key: cord-0005525-xilwey5s
authors: Ji, Xiao W.; Liao, Ya L.; Zhu, Ye F.; Wang, Hai G.; Gu, Ling; Gu, Jiang; Dong, Chen; Ding, Hong L.; Mao, Xu H.; Zhu, Feng C.; Zou, Quan M.
title: Multilocus sequence typing and virulence factors analysis of Escherichia coli O157 strains in China
date: 2011-01-09
journal: J Microbiol
DOI: 10.1007/s12275-010-0132-8
sha: d93f6e90957636cfe8e46fce68c0b66e7e847c61
doc_id: 5525
cord_uid: xilwey5s

Escherichia coli O157:H7, an important food-borne pathogen, has become a major public health concern worldwide. The aim of this study was to investigate the molecular epidemiologic feature of E. coli O157:H7 strains in China. 105 E. coli O157:H7 isolates were collected from various hosts and places over 9 years. A multilocus sequence typing scheme (MLST) was applied for bacteria genotyping and polymerase chain reaction (PCR) was used for virulence factor identification. Seven new MLST sequence types (STs), namely ST836, ST837, ST838, ST839, ST840, ST841, and ST842 were identified, which grouped into two lineages. Phylogenetic analysis suggested that the most two frequent STs in China, ST837 and ST836, may be the derivatives of E. coli O157:H7 Sakai or E. coli O157:H7 EDL933. Geographical diversity and host variety of E. coli O157:H7 were observed in China. In addition, the different distribution of tccp was detected. The data presented herein provide new insights into the molecular epidemiologic feature of E. coli O157:H7, and aid in the investigation of the transmission regularity and evolutionary mechanism of E. coli O157:H7. ELECTRONIC SUPPLEMENTARY MATERIAL: Supplementary material is available for this article at 10.1007/s12275-010-0132-8 and is accessible for authorized users.

Two separate outbreaks of Escherichia coli (E. coli) O157:H7 in Oregon and Michigan, USA, in 1982 were reportedly caused by the consumption of contaminated ground beef (Riley et al., 1983) , and followed by similar large outbreaks in Japan (Michino et al., 1999) and Scotland (Cowden et al., 2001) . In 2006 an outbreak in the USA was associated with polluted spinach (Wendel et al., 2009 ) and a further outbreak in 2009 was attributed to the consumption of raw, refrigerated, pre-packaged cookie dough (http://www.cdc.gov/ecoli/2009/ 0622.html). During 1999 there was a large-scale epidemic in the east of China due to E. coil O157:H7. It is associated with several human conditions including diarrhea, hemorrhagic colitis (HC), and hemolytic uremic syndrome (HUS) (Browning et al., 1990; Armstrong et al., 1996; Dunn et al., 2004) . Results from several studies suggest that E. coil O157:H7 acts in different ways to produce these various human diseases/conditions, and it differs genotypically and in the prevalence of virulence genes (Baker et al., 1997; Kim et al., 1999; Kim et al., 2001; Roe et al., 2003 Roe et al., , 2004 .

The ability of E. coli O157:H7 strains to cause severe diseases in human is primarily related to their capacities to secrete shiga toxin (Stx) and other toxins and to induce attaching and effacing lesion (A/E lesion) (Pradel et al., 2001; Lim et al., 2010) . Stx mainly form two subgroups, Stx1 and Stx2, both of which are toxic to humans and animals for their ability to inhibit protein synthesis (O'Brien and Holmes, 1987; Sandvig et al., 2004) . Enterohemolysin, encoded by hlyA on the large plasmid-borne (about 90 kb) gene, is associated with several severe clinical diseases in humans (Schmidt et al., 1995) . The induction of A/E lesion is an outcome of interplay among effector both from bacteria and human, and TCCP (Tir-cytoskeleton coupling protein) is one of the most important effectors in the crosstalk for its Nck-like coupling activity (Garmendia et al., 2004) . Typically, TCCP consists of an 87 amino acids N-terminus, several almost identical 47 amino acids proline-rich repeats (PRRs) in the middle, and a truncated repeat containing 15 amino acids at the C-terminus (Garmendia et al., 2004) .

The majority of human E. coli O157:H7 infections was caused by the consumption of contaminated food or water (Riley et al., 1983; Armstrong et al., 1996; Muhldorfer et al., 1996; Muller et al., 2001; Dunn et al., 2004) . In China, E. coli O157:H7 has been detected in many different cities, however there is little information available about the prevalence and epidemiological characteristics of this pathogen (Ma et al., 2009) . Multilocus sequence typing (MLST) has been proposed as a nucleotide sequence-based approach that can be applied to many bacterial pathogens (Urwin and Maiden, 2003) . In this study a MLST system was developed using a collection of archived E. coli O157:H7 isolates in China and their virulence genes were identified at the same time.

Bacterial strains used in the study E. coil O157:H7 isolates were obtained from 11 spots located in Jangsu province and Chongqing in China from 1999 to 2007. They were identified by a sorbitol fermentation test and MUG (4methylumbelliferyl-beta-D-glucuronide) assay. Then latex agglutination was used to detect somatic and flagella antigens. Bacteria were stored at -80 o C until required. E. coli O157:H7 Sakai (GenBank: BA000007) and EDL933 (GenBank: AE005174) were used as reference strains in the study.

Bacterial DNA extraction was carried out in accordance with the DNA contamination management guidelines of Millar et al. (2002) . The purified genomic DNA were stored in TE buffer at 4 o C until required. MLST analysis was performed using sequences obtained from seven housekeeping genes, namely aspc, icdA, uidA, clpX, mdh, fadD, and lysp. The primers for DNA amplications are designed according to the recommendations of the E. coli MLST web site (http://mlst.ucc.ie/mlst/dbs/Ecoli) and are shown in Table 1 . The DNA sequences were determined with ABI PRISM 310 DNA sequencer and the program BURST was used for MLST data analysis (Rozas et al., 2003) .

The presence of stx 1 , stx 2 , hlyA, and tccp genes were verified by PCR using the primers shown in Table 1 (De Baets et al., 2004) . About 5 Pl of PCR product was subjected to electrophoresis on a 1.2% agarose gel, stained with ethidium bromide, and examined under a UV illuminator. The number of PRR in tccp was calculated according to the approximate length indicated on the agarose gel.

During a period of about 7 years in 11 monitoring spots, using the biochemical and immunologic assay, 105 E. coli O157:H7 strains were isolated from animals (sheep n=40, cattle n=31, fowl n=12, and pig n=9) and from HUS patients (n=13), as shown in Supplementary data Table 1 . And these isolates were subjected to MLST analysis. Seven loci selected in this study was subjected to stabilizing selection pressure, as the dN/dS ratio (ratio of non-synonymous substitutions to synonymous substitutions) was lower than 1.0. MLST results resolved the 105 E. coli strains into seven new STs, namely ST837, ST841, ST840 ST836, ST838, ST839, and ST842. These STs were new combinations of known alleles except ST841 and ST842, the clxp allele (gene accession number: HM640979) of which was not reported before (Supplementary data Table 1 ). The most frequent STs was ST837 and ST836, represented by 54.3% and 38.1% of total collected isolates, which was followed by ST841 represented by 4 isolates, ST838, ST839, ST840, and ST842 were represented by single isolates (Fig. 1A ).

The relationships between the STs was examined using eBURST, which uses allele profiles to reconstruct the relation- ships between the different clonal lineages. Results showed that the seven STs were grouped into two linkages (Fig. 2) : ST837, ST841, and ST840 belong to one group, which consist of 62 (59.0%) isolates and was represented by ST837, while ST836, ST838, ST839, and ST842 belong to the other group, and it was represented by ST836 (Fig. 1B) .

In order to evaluate the relationship between STs in China and that in EcMLST database, the relationships among members of the different STs are demonstrated in the Fig. 2A . The minimum spanning (MS) tree was generated from the allelic profiles by using EHEC O157:H7 strain EDL933 and Sakai (ST69 and ST66 respectively, http://mlst.ucc.ie/mlst/dbs/ Ecoli) as outgroup. MS tree showed that E. coli O157:H7 STs mainly classied into three clonal complexes, which is represented by ST66 (Sakai strains), ST836 and ST139 respectively. STs isolated in china classied into the same clonal complex, suggesting its relative conserved genetic background. Meanwhile splits tree decomposition demonstrated a similar network among EHEC STs, as indicated by MS tree (Fig. 2B ).

A diverse geographic distribution of STs among different monitoring spots was observed. As shown in Table 2 , The majority of E. coli O157 was isolated in the spot E (30 isolates, 28.6%) and spot F(27 isolates, 25.7%). ST836 and ST837 were the most universal tpye in all monistoring spots. The 4 ST841 stains were only obtained from the spot E. The rare STs, ST 840, ST838, ST839, and ST842, which consist of only one isolated, was observed at the spot B, D, E, and K, respectively.

Host diversity of E. coli O157 was also observed. Sheep and cattle were the main natural reservoir of E. coli O157, isolates from which consist of 38.1% (40 isolates) and 29.5% (31 isolates) of total collections respectively (Table 3) . STs from human containing ST836, ST837, ST841, and ST842. An interesting finding was that the majority of isolates from sheep, cattle fowls and pigs were ST837 and ST836. However, in human the frequency of ST841 was as high as close to that of ST836 and ST837. In addition, the prevalence of allele clpX gene in human isolates was significantly different that in animal isolates, while analyzing the seven housekeeping loci (data not shown).

E. coli O157 isolates was subjected to PCR assays and four genes, namely stx 1 , stx 2 , hylA, and tccp were investigated. Results showed that all E. coli O157 isolates tested carried stx 2 and hylA. And only one isolate proved to carry both stx 1 and stx 2 , which was isolated from human and belonged to ST841 Table 4 ). Interesting findings were obtained in tccp gene distribution. Three different tccp were found in our isolates, namely tccp3, tccp4 and tccp5, which contains 3, 4, and 5 PRRs respectively. The tccp5 was the most frequent gene type, which consist of 81.9% (86 isolates) of total collections. And it was followed by tccp3 and tccp4, This distribution pattern was in consistant with that in ST836 and ST837. The tccp5 appeared in most E. coli O157 isolates from human, but in only approximately 70% (73 isolates) of animal isolates.

The MLST method provides a scalable typing system that reflects the population and evolutionary biology of the bacterium, and makes valid comparisons between results from different laboratories possible. It applies neutral or slowly accumulating genetic variations in housekeeping genes, which are not affected by the rapid evolution detected within genes encoding proteins that influence survival in a particular niche (Selander et al., 1986; Urwin and Maiden, 2003; Cooper and Feil, 2004) . Thus in this study the MLST approach was applied to investigate the clone relationships of E. coli O157:H7 isolates from China.

A total of seven new STs were identified, which were not present in the database of EcMLST systems (http://www.shigatox. net/). The BURST analysis indicates a relative conserved genetic background of E. coli O157:H7 isolates from China, and it revealed that the ST836 may be the ancestor of the 105 strains studied. ST836, ST837 and ST841 appeared in 1999, when an outbreak of E. coli O157 occurred in the east of China. The other STs identified in this study may be the offspring of those originating from the 1999 outbreak. While comparing isolates in china with that from EcMLST database and reference strains Sakai and EDL933, we found a close relationship betweent ST69 (EDL933) and ST66 (Sakai). Thus we proposed that the E. coli Sakai strain (ST66), which is responsible for the outbreak in Japan (ST69), is a descendant of EDL933 with DNA insertion/deletion and recombination. In addition, ST836, which represents the STs in China, is also closely associated with ST66. For ST836 were isolated from Chinese patient in the large outbreak in 1997, we speculate that it may be transferred from Japan after genetic mutations.

In this study, we found an extensive genomic diversity within populations of E. coli O157, even among different monitoring spots. This variety is inconsistent with that in previous reports (Miao and Miller, 1999; Ohnishi et al., 2001; Allison, 2007) . It may because bacteriophage-mediated lateral gene transfer induces numbers of DNA insertion/deletion and recombination events. Meanwhile genetic diversity was also observed between isolates from human and animals. As animals, especially cattle are the natural host for E. coli O157:H7, the bacteria may suffer less evolutionary pressure while colonizing in animals than in human (Blanco et al., 2004a (Blanco et al., , 2004b Zweifel et al., 2004) . This may explain the reason for the genetic variation of E. coli 157:H7 isolated from human.

The production of Stx and induction of A/E lesion was considered as the predominant two ways of EHEC to cause infectious diseases, especially HUS. As observed in our study, All 105 isolates investigated carry the stx (stx 1 or stx 2 ) gene, however just a part of them is pathogenic to human. This finding suggests an indispensable contribution of A/E lesion to E. coli associated diseases. The different distribution of tccp gene in E. coli O157:H7 isolates from human and animals provideadditional evidence. TCCP is an important bacterial effector and stimulates actin polymerization during the formation of A/E lesion (Campellone et al., 2004; Garmendia et al., 2004) . Previous in vitro and in vivo studies have demonstrated that A/E lesion form more efficiently with the increase of PRRs number (Cheng et al., 2008) . The percentage of bacteria carrying tccp5 (tccp gene with 5 PRRs), tccp4, and tccp3 in human isolates is significant higher than in animal isolates, respectively. That may be the reason for Stxproducing E. coli O157:H7 was unable to cause diseases in human.

To summarize, our findings provided novel insights into the The number of isolates was shown in the Table. The tccp3, tccp4, and tccp5 stands for tccp gene contains 3, 4, and 5 prolin rich repeats respectively. molecular epidemiologic feature of E. coli O157:H7 in China. By MLST method, seven new STs which grouped into two clonal complex were identified and the connection between E. coli O157:H7 isolates in China and that in other places of the world was also established. In further studies, we plan to collect and analyze a larger number of isolates to systematically investigate the molecular epidemiology and transmission regularity of E. coli O157:H7.

Stx-phages: Drivers and mediators of the evolution of stec and stec-like pathogens

Emerging foodborne pathogens: Escherichia coli O157:H7 as a model of entry of a new pathogen into the food supply of the developed world

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Escherichia coli O157:H7 haemorrhagic colitis. Report of the first south african case

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Epidemiological investigation of the central scotland outbreak of Escherichia coli O157 infection

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Ancestral divergence, genome diversification, and phylogeographic variation in subpopulations of sorbitolnegative, beta-glucuronidase-negative enterohemorrhagic Escherichia coli O157

A brief overview of Escherichia coli O157:H7 and its plasmid O157

Avian influenza virus, streptococcus suis serotype 2, severe acute respiratory syndromecoronavirus and beyond: Molecular epidemiology, ecology and the situation in china

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Risk assessment models and contamination management: Implications for broad-range ribosomal DNA pcr as a diagnostic tool in medical bacteriology

Regulation of the shiga-like toxin ii operon in Escherichia coli

The occurrence of E. coli O157:H7 in south african water sources intended for direct and indirect human consumption

Shiga and shiga-like toxins

Diversification of Escherichia coli genomes: Are bacteriophages the major contributors?

Heterogeneity of shiga toxin-producing Escherichia coli strains isolated from hemolytic-uremic syndrome patients, cattle, and food samples in central france

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Co-ordinate single-cell expression of lee4-and lee5-encoded proteins of Escherichia coli O157:H7

Heterogeneous surface expression of espa translocon filaments by Escherichia coli O157:H7 is controlled at the posttranscriptional level

DNA polymorphism analyses by the coalescent and other methods

Pathways followed by protein toxins into cells

Molecular analysis of the plasmid-encoded hemolysin of Escherichia coli O157:H7 strain edl 933

Methods of multilocus enzyme electrophoresis for bacterial population genetics and systematics

Multi-locus sequence typing: A tool for global epidemiology

Multistate outbreak of Escherichia coli O157:H7 infection associated with consumption of packaged spinach

Serotypes and virulence genes of ovine non-O157 shiga toxinproducing Escherichia coli in switzerland

We are grateful to Dr. Chen Chen from the Chinese Center for Diseases Control and Prevention and Prof. Ruifu Yang from Beijing Institute of Microbiology and Epidemiology for their help.This work was supported by the National High-tech Research and Development Program (863 project) of China (No. 2007AA02Z409; 2006AA02AZ443), and the National Natural Science Foundation of China (No.30670107).