Genetic diversity of human isolates of Salmonella enterica serovar ...

Journal of Applied Microbiology 2003, 95, 773–780

doi:10.1046/j.1365-2672.2003.02033.x

Genetic diversity of human isolates of Salmonella enterica serovar Enteritidis in Malaysia S.A. Bakeri1, R.M. Yasin2, Y.T. Koh2, S.D. Puthucheary3 and K.L. Thong1,4 1

Institute for Postgraduate Studies, 2Institute for Medical Research, 3Department of Medical Microbiology, Faculty of Medicine, and Institute of Biological Science, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia

4

2003/0291: received 8 April 2003 and accepted 8 May 2003

ABSTRACT S . A . B A K E R I , R . M . Y A S I N , Y . T . K O H , S . D . P U T H U C H E A R Y A N D K . L . T H O N G . 2003.

Aims: The study was undertaken to determine clonal relationship and genetic diversity of the human strains of Salmonella enterica serovar Enteritidis isolated from 1995 to 2002 from different parts of Malaysia. Methods and Results: Antimicrobial susceptibility test, plasmid profiling and pulsed-field gel electrophoresis were applied to analyse 65 human isolates of S. Enteritidis obtained over an eight year period from different parts of Malaysia. Four nonhuman isolates were included for comparison. A total of 14 distinct XbaI–pulsed-field profiles (PFPs) were observed, although a single PFP X1 was predominant and this particular clone was found to be endemic in Malaysia. The incidence of drug resistant S. Enteritidis remained relatively low with only 37% of the strains analysed being resistant to one or more antimicrobial agents. All except one resistant strain carried at least one plasmid ranging in size from 3Æ7 to 62 MDa giving nine plasmid profiles. The three isolates from raw milk and one from well-water had similar PFPs to that of the human isolates. Conclusions: Salmonella Enteritidis strains were more diverse than was previously thought. Fourteen subtypes were noted although one predominant clone persisted in Malaysia. The combination of pulsed-field gel electrophoresis, plasmid profiling and antibiograms provided additional discrimination to the highly clonal strains of S. Enteritidis. Significance and Impact of the Study: This is the first report to assess the genotypes of the predominant clinical S. Enteritidis in different parts of the country. As S. Enteritidis is highly endemic in Malaysia, the data generated would be useful for tracing the source during outbreaks of gastroenteritis in the study area. Keywords: antibiogram, PFGE, plasmids, Salmonella Enteritidis.

INTRODUCTION Human infections with Salmonella enterica serovar Enteritidis continues to be a public health problem worldwide as it is the most frequently reported serovar associated with gastroenteritis (http://www.who.int/salmsurv). Since 1991, S. Enteritidis infections have increased in frequency in Malaysia (Yasin et al. 1995; Lee et al. 1998). The majority Correspondence to: Kwai-Lin Thong, Microbiology Division, Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603, Kuala Lumpur, Malaysia (e-mail: [email protected]).

ª 2003 The Society for Applied Microbiology

of cases were because of unhygienic food handling and consumption of raw or undercooked eggs and poultry products. Despite efforts by the relevant public health authorities to improve food safety, sporadic cases or outbreaks of food poisoning still occur in large populated areas of food eateries especially in institutions, factories and schools. S. Enteritidis is known to have a wide range of animal reservoirs, high potential spread and the ability to survive in the environmental water. Besides poultry and poultry products, S. Enteritidis is also transferred by fresh vegetables, spices, and cheese (D’Aoust 1994; Boonmar et al. 1998;

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Ahmed et al. 2000). Thus, highly discriminating methods are necessary for effective epidemiological surveillance for tracking possible sources of foodborne outbreaks or sporadic cases. The traditional methods of subtyping include biotyping, phage typing, serotyping and antimicrobial susceptibility testing (Threlfall and Frost 1990). These methods often lack discrimination and sometimes these phenotypes are not stable. Although phage typing is the established method of subtyping S. Enteritidis, this method is timeconsuming and restricted to reference laboratories. Therefore, other discriminatory genotypic methods have often been applied to complement phenotypic subtyping. These methods include polymerase chain reaction (PCR) (Hilton and Penn 1998), plasmid profiling, ribotyping (Olsen et al. 1994a; Landeras et al. 1996), pulsed-field gel electrophoresis (Suzuki et al. 1995; Thong et al. 1995; Laconcha et al. 1998; Lukinmaa et al. 1999; Ahmed et al. 2000; Garaizar et al. 2000) and amplified fragment length polymorphism (AFLP) (Laconcha et al. 1998) and more recently, multisequence locus typing (MSLT) (Kotetishvili et al. 2002). There is as yet no gold standard for S. Enteritidis subtyping as each of the currently used techniques has its own limitations (Maslow et al. 1993; Struelens et al. 1998). Among the many molecular approaches, pulsed-field gel electrophoresis (PFGE) in particular, has been widely used in the molecular epidemiological investigation of Salmonella spp. (Suzuki et al. 1995; Thong et al. 1995). A previous study of S. Enteritidis from a local teaching hospital showed that strains were very homogenous by PFGE and ribotyping (Thong et al.1995). There was a close genetic similarity between epidemiologically unrelated and outbreak strains of S. Enteritidis. The continued high prevalence of this Salmonella serovar prompted us to extend the previous study to determine the prevalent pulsotypes among the strains of S. Enteritidis strains collected over an 8-year period (1995–2002) from different parts of Malaysia. Such molecular typing data would allow us to elucidate the clonal relationship and genetic diversity of the strains and would be useful for tracing the source during outbreaks of gastroenteritis in the study area.

Table 1 Resistance patterns and PFGE subtypes of Salmonella Enteritidis strains in Malaysia

Year

Origin

Resistance patterns*

Plasmid type (MDa) (profile)

PFGE type

1995 1995 1995 1995 1996 1996 1996 1996 1996 1996 1997 1997 1998 1999 1999 1999 2000 2000 2000 2000 2001 2001 2001 2001 2002 2002 2002 2002 2002

Kuala Lumpur Kuala Lumpur Seremban Seremban Kuala Lumpur Penang Kuala Terengganu Kuala Lumpur Kuala Lumpur Penang Johor Bharu Kulim Seberang Jaya Kuala Lumpur Penang Kota Bharu Penang Kuala Lumpur Sg. Petani Kuala Lumpur Kuala Lumpur Kuala Lumpur Penang Tumpat Johor Bharu Kuala Lumpur Kuala Lumpur Kuala Lumpur Kota Bharu

T $ $ ST ATCot CKTCot CTCot CKTCot $ ATCot T T TCot A ACot AKS TCot ST TCot T TCot TCot TCot TCot TCot TCot TCot TCot TCot

38,30 (P4) 38 (P1) 38 (P1) 62 (P2) 36 (P3) 62,38 (P5) 62,38 (P5) 62,38,3Æ7 (P6) 38 (P1) 62,36 (P7) 38,36 (P8) 38,36 (P8) 38 (P1) – 62 (P2) 36 (P3) 38 (P1) 38 (P1) 38 (P1) 38,30, 3Æ7, 3 (P9) 38 (P1) 38 (P1) 62,38 (P5) 38,30 (P4) 38 (P1) 38 (P1) 38 (P1) 38 (P1) 62 (P2)

X1 X1 X1f X1c X3 X1 X1g X1 X1a X3 X1 X1 X1d X1 X1b X1I X2 X1c X1 X2 X1 X1 X2a X1 X1 X1 X1 X1j X1c

*A, ampicillin; T, tetracycline; S, streptomycin; C, chloramphenicol; Cot, cotrimoxazole; K, kanamycin ; $, sensitive. Well-water isolate.

carried out at the Salmonella Reference Centre, Institute for Medical Research, Kuala Lumpur. Antimicrobial susceptibility test

M A T E R I A LS A N D M E T H O D S Bacterial strains A total of 65 representative clinical isolates from sporadic cases of gastroenteritis between 1995 and 2002 were analysed. The different geographical locations and dates of isolation are listed in Table 1. Three nonclinical isolates from raw milk (isolated in 1998) and one from well water (isolated in 2002) were included for comparison. The isolation and biochemical identification was carried out according to standard laboratory methods. Serotyping was

A susceptibility test with commonly used antimicrobial agents was performed on Muller–Hinton Agar (Oxoid) by the modified disc diffusion method (National Committee for Clinical Laboratory Standards 2002). Escherichia coli ATCC 25955 and S. aureus ATCC 29213 were used as controls. The antibiotic discs used were ampicillin (A, 10 lg); tetracycline (T, 30 lg); streptomycin (S, 10 lg); chloramphenicol (C, 30 lg); cotrimoxazole (Cot, 25 lg) and kanamycin (K, 30 lg). Interpretation of inhibition zones was according to guidelines by the National Committee for Clinical Laboratory Standards procedure (NCCLS, 2002).

ª 2003 The Society for Applied Microbiology, Journal of Applied Microbiology, 95, 773–780, doi:10.1046/j.1365-2672.2003.02033.x

PFGE ANALYSIS OF SALMONELLA ENTERITIDIS

Plasmid analysis

1

2

3

4

5

6

7

8

9

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10 11 12 13 14 15

Plasmid DNA was extracted according to the method described by Olsen et al. (1994a) and analysed by horizontal agarose electrophoresis in 0Æ8% 0Æ5XTBE gels at 120 V for 3 h. Plasmid sizes were determined with reference to plasmid strains of E. coli 39R and E. coli V517. DNA preparation for PFGE Intact genomic DNA was prepared in agarose plugs as previously described (Thong and Pang 1996; Thong et al. 2001). Genomic DNA was digested with 10 U of restriction enzyme Xba1 (Promega) and PFGE performed using the CHEF-DR II or III system (Bio-Rad Laboratories, Richmond, CA, USA) in gels of 1% agarose in 0Æ5 M TBE buffer (0Æ1 M Tris, 1 M boric acid, 0Æ2 mM EDTA) for 25 h at 200 V at a temperature of 12C, with ramped times of 2–40s. Fragment sizes were estimated in comparison with Lambda ladder (48Æ5 kb concatamers) (New England Biolabs). Data analysis The quantitative difference between two isolates was scored by the Dice coefficient of similarity (F), as previously described (Thong et al. 1995). Clustering analysis was based on the unweighted pair group average method (UPGMA) and was performed with GelCompar software (version 4, Applied Maths). RESULTS A retrospective review of the total numbers of Salmonella spp. submitted to the Institute of Medical Research showed that S. Enteritidis was the most common Salmonella serotype isolated in Malaysia from 1995 to 2000. The total numbers of S. Enteritidis isolated in 1995 through 2000 were 500 (34%), 381 (32%), 220 (25%), 217 (20%), 200 (30%), and 164 (31%), respectively. Of the 65 clinical isolates and the four non-human isolates, 41 of S. Enteritidis were sensitive to all the antimicrobial agents tested. The remaining 24 clinical isolates were resistant to one or more antimicrobial agents. The different antibiograms were: A, T, Cot (n ¼ 1); Cot (n ¼ 2); T, Cot (n ¼ 12); A, Cot (n ¼ 1); A, K, S (n ¼ 1); C, T, Cot (n ¼ 1); A (n ¼ 1); T (n ¼ 4); and A, T, Cot K (n ¼ 1). The environmental well-water isolate was resistant to both tetracycline and cotrimoxazole. Overall, the level of resistant of human S. Enteritidis remained relatively low in Malaysia (Table 1). Results of plasmid screening showed that 24 of the 25 resistant S. Enteritidis carried at least one plasmid ranging

Fig. 1 Representative plasmid profiles of clinical Salmonella Enteritidis strains. Lanes 1–2, 4, 7–11, 13–15: profiles P7, P1, E. coli V517, P4, P2, P1, P5, P5, P5, P3 and E. coli 39R. Lanes 3, 5, 6, 12: plasmid analysis repeated for these strains and had profiles P1, P4, P4 and P3, respectively (data not shown)

in size from 7Æ4 to 62 MDa (Fig. 1). Nine plasmid profiles were observed (Table 1, Fig. 1). The majority of the resistant strains (88% or 22 of 25) had a high molecular weight plasmid of 38 MDa (or 55 kb), alone or in combination with other plasmids. Overall, 18 isolates had one plasmid, nine isolates had two plasmids and one isolate each had three and four plasmids, respectively. Four of the sensitive isolates included for comparison also harboured a single plasmid of 38 MDa (Table 1). Initially, three restriction endonucleases, SpeI, AvrII and XbaI were used to cleave genomic DNAs of 10 different S. Enteritidis strains. Reproducible patterns were obtained when the analysis was repeated three times (data not shown). No further differentiation was provided by both SpeI and AvrII as compared with XbaI (data not shown). Hence, further analysis was performed only with XbaI as it is the most useful restriction endonuclease for Salmonella subtyping (Thong et al. 1995). All the 69 isolates could be typed by PFGE and XbaI digestion yielded 14 different pulsed field profiles (PFPs). The numbers of DNA macrorestriction bands observed in the PFPs ranged from five to 11 (48Æ5–540 kb) (Fig. 2). There was one predominant PFP, X1 and this profile was further differentiated to 10 subtypes (X1a–X1j) with 1–3 bands differences (F ¼ 0Æ8–1Æ0). Of the 88% of the total strains sampled (61 of 69), over the 8-year period and from different localities in Malaysia had very closely related profiles, indicating the homogeneity and clonality of S. Enteritidis strains (Table 2). Three new PFPs were observed that is X2 (three of 69 ¼ 4%), X2a (three of 69 ¼ 4%) and X3 (two of 69 ¼ 3%). Profiles X2, X2a and



M

1

2

3

4

5

6

7

8

9 10 11 12 13 14 15 16 17 18 M

Fig. 2 Representative PFGE-XbaI profiles of clinical and environmental strains of Salmonella Enteritidis. M, lambda DNA concatemer marker; lanes 1–14 clinical strains with profiles: X1, X1a, X1b, X1d, X1e, X1f, X1g, X1h, X1i, X2, X2a and X3, lanes 15–18 environmental strains with profiles X1c, X1, X1 and X1

X3 differed by four bands with respect to X1 and belonged to a genetically diverse group (Fig. 3). The unique X3 pattern was associated with strains isolated only in 1996 and was absent in subsequent years. The other two new PFPs, X2 and X2a were observed only recently, that is in 2000 and 2001 in the urban areas of Kuala Lumpur, Penang and Johor Bharu. The three food isolates were indistinguishable from the clinical isolates having the same predominant profile of X1. The well-water isolate and two clinical isolates had the indistinguishable profile of X1c. The combination of antimicrobial susceptibility patterns, plasmid profiles and pulsotypes further improve the discrimination of these strains (Table 1). For example, 12 strains with a single resistance profiles (TCot) were further subtyped into four plasmid profiles (P1, P2, P4, P5) and five pulsotypes (X1, X1c, X1j, X2, X2a). A dendrogram based on the matrix F values shows the association of the prevalent pulsotypes in Malaysia (Fig. 3). At 69% similarity, two main clusters were noted. The main cluster consisted of 61 isolates with 11 PFPs (X1 and its subtypes) indicating the clonal distribution of these strains whilst the second cluster consisted of six isolates with PFPs X2 and X2. One isolate with PFP X3 was uniquely different from the rest. The four nonclinical strains were grouped together with the clinical isolates in the main cluster.

DISCUSSION Since the 1990s, there has been a steady increase in the total number of clinical Salmonella spp. reported to the Salmonella Reference Center, Institute Medical Research, Malaysia. Overall, the percentages of S. Enteritidis isolates during the last 6 years remained unchanged, ranging from 34% in 1995 to 31% in 2000. It is currently the top Salmonella serovar isolated from cases of human gastroenteritis. According to the WHO Global Salm-Surv., S. Enteritidis is also the most common Salmonella serovars in many countries. Although antimicrobial therapy is not essential for most Salmonella infection, it is necessary for treatment of invasive infections and infections in the immunocompromized patients. The selection of antimicrobial agents for the treatment of these infections has become increasingly restricted because of increasing antimicrobial resistance among Salmonella isolates. In the past ampicillin, chloramphenicol, and trimethoprim-sulfamethoxazole have been the treatment of choice for Salmonella infections. Due to emergence of multidrug-resistant Salmonella, fluoroquinolones and third-generation cephalosporins are the current drugs-of-choice for the treatment of Salmonella infections in adults and children, respectively (Angulo et al. 2000). Antimicrobial-resistant Salmonella results from the use of antimicrobial agents in farm animals, and these resistant



Table 2 PFGE subtypes of representative Salmonella serotype Enteritidis strains from Malaysia during a period of 8 years (1995–2002)*

Year 1995

1996

1997

1998

1998 1999

1999 2000

2000

2001

2002

Geographical area (month/year of isolation)

No. of isolates

PFP (s) (no. of isolates)

GHKL (2/95–3/95) HUKM (2/95–6/95) Seremban (5/95) Terengganu (4/95) GHKL (1/96–3/96 Penang (3/96–4/96) Johor Bahru (3/96) Terengganu (4/95) GHKL.(1/97) Ipoh (1/97) Johor Bahru (1/97) Kulim (1/97) GHKL (1/98) Kota Bharu (2/98) Seremban (1/98) Terengganu (1/98) Penang (1/98) Seberang Jaya (2/98) Klang (2/98) HUKM (1/99) Kulim (1/99) Penang (1/99) Kota Bharu (2/99) Alor Setar (1/99) Klang (6/99) HUKM (1/00) GHKL (1/00) Penang (1/00) Terengganu (1/00) Alor Setar (1/00) Sg. Petani (2/00) UMMC(1/00–11/00) Klang (1/00) GHKL (1/01) Penang (1/01) Alor Setar (1/01) Kelantan (1/01–2/01) Johor Bahru (3/01) Ipoh (3/01) UMMC (1/01–11/01)

32 2 2 1 3 3 1 1 1 1 1 1 1 2 1 1 1 1 2 1 1 1 2 1 1 1 1 1 1 1 1 2 1 3 2 1 2 1 1 8

X1(32) X1(2) X1c(1), X1f(1) X1 (1) X1(1), X1a(1), X3(1) X1(2), X3(1) X1(1) X1g(1) X1(1) X1h(1) X1(1) X1(1) X1d(1) X1(2) X1(1) X1 (1) X1(1) X1d(1) X1(2) X1(1) X1(1) X1b(1) X1(1), X1i(1) X1(1) X1(1) X1c(1) X2(1) X2(1) X1 (1) X1(1) X1(1) X1(1), X1b(1) X1(1) X1(3) X1(1), X2a(1) X1(1) X1(2) X2(1) X1(1) X1(4), X1d(1) X1e(1), X2a(2) X1(2), X1j(1) X1(1) X1c(1)

GHKL (6/02–8/02) Johor Bahru (8/02) Kelantan (6/02)§

3 1 1

GHKL, General Hospital Kuala Lumpur; UMMC, Universiti Malaya Medical Centre; HUKM, Hospital Universiti Kebangsaaan, Kuala Lumpur, Malaysia. *All isolates were from clinical, fecal samples except where indicated. Some of the strains from previous study (Thong et al. 1995). Food isolate (milk). §Environmental isolate (well-water).

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Salmonella are subsequently transmitted to humans via the food chain. In this study, 36Æ2% (25 of 69) of the strains studied showed resistance to one or more antimicrobial agents. A higher resistance rate to tetracyclines (88% or 22 of 25) and trimethoprim–sulfamethoxazole (68% or 17 of 25) was observed. Although the level of multidrug-resistance in S. Enteritidis remained relatively low in this country, continued surveillance and laboratory testing for antimicrobial susceptibility remains the first approach towards detection of emergence of antimicrobial resistant strains. Plasmid profile analysis showed that most of the resistant isolates possessed the 38 MDa (55 kb) plasmid which has been called the Salmonella serovar-specific plasmid (Threlfall et al. 1989; Helmuth and Schroeter 1994; Ridley et al. 1996), is pathogenic as this was reported to be associated with virulence in mice (Nakamura et al. 1985; Stubbs et al. 1994). Different sizes of this plasmid have been reported: 34 MDa (Tassios et al. 1997), 36 MDa (Ridley et al. 1996) or 37 MDa (Ridley et al. 1998). This serovarspecific plasmid was reported to be either found alone or in combination with other plasmids (Threlfall et al. 1989). The absence of plasmid in one of the resistant strains could be caused by plasmid instability, a common phenomenon among salmonellae (Olsen et al. 1994b). There was no distinct association of the PFPs with antibiotic resistant phenotypes as some sensitive and resistant types shared the same patterns. Most of the resistant phenotypes had PFP X1. The strains carrying both ampicillin and tetracycline resistance had the same plasmid 36 MDa and PFGE profile, X3. Although detailed analysis of these plasmids was not done, other researchers have shown that ampicillin-resistant human strains were associated with a 34 MDa conjugated plasmid and that resistance originating in animal strains can be transmitted to humans (Tassios et al. 1997). With PFGE, an extrachromosomal plasmid will not generally be present on the gel if it has no sites for the rare or infrequent restriction enzyme, XbaI being used. If recognition sites are present, the resulting linear band(s) will usually be in the lower range of the PFGE gels as plasmids are often less than 100 kb. In our previous study, PFGE analysis of 35 human isolates from a single teaching hospital showed that the strains were very closely related (Thong et al. 1995). Most of the strains shared one or two PFPs, both in outbreak and sporadic situations. In the present study, more clinical isolates from wider geographical regions in Malaysia and from different years, 1995–2002, were studied in order to determine the true extent of genetic diversity among S. Enteritidis strains. The PFGE approach is a measure of the genetic diversity of the entire genome, which may have arisen as a result of mutation which remove or create recognition sites through insertion, deletion, translocation, inversion or by mobile genetic elements. The assumption is



Dice (Tol 1·0%-1·0%) (H>0·0% S>0·0%) [0·0%-100·0%] PFGE PFGE

No. of strains

100

95

90

85

80

75

70

PFPs

SETX2

3

SETX2a SETX1h

3 1

SETX1i SETX1a

1 1 45

SETX1 SETX1e SETX1d SETX1j SETX1g

1 4 1

SETX1f SETX1b

1 1 2

SETX1c SETX3

3 2

Fig. 3 Dendogram showing cluster analysis of PFGE-Xba1 patterns from 69 strains of Salmonella Enteritidis generated with GelCompar by the UPGMA method based on the matrix of F values. The different PFPs and number of strains tested are indicated

that as time passes and organisms spread, divergence may occur. From a single PFP X1 that was predominant in 1995 (Thong et al. 1995), 14 new PFPs have emerged. Eleven of these DNA profiles differed in 1–3 bands. The original X1 clone could have spread through the environment because of the movement of people. Comparative analysis of clinical isolates obtained 8 years apart, 1995–2002, also indicated that these genotypes were stable and persist over a considerable period of time. Thus the study showed that S. Enteritidis strains from the various hospitals were possibly part of an endemic clone. The present study showed that all the food isolates had identical profile, X1, similar to the human isolates. Strains of S. Enteritidis with PFP X1 were prevalent and recirculating strains were probably responsible for the food poisoning cases in regions of Malaysia during 1995–2001. Whether these strains are more virulent or resistant to the changes in the environment needs further investigation. Two other new PFPs (X2 and X3) were obtained but they were not as widely dispersed as the predominant type. In many developed countries, S. Enteritidis is the most common cause of food-borne gastroenteritis and is associated with contaminated eggs and poultry (Boonmar et al. 1998). Most human Salmonella infections occur as a result of ingestion of contaminated food that is of animal origin. In Malaysia, the continued occurrence of food poisoning because of Salmonella spp. in highly endemic states of Kelantan, Johore and Kedah may indicate environmental sources of Salmonella spp. as demonstrated by the indistinguishable PFGE profiles of the well water and human

isolates. Detailed epidemiological link and identification of sources of contamination would depend on the availability of strains from humans, contaminating foods and the environment at the time of outbreaks of food poisoning. Phage typing is the established method of subtyping Salmonella Enteritidis. However, phage typing is available only in a limited number of reference laboratories. In countries where phage-typing facilities are available, strains with PT1 and PT4 are currently the predominant phage types (Liesbich and Schwarz 1996; Boonmar et al. 1998; Laconcha et al. 1998; Ridley et al. 1998; Tsen and Lin 2001). From other published data, PFGE profiles of these PTs had patterns similar to our PFP X1 obtained in this study (Tassios et al. 1997; Ridley et al. 1998; Lukinmaa et al. 1999; Tsen and Lin 2001). This implies that Salmonella infections due to Enteritidis worldwide is probably caused by a few closely related clones. The high genetic homogeneity among the strains of S. Enteritidis has been noted previously (Thong et al. 1995) and by others (Tassios et al. 1997; Tsen and Lin 2001). In the present study, PFGE was useful in identifying clusters of S. Enteritidis cases and was able to detect minor variations among this clonal species. The multiple subtypes of the predominant profile (X1) demonstrated that PFGE is sufficiently discriminatory to reveal small differences at the DNA level, even within closely related clones. It has also been proposed that a combination of one or two molecular typing methods such as PCR and ribotyping are needed to enhance the ability to discriminate these highly clonal pathogenic strains (Garaizar et al. 2000). In the present study, the combination of plasmid profiles,



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