Letters in Applied Microbiology ISSN 0266-8254
ORIGINAL ARTICLE
Putative classification of clades of enterohemorrhagic Escherichia coli O157 using an IS-printing system S. Hirai1,2, E. Yokoyama1, Y. Etoh3, J. Seto4, S. Ichihara3, Y. Suzuki4, E. Maeda3, N. Sera3, K. Horikawa3, S. Sato5 and T. Yamamoto2 1 2 3 4 5
Division of Bacteriology, Chiba Prefectural Institute of Public Health, Chiba, Japan Department of Microbiology and Molecular Genetics, Graduate School of Pharmaceutical Science, Chiba University, Chiba, Japan Division of Pathology and Bacteriology, Fukuoka Institute of Health and Environmental Sciences, Fukuoka, Japan Department of Microbiology, Yamagata Prefectural Institute of Public Health, Yamagata, Japan Chiba Prefectural Institute of Public Health, Chiba, Japan
Significance and Impact of the Study: This study demonstrated that enterohemorrhagic E. coli O157 (O157) strains could be putatively classified in clades using an IS-printing system. IS-printing was previously developed as a relatively quick and easy tool for analysis of insertion sequence 629 in the O157 genome. Since most local government public health institutes in Japan carry out IS-printing for early detection of O157 outbreaks, these data should be useful for putative classification of O157 strains in each area.
Keywords enterohemorrhagic Escherichia coli O157, IS-printing system, population genetic analysis, putative classification of clades, Relative Likelihood. Correspondence Shinichiro Hirai, Division of Bacteriology, Chiba Prefectural Institute of Public Health, 666-2 Nitona, Chuo, Chiba City, Chiba 260-8715, Japan. E-mail: [email protected] 2015/0379: received 24 February 2015, revised 11 May 2015 and accepted 25 May 2015 doi:10.1111/lam.12448
Abstract Enterohemorrhagic Escherichia coli O157 (O157) strains can be classified in clades by single nucleotide polymorphisms (SNPs), but this analysis requires significant laboratory effort. As the distribution of insertion sequence (IS) 629 insertions has been reported to be biased among different clades, O157 isolates can be putatively classified in clades by comparison with an IS629 distribution database. A database of the IS629 distribution in O157 strains isolated in Chiba Prefecture and their classification in clades was determined by SNP analysis and IS-printing, an easy and quick analytical tool for IS629 in the O157 genome. The IS629 distribution in O157 strains isolated in Fukuoka and Yamagata Prefectures was determined by IS-printing. These strains were putatively classified in clades by Relative Likelihood calculations that compared the IS-printing data and the IS629 distribution database. Concordance Ratios were calculated, which compared the number of strains putatively classified in a clade by Relative Likelihood to the number of strains classified in that clade by SNP analysis. For the Fukuoka and Yamagata strains, the Concordance Ratios for clades 3, 6 and 8 were 97–100%, for clade 7 about 88%, and for clades 2 and 12 over 90%. In conclusion, O157 clade 2, 3, 6, 7, 8 and 12 strains could be putatively classified by IS-printing.
Introduction Recent studies showed that enterohemorrhagic Escherichia coli O157 (O157) strains can be divided into several phylogenetic groups; i.e., lineages (Kim et al. 2001), subgroups and clusters (Shaikh and Tarr 2003), and clades (Manning et al. 2008). Yokoyama et al. (2012) showed that the hierarchical relationship of O157 phylogenetic groups was, in descending order, lineage, subgroup,
cluster, and clade, and proposed a paraphyletic model for O157 evolution. As shown in Table S1, this model has been further modified with new clade designations based on lineage analysis data (Hirai et al. 2013). Different distributions of strains in O157 phylogenetic groups have been reported in some countries. Clade 2, 3, 7 and 12 strains were prevalent in some prefectures in Japan (Hirai et al. 2014), clade 2 and 8 strains in Michigan, U.S. (Manning et al. 2008), and clade 6 and 7 strains
Letters in Applied Microbiology 61, 267--273 © 2015 The Society for Applied Microbiology
267
S. Hirai et al.
Identification of EHEC O157 clades
in Australia (Mellor et al. 2012). Phylogenetic differences were also reported in the strains in some regions within a country. Different lineage distributions of O157 strains were found between the North and South Islands of New Zealand (Franz et al. 2012; Jaros et al. 2013). Since each lineage includes specific clades, the clade distribution of O157 strains may be different within New Zealand. Differences have been reported in the pathogenicity of O157 strains in different clades. For example, more clade 8 strains were isolated from patients with hemolytic uremic syndrome (HUS) than strains in other clades, suggesting strong HUS pathogenicity of clade 8 strains (Manning et al. 2008). In contrast, fewer clade 12 strains were isolated from hemorrhagic colitis patients than strains in other clades, indicating that clade 12 strains may be weakly pathogenic for this disease (Etoh et al. 2014). HUS in O157-infected patients in Argentina, where clade 8 is prevalent (Mellor et al. 2012), has been reported to be ten times higher than in other developed counties (Rivas et al. 2006; Leotta et al. 2008). Therefore, information on the clade distribution of O157 strains in an area should be useful for analysis of the public health risk of O157 infections in that area (Hirai et al. 2014). Classification of O157 strains in clades has been based on two types of analysis: detection of single nucleotide polymorphisms (SNPs) at 32 loci in the O157 genome (Manning et al. 2008) and lineage specific polymorphism assay-6 (LSPA-6) (Kim et al. 2001). Yokoyama et al. (2012) used a low cost ARMS-PCR method to study the distribution of O157 strains in clades, but this method was not suitable for a large scale study because it required agarose gel electrophoresis analysis of the PCR products. We developed a method for classification of O157 strains in clades using capillary gel sequence (CES) analysis and carried out a large scale study (Hirai et al. 2014). However, since CES analysis requires very expensive equipment, research institutes without CES capability cannot carry out this analysis. We previously reported linkage disequilibrium of insertion sequence (IS) 629 among O157 strains in different clades (Hirai et al. 2013). An IS-printing system was developed as a molecular epidemiological tool to investigate the distribution of IS629 in the O157 genome (Ooka et al. 2009). IS-printing can be carried out easily and quickly using two sets of multiplex PCRs, each for 16 loci, followed by analysis of the PCR amplicons by agarose gel electrophoresis. Therefore, IS-printing data for O157 strains should enable putative strain classification in clades by comparison with a database of the biased IS629 distribution in strains in O157 clades. For data analysis, the Relative Likelihood, a numerical measure of the probability that an organism is a member of a biological group, was used. The Relative Likelihood 268
can be calculated by comparing some characters of an organism with those of members of a biological group (Bascomb et al. 1973; Willcox et al. 1973), and has been used for the classification of unknown bacteria in some commercial kits; e.g., the API series (Sysmex Biomerieux, Lyon, France) and the BD BBLCrystal series (Becton, Dickinson and Company, Franklin Lakes, NJ). Therefore, O157 strains can be putatively classified in clades by Relative Likelihood using a database of the IS629 distribution in the strains in O157 clades. In this study, the IS629 distribution in O157 strains isolated in Chiba Prefecture, Japan, was determined by IS-printing, and these strains were classified in clades by SNP analysis and LSPA-6 to construct a database of the IS629 distribution in strains in each clade. The IS629 distribution in O157 strains isolated in Fukuoka and Yamagata Prefectures was determined by IS-printing, and these strains were putatively classified in clades by Relative Likelihood calculations that compared the IS-printing data and the IS629 distribution database. The Concordance Ratio, the ratio of the number of strains putatively classified in a clade by Relative Likelihood from IS-printing analysis relative to the number classified by SNP analysis and LSPA-6, was calculated for the O157 strains isolated in Fukuoka and Yamagata Prefectures. The Relative Likelihood was shown to be useful for the putative classification of O157 strains in clades. Results and discussion A database of the IS629 distribution at loci in O157 strains isolated in Chiba Prefecture and classified in major clades 2, 3, 6, 7a, 7b, 8a, 8b and 12 was constructed by IS-printing, SNP analysis and LSPA-6 (Table 1). The IS629 distributions in the O157 strains isolated in Fukuoka and Yamagata Prefectures were determined by ISprinting and the strains were putatively classified in clades by Relative Likelihood using the database for the Chiba O157 strains. Concordance Ratios were calculated for strains in the major clades to evaluate the applicability of the IS-printing system for putative identification of the clades of O157 strains (Table 2). For O157 strains isolated in Fukuoka and Yamagata Prefectures, the Concordance Ratios for strains in clades 3, 6 and 8 were 97–100%, in clade 7 about 88%, and in clade 12 over 90%. However, the Concordance Ratio for clade 2 strains isolated in Fukuoka was 966%, and for clade 2 strains isolated in Yamagata was 813%. Three clade 2 strains isolated in Yamagata had the clade 3 IS629 distribution (Fig. 1) and, therefore, were incorrectly classified as clade 3. An O157 strain with an atypical IS629 distribution for its clade could be incorrectly classified by Relative Likeli-
Letters in Applied Microbiology 61, 267--273 © 2015 The Society for Applied Microbiology
S. Hirai et al.
Identification of EHEC O157 clades
Table 1 Fraction of strains isolated in Chiba Prefecture in each major O157 clade with IS629 at each locus IS629 insertion locus* Clade
1–01
1–02
1–03
1–04
1–05
1–06
1–07
1–08
1–09
1–10
1–11
1–12
1–13
1–14
1–15
1–16
2 3 6 7a 7b 8a 8b 12
075 089 095 029 040 098 077 080
090 075 087 022 094 085 088 015
086 007 000 001 038 000 000 002
089 076 087 068 098 006 000 074
000 000 000 001 000 000 000 010
003 002 003 002 000 083 007 006
094 091 097 032 092 100 098 074
092 092 003 060 002 002 002 003
068 095 021 010 098 092 088 089
095 094 097 003 000 000 000 000
000 000 000 000 000 000 000 008
091 087 097 014 100 098 087 008
097 097 095 089 100 100 095 051
062 078 003 000 000 000 000 000
096 095 097 089 100 094 093 097
074 073 077 083 087 081 092 079
IS629 insertion locus† Clade
2–01
2–02
2–03
2–04
2–05
2–06
2–07
2–08
2–09
2–10
2–11
2–12
2–13
2–14
2–15
2–16
2 3 6 7a 7b 8a 8b 12
000 000 092 029 098 077 025 088
090 093 090 085 100 096 100 094
089 074 041 002 000 004 083 009
092 074 090 088 096 091 097 014
003 003 000 000 000 000 000 000
000 000 000 002 000 002 000 010
070 094 018 011 100 089 092 092
000 000 000 007 000 000 000 070
000 001 000 041 000 000 002 003
008 073 000 000 000 000 000 000
089 080 000 003 098 089 092 008
094 094 095 085 100 096 075 082
074 092 092 023 098 098 002 009
000 000 000 002 000 000 000 004
094 092 000 083 100 092 090 093
079 089 095 068 000 004 000 084
*These are the 16 loci analysed by multiplex PCR using first set of primers in the IS-printing system. †These are the 16 loci analysed by multiplex PCR using second set of primers in the IS-printing system. Table 2 Number of strains isolated in Fukuoka and Yamagata Prefectures and putatively classified in major O157 clades by Relative Likelihood Clade putatively identified by Relative Likelihood Major clade (a) Fukuoka 2 3 6 7 8 12 (b) Yamagata 2 3 6 7 8 12
2
3
6
7a
7b
8a
8b
12
ND*
Total
Concordance ratio (%)
143 1 0 0 0 0
3 198 0 2 0 0
0 0 26 1 0 0
0 0 0 64 0 3
2 0 0 21 0 0
0 0 0 0 25 0
0 0 0 0 23 0
0 0 0 8 0 46
5 1 0 2 1 0
153 200 26 98 49 49
966 995 1000 885 1000 939
13 1 0 0 0 0
3 33 0 0 0 0
0 0 4 0 0 0
0 0 0 5 0 0
0 0 0 2 0 0
0 0 0 0 4 0
0 0 0 0 5 0
0 0 0 1 0 5
1 1 0 1 0 0
17 35 4 9 9 5
813 971 1000 875 1000 1000
*The Relative Likelihood cut-off was
ORIGINAL ARTICLE
Putative classification of clades of enterohemorrhagic Escherichia coli O157 using an IS-printing system S. Hirai1,2, E. Yokoyama1, Y. Etoh3, J. Seto4, S. Ichihara3, Y. Suzuki4, E. Maeda3, N. Sera3, K. Horikawa3, S. Sato5 and T. Yamamoto2 1 2 3 4 5
Division of Bacteriology, Chiba Prefectural Institute of Public Health, Chiba, Japan Department of Microbiology and Molecular Genetics, Graduate School of Pharmaceutical Science, Chiba University, Chiba, Japan Division of Pathology and Bacteriology, Fukuoka Institute of Health and Environmental Sciences, Fukuoka, Japan Department of Microbiology, Yamagata Prefectural Institute of Public Health, Yamagata, Japan Chiba Prefectural Institute of Public Health, Chiba, Japan
Significance and Impact of the Study: This study demonstrated that enterohemorrhagic E. coli O157 (O157) strains could be putatively classified in clades using an IS-printing system. IS-printing was previously developed as a relatively quick and easy tool for analysis of insertion sequence 629 in the O157 genome. Since most local government public health institutes in Japan carry out IS-printing for early detection of O157 outbreaks, these data should be useful for putative classification of O157 strains in each area.
Keywords enterohemorrhagic Escherichia coli O157, IS-printing system, population genetic analysis, putative classification of clades, Relative Likelihood. Correspondence Shinichiro Hirai, Division of Bacteriology, Chiba Prefectural Institute of Public Health, 666-2 Nitona, Chuo, Chiba City, Chiba 260-8715, Japan. E-mail: [email protected] 2015/0379: received 24 February 2015, revised 11 May 2015 and accepted 25 May 2015 doi:10.1111/lam.12448
Abstract Enterohemorrhagic Escherichia coli O157 (O157) strains can be classified in clades by single nucleotide polymorphisms (SNPs), but this analysis requires significant laboratory effort. As the distribution of insertion sequence (IS) 629 insertions has been reported to be biased among different clades, O157 isolates can be putatively classified in clades by comparison with an IS629 distribution database. A database of the IS629 distribution in O157 strains isolated in Chiba Prefecture and their classification in clades was determined by SNP analysis and IS-printing, an easy and quick analytical tool for IS629 in the O157 genome. The IS629 distribution in O157 strains isolated in Fukuoka and Yamagata Prefectures was determined by IS-printing. These strains were putatively classified in clades by Relative Likelihood calculations that compared the IS-printing data and the IS629 distribution database. Concordance Ratios were calculated, which compared the number of strains putatively classified in a clade by Relative Likelihood to the number of strains classified in that clade by SNP analysis. For the Fukuoka and Yamagata strains, the Concordance Ratios for clades 3, 6 and 8 were 97–100%, for clade 7 about 88%, and for clades 2 and 12 over 90%. In conclusion, O157 clade 2, 3, 6, 7, 8 and 12 strains could be putatively classified by IS-printing.
Introduction Recent studies showed that enterohemorrhagic Escherichia coli O157 (O157) strains can be divided into several phylogenetic groups; i.e., lineages (Kim et al. 2001), subgroups and clusters (Shaikh and Tarr 2003), and clades (Manning et al. 2008). Yokoyama et al. (2012) showed that the hierarchical relationship of O157 phylogenetic groups was, in descending order, lineage, subgroup,
cluster, and clade, and proposed a paraphyletic model for O157 evolution. As shown in Table S1, this model has been further modified with new clade designations based on lineage analysis data (Hirai et al. 2013). Different distributions of strains in O157 phylogenetic groups have been reported in some countries. Clade 2, 3, 7 and 12 strains were prevalent in some prefectures in Japan (Hirai et al. 2014), clade 2 and 8 strains in Michigan, U.S. (Manning et al. 2008), and clade 6 and 7 strains
Letters in Applied Microbiology 61, 267--273 © 2015 The Society for Applied Microbiology
267
S. Hirai et al.
Identification of EHEC O157 clades
in Australia (Mellor et al. 2012). Phylogenetic differences were also reported in the strains in some regions within a country. Different lineage distributions of O157 strains were found between the North and South Islands of New Zealand (Franz et al. 2012; Jaros et al. 2013). Since each lineage includes specific clades, the clade distribution of O157 strains may be different within New Zealand. Differences have been reported in the pathogenicity of O157 strains in different clades. For example, more clade 8 strains were isolated from patients with hemolytic uremic syndrome (HUS) than strains in other clades, suggesting strong HUS pathogenicity of clade 8 strains (Manning et al. 2008). In contrast, fewer clade 12 strains were isolated from hemorrhagic colitis patients than strains in other clades, indicating that clade 12 strains may be weakly pathogenic for this disease (Etoh et al. 2014). HUS in O157-infected patients in Argentina, where clade 8 is prevalent (Mellor et al. 2012), has been reported to be ten times higher than in other developed counties (Rivas et al. 2006; Leotta et al. 2008). Therefore, information on the clade distribution of O157 strains in an area should be useful for analysis of the public health risk of O157 infections in that area (Hirai et al. 2014). Classification of O157 strains in clades has been based on two types of analysis: detection of single nucleotide polymorphisms (SNPs) at 32 loci in the O157 genome (Manning et al. 2008) and lineage specific polymorphism assay-6 (LSPA-6) (Kim et al. 2001). Yokoyama et al. (2012) used a low cost ARMS-PCR method to study the distribution of O157 strains in clades, but this method was not suitable for a large scale study because it required agarose gel electrophoresis analysis of the PCR products. We developed a method for classification of O157 strains in clades using capillary gel sequence (CES) analysis and carried out a large scale study (Hirai et al. 2014). However, since CES analysis requires very expensive equipment, research institutes without CES capability cannot carry out this analysis. We previously reported linkage disequilibrium of insertion sequence (IS) 629 among O157 strains in different clades (Hirai et al. 2013). An IS-printing system was developed as a molecular epidemiological tool to investigate the distribution of IS629 in the O157 genome (Ooka et al. 2009). IS-printing can be carried out easily and quickly using two sets of multiplex PCRs, each for 16 loci, followed by analysis of the PCR amplicons by agarose gel electrophoresis. Therefore, IS-printing data for O157 strains should enable putative strain classification in clades by comparison with a database of the biased IS629 distribution in strains in O157 clades. For data analysis, the Relative Likelihood, a numerical measure of the probability that an organism is a member of a biological group, was used. The Relative Likelihood 268
can be calculated by comparing some characters of an organism with those of members of a biological group (Bascomb et al. 1973; Willcox et al. 1973), and has been used for the classification of unknown bacteria in some commercial kits; e.g., the API series (Sysmex Biomerieux, Lyon, France) and the BD BBLCrystal series (Becton, Dickinson and Company, Franklin Lakes, NJ). Therefore, O157 strains can be putatively classified in clades by Relative Likelihood using a database of the IS629 distribution in the strains in O157 clades. In this study, the IS629 distribution in O157 strains isolated in Chiba Prefecture, Japan, was determined by IS-printing, and these strains were classified in clades by SNP analysis and LSPA-6 to construct a database of the IS629 distribution in strains in each clade. The IS629 distribution in O157 strains isolated in Fukuoka and Yamagata Prefectures was determined by IS-printing, and these strains were putatively classified in clades by Relative Likelihood calculations that compared the IS-printing data and the IS629 distribution database. The Concordance Ratio, the ratio of the number of strains putatively classified in a clade by Relative Likelihood from IS-printing analysis relative to the number classified by SNP analysis and LSPA-6, was calculated for the O157 strains isolated in Fukuoka and Yamagata Prefectures. The Relative Likelihood was shown to be useful for the putative classification of O157 strains in clades. Results and discussion A database of the IS629 distribution at loci in O157 strains isolated in Chiba Prefecture and classified in major clades 2, 3, 6, 7a, 7b, 8a, 8b and 12 was constructed by IS-printing, SNP analysis and LSPA-6 (Table 1). The IS629 distributions in the O157 strains isolated in Fukuoka and Yamagata Prefectures were determined by ISprinting and the strains were putatively classified in clades by Relative Likelihood using the database for the Chiba O157 strains. Concordance Ratios were calculated for strains in the major clades to evaluate the applicability of the IS-printing system for putative identification of the clades of O157 strains (Table 2). For O157 strains isolated in Fukuoka and Yamagata Prefectures, the Concordance Ratios for strains in clades 3, 6 and 8 were 97–100%, in clade 7 about 88%, and in clade 12 over 90%. However, the Concordance Ratio for clade 2 strains isolated in Fukuoka was 966%, and for clade 2 strains isolated in Yamagata was 813%. Three clade 2 strains isolated in Yamagata had the clade 3 IS629 distribution (Fig. 1) and, therefore, were incorrectly classified as clade 3. An O157 strain with an atypical IS629 distribution for its clade could be incorrectly classified by Relative Likeli-
Letters in Applied Microbiology 61, 267--273 © 2015 The Society for Applied Microbiology
S. Hirai et al.
Identification of EHEC O157 clades
Table 1 Fraction of strains isolated in Chiba Prefecture in each major O157 clade with IS629 at each locus IS629 insertion locus* Clade
1–01
1–02
1–03
1–04
1–05
1–06
1–07
1–08
1–09
1–10
1–11
1–12
1–13
1–14
1–15
1–16
2 3 6 7a 7b 8a 8b 12
075 089 095 029 040 098 077 080
090 075 087 022 094 085 088 015
086 007 000 001 038 000 000 002
089 076 087 068 098 006 000 074
000 000 000 001 000 000 000 010
003 002 003 002 000 083 007 006
094 091 097 032 092 100 098 074
092 092 003 060 002 002 002 003
068 095 021 010 098 092 088 089
095 094 097 003 000 000 000 000
000 000 000 000 000 000 000 008
091 087 097 014 100 098 087 008
097 097 095 089 100 100 095 051
062 078 003 000 000 000 000 000
096 095 097 089 100 094 093 097
074 073 077 083 087 081 092 079
IS629 insertion locus† Clade
2–01
2–02
2–03
2–04
2–05
2–06
2–07
2–08
2–09
2–10
2–11
2–12
2–13
2–14
2–15
2–16
2 3 6 7a 7b 8a 8b 12
000 000 092 029 098 077 025 088
090 093 090 085 100 096 100 094
089 074 041 002 000 004 083 009
092 074 090 088 096 091 097 014
003 003 000 000 000 000 000 000
000 000 000 002 000 002 000 010
070 094 018 011 100 089 092 092
000 000 000 007 000 000 000 070
000 001 000 041 000 000 002 003
008 073 000 000 000 000 000 000
089 080 000 003 098 089 092 008
094 094 095 085 100 096 075 082
074 092 092 023 098 098 002 009
000 000 000 002 000 000 000 004
094 092 000 083 100 092 090 093
079 089 095 068 000 004 000 084
*These are the 16 loci analysed by multiplex PCR using first set of primers in the IS-printing system. †These are the 16 loci analysed by multiplex PCR using second set of primers in the IS-printing system. Table 2 Number of strains isolated in Fukuoka and Yamagata Prefectures and putatively classified in major O157 clades by Relative Likelihood Clade putatively identified by Relative Likelihood Major clade (a) Fukuoka 2 3 6 7 8 12 (b) Yamagata 2 3 6 7 8 12
2
3
6
7a
7b
8a
8b
12
ND*
Total
Concordance ratio (%)
143 1 0 0 0 0
3 198 0 2 0 0
0 0 26 1 0 0
0 0 0 64 0 3
2 0 0 21 0 0
0 0 0 0 25 0
0 0 0 0 23 0
0 0 0 8 0 46
5 1 0 2 1 0
153 200 26 98 49 49
966 995 1000 885 1000 939
13 1 0 0 0 0
3 33 0 0 0 0
0 0 4 0 0 0
0 0 0 5 0 0
0 0 0 2 0 0
0 0 0 0 4 0
0 0 0 0 5 0
0 0 0 1 0 5
1 1 0 1 0 0
17 35 4 9 9 5
813 971 1000 875 1000 1000
*The Relative Likelihood cut-off was
