the two coun- tries were not identical (1 8).

In the present study the few food isolates and the strains of human origin did not share the same profile. There is an urgent need for cleav- age of more L. rnonocytogenes isolates from food purchased in Sweden.

The other enzymes tested in this study (e.g. Eco RI, Fig. 1) were of little use for discrimi- nation between the isolates under the electro- phoretic conditions used. The profiles obtained after cleavage with Xho I did not change after a second and alternative preparation of total DNA.

TABLE 1. Origin and restriction profiles of 31 L. monocytogenes strains serovar 46, phagovar

2389:2425:3274:2671:47:108:340 using Xho I Strain Origin Restriction - designation profile SLU 24 Cheese A I SLU 21 Cheese A V SLU 71 Cheese B I1 SLU 78-80 Cheese C (one sample) I1 SLU 68 Cheese D V

SLU 204208 Sheep B (one sheep) I1

SLU 199-203 Sheep D (one sheep) I11 SLU 8 Human A 111 SLU 46 Human B I11 SLU 47 Human C 111 SLU 48 Human D I11 SLU 151 Human E 111 SLU 153 Human F 111 SLU 154 Human G 111 SLU 156 Human H I11 SLU 159 Human I I11 SLU 161 Human J 111 SLU 126 Cattle IV SLU 164 Deep litter 111

SLU 184 Sheep A I1

SLU 137 Sheep C 111

Fig. I . REA profiles of L. monocytogenes strains pro- duced by cleavage of DNA with Xho I and Eco RI. Lanes a, j, s: lambda Hind I11 markers; lanes b-i: profiles obtained by using Xho I; lane b: profile 111 (SLU 156); lane c: profile I1 (SLU 80); lane d: profile I (SLU 24); lane e: profile V (SLU 68); lane f: profile IV (SLU 126); lane g: profile VI (NCTC 11994); lane h: profile VII (SLU 149); lane i: profile VIII (ATCC 191 11); lanes k-r: profiles obtained by using Eco RI for strains SLU 156, SLU 80, SLU 24, SLU 68, SLU 126, NCTC 11994, SLU 149, ATCC 19111.

CONCLUSION

Xho I cleavage of Listeriu DNA appears to be a useful and cheap method for demonstration of polymorphism in L. rnonocytogenes strains serovar 4b, phagovar 2389:2425:3274:267 1 :47: 108:340. This further characterization may be of epidemiological importance since this phagovar has been associated with at least two outbreaks of human listeriosis in Europe.

The financial support provided by the Swedish Coun- cil for Forestry and Agricultural Research is gratefully acknowledged. We thank H. P R. Seeliger, J. Bille and E. Bannerman for serotyping and J. Rocourt for phage typing.

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