J Med Microbiol 57 (2008), 58-63; DOI: 10.1099/jmm.0.47213-0
© 2008 Society for General Microbiology
ISSN 1473-5644
Molecular typing of Japanese Escherichia coli O157 : H7 isolates from clinical specimens by multilocus variable-number tandem repeat analysis and PFGE
Fumihiko Kawamori1,2,
Midori Hiroi1,2,
Tetsuya Harada1,
Katsuhiko Ohata1,
Kanji Sugiyama1,
Takashi Masuda1 and
Norio Ohashi2,3
1 Department of Microbiology, Shizuoka Institute of Environment and Hygiene, Kita'ando, Aoi-ku, Shizuoka-shi, Shizuoka 420-8637, Japan
2 Laboratory of Environmental Microbiology, Institute for Environmental Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shzuoka-shi, Shizuoka 422-8526, Japan
3 Global COE Program, Graduate School of Nutritional and Environmental Sciences, University of Shizuoka, Shizuoka, Japan
Correspondence
Fumihiko Kawamori
fumihiko1_kawamori{at}pref.shizuoka.lg.jp
Received 6 February 2007
Accepted 16 September 2007
The multilocus variable-number tandem repeat analysis (MLVA) method to target eight variable-number tandem repeat loci, based on agarose gel electrophoresis separation of multiplexed PCR products, and the PFGE method were applied to clinical isolates of Escherichia coli O157 : H7 with the aim of comparing their performance as methods of typing this bacterium. Using MLVA, a total of 57 isolates from patients in Shizuoka prefecture, Japan, were divided into 20 types and classified into 23 PFGE types. Twenty-four isolates from four sporadic infections, four household contact infections and one outbreak that occurred in central parts of Shizuoka prefecture during August to November in 2005 were shown to be the same MLVA type, and most of the isolates had identical PFGE banding patterns, suggesting the diffuse outbreak in these parts of Japan. Thus, there was a good correlation between MLVA types and PFGE types, with both methods displaying broadly similar discriminatory powers. However, the MLVA typing proved to be a much easier and more rapid method for the analysis of E. coli O157 : H7 strain relatedness to identify transmission routes. Hence, our MLVA method would be a suitable technique for routine typing in many laboratories, including public health agencies, and even in hospitals.
Abbreviations: MLVA, multilocus variable-number tandem repeat analysis; TR, tandem repeat, VNTR, variable-number tandem repeat.
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INTRODUCTION
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Escherichia coli O157 : H7 has emerged as a food-borne pathogen causing severe illness with bloody diarrhoea and haemolytic uraemic syndrome (Besser et al., 1999), and is of public health significance worldwide. Most E. coli O157 : H7 infections are caused by exposure to bovine faeces-contaminated food or water, and by person-to-person transmission in household contact infections or outbreaks (Keen et al., 2006; Parry & Salmon, 1998; Rangel et al., 2005). In Japan, the extraordinarily large outbreaks of E. coli O157 : H7 that occurred in 1990 and 1996 resulted in the infection of thousands of people and the death of seven patients by haemolytic uraemic syndrome (Izumiya et al., 1997; Watanabe et al., 1996). Since then, more than 2000 cases per year from sporadic infections, household contact infections and outbreaks have been documented in Japan.
Molecular typing methods are increasingly used to assess the relatedness of E. coli O157 : H7 strains, in particular to identify their transmission routes. PFGE-based typing is highly discriminative (Izumiya et al., 1997; Swaminathan et al., 2001), but it is time-consuming and cannot easily handle a large number of sample sets.
Multilocus variable-number tandem repeat analysis (MLVA) is a recently developed PCR-based technique (Lindstedt, 2005) used to target tandem repeats (TRs), which are areas of the bacterial genome that evolve rapidly (the definition of a variable-number TR (VNTR)] (van Belkum et al., 1998). Targeting of these elements, which often vary in number among different strains of the same species, has been successfully used to discriminate between strains of prokaryotes such as Bacillus anthracis (Keim et al., 2000), Yersinia pestis (Klevytska et al., 2001), Salmonella enterica (Lindstedt et al., 2003), Neisseria meningitidis (Yazdankhah et al., 2005), Mycobacterium tuberculosis (Cowan et al., 2002; Savine et al., 2002) and E. coli O157 : H7 (Noller et al., 2003; Lindstedt et al., 2004; Keys et al., 2005). However, in E. coli O157 : H7, MLVA-based typing requires sequencing to determine the number of short repeats (mostly 6 bp) at TR loci. Here, we describe a much simpler MLVA method based on the agarose gel electrophoresis separation of multiplexed PCR products for the analysis of E. coli O157 : H7 strain relatedness, with the objective of identifying the transmission routes. Furthermore, we compared its performance with that of PFGE-based typing.
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METHODS
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E. coli O157 : H7 isolates and culture.
A total of 57 E. coli O157 : H7 isolates were obtained from 20 patients with sporadic infections, 6 from 1 outbreak and 31 from 11 household contact infections (2–5 patients per case) in Shizuoka prefecture, Japan, in 2005. Production of Stx was examined by the reversed passive latex agglutination test according to the manufacturer's instructions (Denka Seiken). Each isolate was cultured on a sorbitol MacConkey agar plate (Oxoid) for 18 to 24 h at 37 °C, and a single colony was transferred and spread onto a heart infusion agar plate and further incubated for 18 to 24 h at 37 °C.
MLVA.
The entire E. coli O157 : H7 EDL933 genome sequence (GenBank accession no. AE005174) was analysed to find the genetic loci of VNTR using Tandem Repeats Finder software (Benson, 1999). Of 112 VNTR loci detected in the genome, 19 were selected for designing PCR primers. From preliminary PCR experiments using these primers, we eventually chose eight VNTR loci as targets for MLVA typing (Table 1
). Multiplexed PCR was performed using primer-pair sets for four combinations of two VNTR loci (VR1 and VR7, VR2 and VR5, VR3 and VR8, and VR4 and VR6). PCR reaction mixtures (20 µl) contained template DNA, 0.5 µM each primer, 2.5 mM dNTPs, 2.0 µl 10x PCR buffer, and 1 unit Ex Taq hot start version (Takara-Bio). The mixture tubes were placed in a thermal cycler (GeneAmp PCR system 9700; Applied Biosystems) and the temperature was raised to 94 °C for 5 min, followed by 30 cycles of 95 °C for 30 s, 55 °C for 30 s and 72 °C for 30 s. The final hold was for 5 min at 72 °C. After PCR, the amplicons were electrophoresed in a 20 cm long 3 % agarose gel (MetaPhor agarose; Cambrex) in a Multi-submerge agar 30 (Atto) apparatus at 8 V cm–1 for 3 h at 15 °C. Two DNA size markers of 20 and 100 bp ladders were mixed, and used for the estimation of PCR product sizes. After electrophoresis, the gels were stained and visualized with 0.5 µg ethidium bromide ml–1. Based on the PCR product sizes, the number of repetitive DNAs at each VNTR locus was estimated, and the E. coli O157 : H7 clinical isolates were classified based on the variation of the number of repeats at eight VNTR loci.
PFGE.
PFGE analysis was performed according to the procedure described by Ribot et al. (2001). Briefly, 100 µl bacterial suspension, 20 mg proteinase K ml–1 (Roche) and 110 µl 1 % (w/v) SeaKem Gold agarose (Cambrex) kept at 60 °C were mixed. The solution was poured into the wells of a sample plug caster (Bio-Rad) and kept at 4 °C to solidify. The plugs were incubated with 5 ml lysis buffer (50 mM EDTA pH 8.0, 50 mM Tris, 1 % N-lauroyl sarcosine and 0.2 mg proteinase K ml–1) at 54 °C for 60 min, washed with distilled water at 54 °C for 15 min and washed three times with TE buffer (10 mM Tris/HCl, 1 mM EDTA, pH 8.0) at 54 °C for 10 to 60 min. Then, the plugs were cut and treated with XbaI restriction enzyme (30 units per sample) at 37 °C for 2 to 3 h. Electrophoresis was performed in a 1 % SeaKem Gold agarose gel with 0.5x TBE (50 mM Tris, 45 mM boric acid, 0.5 mM EDTA) using the CHEF-DR III system (Bio-Rad) at pulse time of 2.2–54.2 s and 6 V cm–1 for 20 h at 12 °C. After electrophoresis, the gel was stained with ethidium bromide solution and photographed under a transilluminator. Computer-assisted analysis for the comparison of PFGE banding patterns was performed with FingerPrinting II software (Bio-Rad) using the Dice coefficient according to the manufacturer's instructions. A dendrogram was constructed by UPGMA, and a tolerance of 1 % in the band position was applied to the clustering.
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RESULTS AND DISCUSSION
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A total of 57 E. coli O157 : H7 clinical isolates obtained from patients in Shizuoka prefecture, Japan, were examined by MLVA to target the eight VNTR loci of the genome. Multiplexed PCR revealed the targeted amplicons at the eight loci in all isolates tested, except in two isolates of each at the VR1 and VR4 loci (Table 2). Although the repeat array size is a very short sequence of 6 bp at VR1, VR 3, VR4, VR5 and VR8 loci (Table 1
), we could successfully distinguish the number of repetitive DNAs among the isolates on the agarose gel (Fig. 1). As shown in Table 2, the 57 isolates were divided into eight types with 4 to 14 repeats at VR1, three types with 4 to 7 repeats at VR2, six types with 4 to 10 repeats at VR3, eight types with 6 to 17 repeats at VR4, six types with 5 to 16 repeats at VR5, four types with 6 to 9 repeats at VR6, four types with 4 to 8 repeats at VR7, and five types with 3 to 8 repeats at VR8. Eventually, all isolates tested could be classified into 20 types based on the variation of the number of repetitive DNAs at the 8 loci (MLVA types A to T in Table 2). Twenty-four isolates from four sporadic infections, four household contact infections (related cases 8, 9, 10 and 11 in Fig. 2) and one outbreak were shown to be the same MLVA type N, suggesting the predominance of E. coli O157 : H7 during 2005 in Shizuoka prefecture of Japan.
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Fig. 1. Representative agarose gel MLVA profiles of Japanese E. coli O157 : H7 clinical isolates. Lanes: M, DNA size markers (20+100 bp ladders); 1, S05-V9 (MLVA type C); 2, S05-V19 (MLVA type D); 3, S05-V42 (MLVA type L); 4, S05-V33 (MLVA type N).
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Fig. 2. Dendrogram of Japanese E. coli O157 : H7 clinical isolates constructed based on PFGE with XbaI and the corresponding MLVA types. Clinical isolates from each case of household contact infection (corresponding to ‘related case’) and one outbreak are enclosed with a line, and the others are from sporadic infections. Identical PFGE types which were considered by cluster analysis with 1 % tolerance in the band positions are indicated by brackets on the right of PFGE photos. Isolates which have banding patterns considered as unique PFGE and MLVA types are indicated by asterisks after the isolate and MLVA type, respectively. STX, Shiga toxin.
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The strain relatedness of 57 Japanese E. coli O157 : H7 isolates was further analysed by PFGE typing (Fig. 2). A total of 19–24 DNA bands of the 57 isolates separated by PFGE were detected in the 30 to 500 kb range. By cluster analysis with 1 % tolerance in band positions, isolates with identical PFGE banding patterns were considered as ‘identical PFGE types’. We also considered any isolate with more than three different (in size or in number) bands in comparison with any other isolates as a ‘unique PFGE type’, and isolates with one or two different bands in comparison with the other isolates as ‘similar PFGE types’. As shown in Fig. 2, the tree branched into two major clusters: one (10 isolates) consisting of 8 STX2-producing and 2 STX1-STX2-producing isolates, and another (47 isolates) composed of 46 STX1-STX2-producing and 1 STX2-producing isolates. By PFGE, the total 57 clinical isolates were classified into 23 types, and the isolates with identical PFGE banding patterns were further grouped into 8 identical PFGE types (indicated by the brackets on the right of the PFGE photos in Fig. 2). Twenty-two isolates from three sporadic infections, four household contact infections (related cases 8, 9, 10 and 11 in Fig. 2) and one outbreak, which had identical PFGE banding patterns, were considered as the major identical PFGE types. This corresponds well to the results obtained by the MLVA method (MLVA type N), except for two isolates (SV05-V25 and SV05-V30). Two to five isolates from each of the eleven household contact infections (related cases 1 to 11), except for S05-V25, were genetically related by the PFGE method.
The PFGE profiles were well correlated with the results of MLVA as follows: (i) 41 out of 46 isolates with MLVA types C, D, F, N, O, Q, R, S and T had PFGE banding patterns identical to those of other isolates with the same MLVA types (indicated by the brackets on the right of the PFGE photos in Fig. 2), except for S05-V31, S05-V24, S05-V13, S05-V25 and S05-V30; (ii) 8 out of 11 isolates with ‘unique MLVA types’ (MLVA types marked with asterisks in Fig. 2), except for S05-V47, S05-V4 and S05-V12, from sporadic infections had unique PFGE banding patterns (isolate with asterisks in Fig. 2); (iii) 3 isolates, S05-V31, S05-V13 and S05-V25, had only 1 or 2 different bands on PFGE from those of other isolates with MLVA types D, R and N, respectively (Fig. 2); (iv) 1 isolate with MLVA type R, S05-V24, was different only in the number of repeats at VR4 locus from those of 2 other isolates with MLVA type Q, although all 3 isolates in the related case 6 had identical PFGE banding patterns (Table 2, Fig. 2); (v) 3 isolates of S05-V47 (MLVA type G), S05-V4 (MLVA type J) and S05-V12 (MLVA type P), had only 1 or 2 different bands on PFGE and only 1 or 2 different numbers of repeats at the VR4 locus from those of the isolates with MLVA types J, G and N, respectively (Table 2, Fig. 2). The only striking discrepancy between the results of MLVA and PFGE was found in one isolate, S05-V30, which had a unique PFGE type with more than 5 different bands in comparison with any other isolates, but it was grouped into MLVA type N consisting of 24 isolates. The identity between the types of E. coli O157 : H7 isolates in MLVA and PFGE was 93.0 % (53/57).
The MLVA method, which was developed recently, is useful for bacterial typing targeting VNTR loci with less than 100 bp TR sequences distributed throughout the genome. Because the number of repetitive DNAs with a repeat array size of 6 bp varies on the E. coli O157 : H7 genome, the repeats have been frequently used as targets of MLVA (Keys et al., 2005; Noller et al., 2003). To date, the number of such short repeats at loci has been determined by sequencing (Keys et al., 2005; Lindstedt et al., 2004; Noller et al., 2003). However, we could estimate the number of such short repetitive DNAs more easily on agarose gel using the MLVA method described in this study. An important aspect of this technique is that the PCR target length at five VNTR loci with a repeat array size of 6 bp (VR1, VR3, VR4, VR5 and VR8) should be less than 200 bp, because the small size differences of the 6 bp repeats between PCR products of more than 200 bp might not separate well on agarose gel.
Noller et al. (2003) reported that MLVA to target seven VNTR loci has a discriminatory power higher than that of PFGE, because one of seven VNTR loci (Noller's TR2) with a repeat array size of 6 bp at 3 559 120–3 559 225 bp of E. coli O157 : H7 EDL933 strain has a large variation in the number of repeats among isolates. According to their report, Noller's TR2 locus has been shown to vary between 4 and 58 repeats among 58 strains. Lindstedt et al. (2004) and Keys et al. (2005) confirmed such a large variation of Noller's TR2 by their MLVA. Noller et al. (2006) further reported that the number of repeats at one of seven VNTRs (Noller's TR2) was highly changed in 35 of 534 colonies after one to ten times broth-culture-passages from an E. coli O157 : H7 single strain, but the number of repetitive DNAs at other loci had changed only in 0 to 4 colonies. In this study, however, we did not target Noller's TR2 locus for our MLVA typing, because the variation of the number of repeats at that locus is too large for our typing. It is almost impossible to design PCR primers to obtain less than a 200 bp product at Noller's TR2 locus. Hence, the discriminatory power of PFGE seems to be slightly superior to that of our MLVA typing, but the results obtained by MLVA and PFGE in this study were well correlated. In both MLVA and PFGE, the E. coli O157 : H7 clinical isolates from respective sporadic infections were mostly a unique type, and the isolates from household contact infections and from an outbreak were clustered in each case with some exceptions. A public health significance of this finding is that 24 isolates with MLVA type N were predominant (42.1 %, 24/57) and 23 of those 24 isolates had identical or similar PFGE types. A total of 23 out of 24 isolates, not including S05-V30, were obtained from 8 cases of 3 sporadic infections, 4 household contact infections and 1 outbreak that occurred in central parts of Shizuoka prefecture during August to November in 2005, suggesting that a diffuse outbreak might have occurred in these parts of Japan during that season.
In Japan, the National Institute of Infectious Diseases and prefectural public health institutes have made plans to set up a PFGE-based national network (PulseNet Japan) for monitoring the cross-prefectural spread of Japanese O157 strains (Matsumoto et al., 2005). If the PulseNet national database eventually becomes available, the use of two or more endonucleases rather than a single enzyme in PFGE would be more useful for the precise classification of the strains as suggested by Gupta et al. (2004). Without the organization of the PulseNet surveillance system, the use of multiple enzymes will probably further complicate and confuse the interpretation of PFGE results.
As mentioned above, MLVA typing based on the agarose gel electrophoresis separation of multiplexed PCR products, as well as PFGE typing based on XbaI digestion, is available for the analysis of E. coli O157 : H7 strain relatedness in order to identify transmission routes. Because our MLVA method is a much easier and more rapid technique, it will facilitate the typing and molecular epidemiology of E. coli O157 : H7 clinical isolates in research institutes, public health agencies and even hospitals.
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ACKNOWLEDGEMENTS
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This work was supported in part by a grant, H18-Shinkou-Ippan-016, to F. K. for research on emerging and re-emerging infectious diseases, from the Japanese Ministry of Health, Labor and Welfare.
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INTRODUCTION
METHODS
