[Multilocus sequence typing analysis of enteropathogenic escherichia coli isolates in 8 provinces of China, 2006-2014].

OBJECTIVE: To investigate the molecular typing feature of enteropathogenic Escherichia coli (EPEC) strains isolated from different reservoirs in eight provinces of China from 2006 to 2014. METHODS: According to the time, place, reservoir, and PFGE pattern of the EPEC strains isolated from stools of humans with diarrhea, animal feces, and foods in eight provinces of China between 2006 and 2014, 149 EPEC strains were selected and characterized by multilocus sequence typing (MLST) using seven housekeeping genes provided by E.coli MLST database. Strain analysis demonstrated 56 different sequence types (STs). SeqMan II, MEGA 5.05, and eBURST V3 were applied to analyze the genetic relationships of domestic and forein existing 392 strains (243 EPEC strains included in the E.coli MLST database and 149 EPEC strains comprised in the present study). RESULTS: Among the 56 different STs, the prevalent ST was ST-40, which included 19 (19/149, 12.8%) isolates. Nineteen new STs were identified. Eleven new alleles were detected in six house-keeping genes (adk, fumC, gyrB, icd, mdh, and purA). Six STs were simultaneously detected among EPEC strains isolated from patients with diarrhea and animals. And these EPEC strains were all aEPEC strains. Two STs were simultaneously identified among EPEC strains isolated from patients with diarrhea and foods. Also, these EPEC strains were all aEPEC strains. 33 out of 173 STs were divided into five major clone complexes by eBURST, STC-29, STC-10, STC-20, STC-28, and STC-517. The remaining EPEC strains included in the other 140 STs were part of the other clone complexes or just were singletons. CONCLUSION: A high degree of phylogenetic heterogeneity was observed among the EPEC strains isolated in eight provinces of China. The EPEC strains with same STs of human isolates isolated from animal feces and foods were all aEPEC strains.

Characterization of Shiga toxin-producing Escherichia coli isolated from healthy pigs in China.

BACKGROUND: Shiga toxin-producing Escherichia coli (STEC) is recognized as an important human diarrheal pathogen. Swine plays an important role as a carrier of this pathogen. In this study we determined the prevalence and characteristics of STEC from healthy swine collected between May 2011 and August 2012 from 3 cities/provinces in China. RESULTS: A total of 1003 samples, including 326 fecal, 351 small intestinal contents and 326 colon contents samples, was analyzed. Two hundred and fifty five samples were stx-positive by PCR and 93 STEC isolates were recovered from 62 stx-positive samples. Twelve O serogroups and 19 O:H serotypes including 6 serotypes (O100:H20/[H20], O143:H38/[H38], O87:H10, O172:H30/[H30], O159:H16, O9:H30/[H30]) rarely found in swine and ruminants were identified. All 93 STEC isolates harbored stx2 only, all of which were stx2e subtype including 1 isolate being a new variant of stx2e. 53.76%, 15.05% and 2.15% STEC isolates carried astA, hlyA and ehxA respectively. Four STEC isolates harbored the high-pathogenicity island. Of the 15 adherence-associated genes tested, 13 (eae, efa1, iha, lpfAO113, lpfAO157/OI-154, lpfAO157/OI-141, toxB, saa, F4, F5, F6, F17 or F41) were all absent while 2 (paa and F18) were present in 7 and 4 STEC isolates respectively. The majority of the isolates were resistant to tetracycline (79.57%), nalidixic acid (78.49%), trimethoprim-sulfamethoxazole (73.12%) and kanamycin (55.91%). The STEC isolates were divided into 63 pulsed-field gel electrophoresis patterns and 21 sequence types (STs). Isolates of the same STs generally showed the same or similar drug resistance patterns. A higher proportion of STEC isolates from Chongqing showed multidrug resistance with one ST (ST3628) resistant to 14 antimicrobials. CONCLUSIONS: Our results indicate that swine is a significant reservoir of STEC strains in China. Based on comparison by serotypes and sequence types with human strains and presence of virulence genes, the swine STEC may have a low potential to cause human disease.

Effect of Stenotrophomonas maltophilia on Tuberculosis.

Tuberculosis (TB) is an important infectious disease suffered by many countries, including China. In this stage, accurate diagnosis and treatment are key measures for the prevention and control of TB. Stenotrophomonas maltophilia is a global emerging Gram-negative, multidrug-resistant (MDR) organism characterized by its high contribution to the increase in crude mortality rates. By single cell preparation and strain identification, we isolated S. maltophilia from stored cultures of Mycobacterium tuberculosis (Mtb). We found that S. maltophilia could not be removed from sputum by alkali treatment or inhibited by antibiotic mixture added to MGIT 960 indicator tubes. When co-cultured with Mtb on a Löwenstein-Jensen (L-J) slant, it could inhibit the growth of Mtb and liquefy the medium. More seriously, it was resistant to 10 of the 12 anti-TB drugs, including isoniazid and rifampin, and made the mixed samples display multidrug-resistant Mtb (MDR-TB) results in the drug sensitivity test, which might change a treatment regimen and increase disease burden. Following, we conducted a small-scale surveillance which showed that the isolation rate of S. maltophilia in TB patients was 6.74%, but these patients had no special characteristics and the presence of S. maltophilia was hidden. The effect of S. maltophilus on TB and its mechanism are unclear and require more attention. IMPORTANCE China is a high-burden country for tuberculosis (TB), multidrug-resistant/rifampicin-resistant tuberculosis (MDR/RR-TB), and HIV-associated TB. Increasing the positive rate of culture and the accuracy of antibiotic susceptibility testing (AST) are important for diagnosis, treatment, and control of TB. In our study, we found that the isolation rate of Stenotrophomonas maltophilia in TB patients was not neglectable and that this bacterium affects the isolation and AST results of TB. Due to a lack of relevant research, the impact of S. maltophilia on the course and outcome of TB is unclear. However, the characteristics of S. maltophilia that increase disease mortality require attention. Therefore, in the clinical testing of TB, in addition to mycobacteria, it is recommended to increase the detection of co-infected bacteria and improve the awareness of TB clinicians of these bacteria.

Genetic diversity and molecular typing of Listeria monocytogenes in China.

BACKGROUND: Listeria monocytogenes can cause invasive diseases in humans and farm animals and is frequently isolated from dairy products and poultry. Listeriosis is uncommon in China but L. monocytogenes has been isolated from foods and food processing environments in China. However little is known of genetic diversity of Chinese L. monocytogenes isolates and their relationships with global isolates. RESULTS: Two hundred and twelve isolates of L. monocytogenes from food sources from 12 provinces/cities in China were analysed by serotyping, Pulsed Field Gel Electrophoresis (PFGE) and Multi-locus Sequence Typing (MLST). The predominant serotypes are 1/2a, 1/2b and 1/2c accounting for 90.1% of the isolates. PFGE divided the isolates into 61 pulse types (PTs). Twenty nine PTs were represented by more than one isolates with PT GX6A16.0004 containing the most number of isolates. MLST differentiated the isolates into 36 STs, among which 15 were novel. The 3 most common STs were ST9 (29.1%), ST8 (10.7%) and ST87 (9.2%), accounting for 49.0% of the isolates. CONCLUSIONS: STs prevalent in other parts of the world are also prevalent in China including 7 STs (ST1-ST3, ST5, ST6, ST8, ST9) which caused maternal fetal infections or outbreaks, suggesting that these STs potentially can also cause severe human infections or outbreaks in China. Surveillance of these STs will provide important information for prevention of listeriosis. This study also enhances our understanding of genetic diversity of L. monocytogenes in China.

Development of a multiplex PCR assay targeting O-antigen modification genes for molecular serotyping of Shigella flexneri.

Shigella flexneri is the major Shigella species that causes diarrheal disease in developing countries. It is further subdivided into 15 serotypes based on O-antigen structure. Serotyping of S. flexneri is important for epidemiological purposes. In this study, we developed a multiplex PCR assay targeting the O-antigen synthesis gene wzx and the O-antigen modification genes gtrI, gtrIC, gtrII, oac, gtrIV, gtrV, and gtrX for molecular serotyping of S. flexneri. The multiplex PCR assay contained eight sets of specific PCRs in a single tube and can identify 14 of the 15 serotypes (the exception being serotype Xv) of S. flexneri recognized thus far. A nearly perfect concordance (97.8%) between multiplex PCR assay and slide agglutination was observed when 358 S. flexneri strains of various serotypes were analyzed, except that 8 strains were carrying additional cryptic and/or defective serotype-specific genes. The multiplex PCR assay provides a rapid and specific method for the serotype identification of S. flexneri.

pO157_Sal, a novel conjugative plasmid detected in outbreak isolates of Escherichia coli O157:H7.

In addition to the large virulence plasmid pO157, a novel 38-kb conjugative plasmid, pO157_Sal, was identified and sequenced from an Escherichia coli O157:H7 outbreak-associated Chinese isolate that shares high similarity with a plasmid in Salmonella enterica serovar Agona. The plasmid was found in 15 of 326 isolates, 12 of which were of the same pulsed-field gel electrophoresis type.

Emergence of a new multidrug-resistant serotype X variant in an epidemic clone of Shigella flexneri.

Shigella spp. are the causative agent of shigellosis with Shigella flexneri serotype 2a being the most prevalent in developing countries. Epidemiological surveillance in China found that a new serotype of S. flexneri appeared in 2001 and replaced serotype 2a in 2003 as the most prevalent serotype in Henan Province. The new serotype also became the dominant serotype in 7 of the 10 other provinces under surveillance in China by 2007. The serotype was identified as a variant of serotype X. It differs from serotype X by agglutination to the monovalent anti-IV type antiserum and the group antigen-specific monoclonal antibody MASF IV-I. Genome sequencing of a serotype X variant isolate, 2002017, showed that it acquired a Shigella serotype conversion island, also as an SfX bacteriophage, containing gtr genes for type X-specific glucosylation. Multilocus sequence typing of 15 genes from 37 serotype X variant isolates and 69 isolates of eight other serotypes, 1a, 2a, 2b, 3a, 4a, 5b, X, and Y, found that all belong to a new sequence type (ST), ST91. Pulsed-field gel electrophoresis revealed 154 pulse types with 655 S. flexneri isolates analyzed and identified 57 serotype switching events. The data suggest that S. flexneri epidemics in China have been caused by a single epidemic clone, ST91, with frequent serotype switching to evade infection-induced immunity to serotypes to which the population was exposed previously. The clone has also acquired resistance to multiple antibiotics. These findings underscore the challenges to the current vaccine development and control strategies for shigellosis.

Spread of Streptococcus suis sequence type 7, China.

Streptococcus suis sequence type (ST) 7 has been spreading throughout China. To determine events associated with its emergence, we tested 114 isolates. In all 106 ST7 strains responsible for human outbreaks and sporadic infections, the tetracycline-resistance gene, tetM, was detected on the conjugative transposon Tn916. Horizontal transmission of tetM is suspected.

Genome sequence of Shigella flexneri 2a: insights into pathogenicity through comparison with genomes of Escherichia coli K12 and O157.

We have sequenced the genome of Shigella flexneri serotype 2a, the most prevalent species and serotype that causes bacillary dysentery or shigellosis in man. The whole genome is composed of a 4 607 203 bp chromosome and a 221 618 bp virulence plasmid, designated pCP301. While the plasmid shows minor divergence from that sequenced in serotype 5a, striking characteristics of the chromosome have been revealed. The S.flexneri chromosome has, astonishingly, 314 IS elements, more than 7-fold over those possessed by its close relatives, the non-pathogenic K12 strain and enterohemorrhagic O157:H7 strain of Escherichia coli. There are 13 translocations and inversions compared with the E.coli sequences, all involve a segment larger than 5 kb, and most are associated with deletions or acquired DNA sequences, of which several are likely to be bacteriophage-transmitted pathogenicity islands. Furthermore, S.flexneri, resembling another human-restricted enteric pathogen, Salmonella typhi, also has hundreds of pseudogenes compared with the E.coli strains. All of these could be subjected to investigations towards novel preventative and treatment strategies against shigellosis.

Genetic Diversity of Intimin Gene of Atypical Enteropathogenic Escherichia coli Isolated from Human, Animals and Raw Meats in China.

Atypical enteropathogenic Escherichia coli (aEPEC) is considered to be an emerging enteropathogen that is more prevalent than typical EPEC in developing and developed countries. The major adherence factor, intimin, an outer membrane protein encoded by eae, plays a pivotal role in the pathogenesis of aEPEC. This study investigated the distribution and polymorphisms of intimin subtypes of 143 aEPEC strains from diarrheal patients, healthy carriers, animals, and raw meats in China. These aEPEC strains belonged to more than 71 different serotypes, which comprised 52 O serogroups and 24 H types. Sixty-eight different eae genotypes and 19 intimin subtypes were detected. Eighteen, eight, seven, and five intimin subtypes were identified from 86 diarrheal patients, 14 healthy carriers, 19 animals, and 24 raw meats strains, respectively. Intimin ß1 was the most prevalent subtype in strains from diarrheal patients (34.88%) and animals (47.37%). There was a statistically significant difference in the distribution of eae-ß1 between diarrheal patients and healthy carriers (P = 0.004). Intimin-θ was more predominant among raw meat strains (50%) than among diarrheal patients strains (12.79%, P = 0.0003), healthy carrier strains (7.14%, P = 0.007), or animal strains (15.79%, P = 0.020). The two predominant subtypes (eae-ß1 and eae-θ) had considerable polymorphisms with no significant differences among the four sources. PFGE analysis revealed 119 distinct patterns and the strains were clustered into 11 groups with similarity indices ranging from 63% to 100%. These results suggest that in China, aEPEC strains from different sources are highly heterogeneous. Animals and raw meats are important sources of genetically diverse intimin-harboring aEPEC, which might serve as important transmission vehicles of these bacteria.

Molecular and Phylogenetic Characterization of Non-O157 Shiga Toxin-Producing Escherichia coli Strains in China.

Shiga toxin-producing Escherichia coli (STEC) causes diarrhea and hemorrhagic colitis with life-threatening complications, such as hemolytic uremic syndrome. The aim of this study was to assess the molecular epidemiologic features of non-O157 STEC strains from different resources in China and illustrate the role of animal reservoirs or animal-derived foodstuffs in human STEC infections. A collection of 301 non-O157 STEC isolates from domestic and wild animals (i.e., cattle, goat, pig, yak, pika, and antelope), raw meats (i.e., beef, pork, mutton, chicken, and duck), diarrheal patients, and healthy carriers in different regions of China were selected in this study. Of the 301 analyzed STEC isolates, 67 serogroups, and 118 serotypes were identified; this included some predominant serogroups associated with human disease, such as O26, O45, O103, O111, and O121. Eighteen different combinations of stx subtypes were found. Eleven isolates carried the intimin gene eae, 93 isolates contained ehxA, and 73 isolates carried astA. The prevalence of other putative adhesion genes saa, paa, efa1, and toxB was 28.90% (87), 6.98% (21), 2.31% (7), and 1% (3), respectively. The phylogenetic distribution of isolates was analyzed by multilocus sequence typing (MLST). Ninety-four sequence types were assigned across the 301 isolates. A subset of isolates recovered from yak and pika residing in the similar wild environments, Qinghai-Tibetan plateau, showed similar genetic profiles and more tendencies to cluster together. Isolates from goat and mutton exhibited close genetic relatedness with those from human-derived isolates, providing evidence that transmission may have occurred locally within intraspecies or interspecies, and importantly, from animal reservoirs, or raw meats to humans. Comparing isolates in this study with highly virulent strains by MLST, along with serotyping and virulence profiles, it is conceivable that some of isolates from goat, yak, or raw meats may have potential to cause human diseases.

Prevalence and characteristics of Shiga toxin-producing Escherichia coli isolated from retail raw meats in China.

Shiga toxin-producing Escherichia coli (STEC) causes diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome in humans. Most human infections are attributed to consumption of STEC-contaminated foodstuffs of animal origin. In this study, we evaluated the prevalence of STEC from retail raw meats collected from two geographical regions in China. The results revealed that 166 out of 853 samples were stx-positive; 63 STEC isolates were recovered from 58 stx-positive samples including pork (4.4%, 14/318), beef (11.0%, 21/191), mutton (20.6%, 26/126), chicken (0.5%, 1/205), and duck (7.7%, 1/13). Twenty-six O serogroups and 33 O:H serotypes were identified. All three stx1 subtypes and five stx2 subtypes (2a to 2e) were found in the 63 STEC isolates, among which stx2e-positive STEC isolates were the most predominant (39.7%), followed by stx1c only (20.6%), stx1c+stx2b (14.3%), and stx1a only (9.5%). STEC isolates carried virulence genes eae (6.3%), ehxA (36.5%), katP (4.8%), astA (11.1%), and subA (36.5%). Of the four adherence-associated genes tested, toxB was absent, whereas saa, paa, and efa1 were present in 28, three, and one STEC isolates respectively. The STEC isolates were divided into 50 PFGE patterns and 33 sequence types. STEC from different sources and geographical regions were separated by PFGE and MLST. Our results revealed that there is a high genetic diversity of STEC in retail raw meats, some of which have potential to cause human diseases.

[Surveillance for diarrheagenic Escherichia coli in Shanghai, 2012-2013].

OBJECTIVE: To understand the distribution of diarrheagenic Escherichia (E.) coli in population in Shanghai and discuss the practice model of cooperation in enteric infectious disease prevention and control between public health institution and hospital. METHODS: Sentinel hospitals were assigned, standard detection and identification of diarrheagenic E. coli were conducted, incidence curve of diarrheagenic E. coli infection was drawn and epidemiologic survey and laboratory detection were conducted for suspect diarrheagenic E. coli infection outbreaks. RESULTS: A total of 7 204 stool specimens were collected from diarrhea patients in 4 hospitals during 2012-2013, in which 712 (9.9% ) were diarrheagenic E. coli positive, including 351 enteropathogenic E. coli (EPEC) strains, 292 enterotoxigenic E. coli (ETEC) strains, 32 enteroinvasive E. coli(EIEC) strains and 6 Shiga toxin-producing E. coli (STEC/EHEC) strains, as well as 31 mixed strains. EPEC infection mainly occurred in children aged 1-5 years; and all of these infections were caused by aEPEC. The incidence peak of ETEC infection was during August, the positive rate was >20%. The ETEC infection mainly occurred in infants aged 1-28 days in 2012 and in people aged 20-60 years in 2013 (P<0.05). ST was the major type (59.6%), followed by LT (27.8%) and ST/LT (12.6%). EIEC infection increased in children obviously in 2013 (P<0.01). No EHEC O157:H7 case was detected, but two EHEC O26:H11 (eae-hlyA-stx1a) cases in children were reported for the first time in Shanghai. The survey result indicated that the multidrug-resistant ETEC (STh-CS21-CFA/I-ClyA-EatA-ST2332-SHNL0005) strain causing outbreak in 15 newborns in Shanghai in 2012 was in the same clone as the strain detected in Zigong in Sichuan province. CONCLUSION: Significant change has occurred in diarrheagenic E. coli distribution in Shanghai in recent years, ETEC has potential risk to cause outbreak of hospital acquired infection in neonates and food borne infection. The active surveillance on ETEC and other enteric pathogens by both public health institutions and hospitals need to be improved.

Sequential isolation in a patient of Raoultella planticola and Escherichia coli bearing a novel ISCR1 element carrying blaNDM-1.

BACKGROUND: The gene for New Delhi metallo-ß-lactamase 1 (NDM-1) has been reported to be transmitted via plasmids which are easily transferable and capable of wide distribution. We report the isolation of two NDM-1 producing strains and possible in vivo transfer of blaNDM-1 in a patient. METHODS: Clinical samples were collected for bacterial culture and antibiotic susceptibility testing from a patient during a 34-day hospitalization. The presence of blaNDM-1 was detected by PCR and sequencing. Plasmids of interest were sequenced. Medical records were reviewed for evidence of association between the administration of antibiotics and the acquisition of the NDM-1 resistance. RESULTS: A NDM-1 positive Raoultella planticola was isolated from blood on the ninth day of hospitalization without administration of any carbapenem antibiotics and a NDM-1 positive Escherichia coli was isolated from feces on the 29th day of hospitalization and eight days after imipenem administration. The blaNDM-1 was carried by a 280 kb plasmid pRpNDM1-1 in R. planticola and a 58 kb plasmid pEcNDM1-4 in E. coli. The two plasmids shared a 4812 bp NDM-1-ISCR1 element which was found to be excisable from the plasmid as a free form and transferrable in vitro to a NDM-1 negative plasmid from E. coli. CONCLUSION: blaNDM-1 was embedded in an ISCR1 complex class 1 integron as a novel 4812 bp NDM-1-ISCR1 element. The element was found to be able to self excise to become a free form, which may provide a new vehicle for NDM-1 dissemination. This mechanism could greatly accelerate the spread of NDM-1 mediated broad spectrum ß-lactam resistance.

An O island 172 encoded RNA helicase regulates the motility of Escherichia coli O157:H7.

Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 is a major cause of zoonotic food- and water-borne intestinal infections worldwide with clinical consequences ranging from mild diarrhoea to hemolytic uraemic syndrome. The genome of EHEC O157:H7 contains many regions of unique DNA that are referred to as O islands including the Shiga toxin prophages and pathogenicity islands encoding key virulence factors. However many of these O islands are of unknown function. In this study, genetic analysis was conducted on OI-172 which is a 44,434 bp genomic island with 27 open reading frames. Comparative genome analysis showed that O1-72 is a composite island with progressive gain of genes since O157:H7 evolved from its ancestral O55:H7. A partial OI-172 island was also found in 2 unrelated E. coli strains and 2 Salmonella strains. OI-172 encodes several putative helicases, one of which (Z5898) is a putative DEAH box RNA helicase. To investigate the function of Z5898, a deletion mutant (EDL933ΔZ5898) was constructed in the O157:H7 strain EDL933. Comparative proteomic analysis of the mutant with the wild-type EDL933 found that flagellin was down-regulated in the Z5898 mutant. Motility assay showed that EDL933ΔZ5898 migrated slower than the wild-type EDL933 and electron microscopy found no surface flagella. Quantitative reverse transcription PCR revealed that the fliC expression of EDL933ΔZ5898 was significantly lower while the expression of its upstream regulator gene, fliA, was not affected. Using a fliA and a fliC promoter - green fluorescent protein fusion contruct, Z5898 was found to affect only the fliC promoter activity. Therefore, Z5898 regulates the flagella based motility by exerting its effect on fliC. We conclude that OI-172 is a motility associated O island and hereby name it the MAO island.

[Pulsed-field gel electrophoresis typing on non-O157 Shiga toxin-producing Escherichia coli isolates].

OBJECTIVE: To establish a database and to understand the molecular epidemiological features of non-O157 Shiga toxin-producing Escherichia coli (STEC) isolates from different animal reservoirs and patients. METHODS: Pulsed-field gel electrophoresis (PFGE) was performed according to the PulseNet protocol with minor modifications. A dendrogram was constructed using the BioNumerics. RESULTS: Under the PulseNet protocol, 62 PFGE patterns were obtained from 76 non-O157 STEC isolates and then divided into A to M groups. Isolates from different sources were widely distributed in different groups, but were predominant seen in certain groups. CONCLUSION: The non-O157 STEC isolates in China were highly polymorphic. PulseNet protocol seemed to be suitable for the typing of Chinese non-O157 STEC isolates.

Shiga toxin-producing Escherichia coli in yaks (Bos grunniens) from the Qinghai-Tibetan Plateau, China.

Shiga toxin (Stx)-producing Escherichia coli (STEC) are recognized as important human pathogens of public health concern. Many animals are the sources of STEC. In this study we determined the occurrence and characteristics of the STEC in yaks (Bos grunniens) from the Qinghai-Tibetan plateau, China. A total of 728 yak fecal samples was collected from June to August, 2012 and was screened for the presence of the stx 1 and stx 2 genes by TaqMan real-time PCR after the sample was enriched in modified Tryptone Soya Broth. Of the 138 (18.96%) stx 1 and/or stx 2-positive samples, 85 (61.59%) were confirmed to have at least 1 STEC isolate present by culture isolation, from which 128 STEC isolates were recovered. All STEC isolates were serotyped, genotyped by pulsed-field gel electrophoresis (PFGE) and characterized for the presence of 16 known virulence factors. Fifteen different O serogroups and 36 different O:H serotypes were identified in the 128 STEC isolates with 21 and 4 untypable for the O and H antigens respectively. One stx 1 subtype (stx 1a) and 5 stx 2 subtypes (stx 2a, stx 2b, stx 2c, stx 2d and stx 2g) were present in these STEC isolates. Apart from lpfA O157/OI-141, lpfA O157/OI-154, lpfA O113, katP and toxB which were all absent, other virulence factors screened (eaeA, iha, efa1, saa, paa, cnf1, cnf2, astA, subA, exhA and espP) were variably present in the 128 STEC isolates. PFGE were successful for all except 5 isolates and separated them into 67 different PFGE patterns. For the 18 serotypes with 2 or more isolates, isolates of the same serotypes had the same or closely related PFGE patterns, demonstrating clonality of these serotypes. This study was the first report on occurrence and characteristics of STEC isolated from yaks (Bos grunniens) from the Qinghai-Tibetan plateau, China, and extended the genetic diversity and reservoir host range of STEC.

SNP genotyping of enterohemorrhagic Escherichia coli O157:H7 isolates from China and genomic identity of the 1999 Xuzhou outbreak.

Enterohemorrhagic Escherichia coli O157:H7 is a well-known pathogen as a cause of diarrhea, hemorrhagic colitis (HC), and hemolytic uremic syndrome (HUS). Single nucleotide polymorphisms (SNPs) have been widely used to determine genetic relatedness and epidemiological relationship of O157:H7. Little is known of genetic diversity of Chinese O157:H7 isolates and their relationships with global isolates. The minimum sets of 32 SNPs each from Manning et al. and Clawson et al. were used to type 325 Chinese O157:H7 isolates. The 64 SNPs divided the Chinese O157:H7 isolates into 5 SNP genotypes (SG-1-SG-5). The most common SGs were SG-5 (79.69%) and SG-1 (14.46%). Human isolates concentrated in SG-1 and SG-5, and there is only 1 human isolates in SG-3. The 47 isolates in SG-1 were further divided by an additional SNP sourced from Xuzhou21 genome into 2 subtypes (SG-1.1 and SG-1.2). Strains in SG-1.1 caused the 1999 Xuzhou deadly outbreak. Our Chinese isolates have been found to belong to a limited number of SNP genotypes and are represented by distantly related clades in Manning et al. and lineages in Claswon et al., suggesting parallel spread of these SNP genotypes in China.

Global transcriptional and phenotypic analyses of Escherichia coli O157:H7 strain Xuzhou21 and its pO157_Sal cured mutant.

Escherichia coli O157:H7 is an important food-borne pathogen that can cause hemorrhagic colitis and hemolytic-uremic syndrome in humans. pO157_Sal, a novel conjugative plasmid is present in a Chinese O157:H7 outbreak strain Xuzhou21. Here we investigated the phenotypic and transcriptional differences between the wild type strain Xuzhou21 and the pO157_Sal cured mutant strain Xuzhou21m. RNA-Seq analysis found that all 52 ORFs encoded on pO157_Sal were transcribed. One hundred and sixty eight chromosomal and pO157 genes were differentially expressed (≥2 fold difference) between Xuzhou21 and Xuzhou21m. Sixty-seven and 101 genes were up-regulated and down-regulated respectively by pO157_Sal including genes related to stress response, adaption and virulence. The plasmid-cured mutant Xuzhou21m grew slower than wild type Xuzhou21 and pO157_Sal plasmid complemented strain Xuzhou21c in M9 medium under the condition of high NaCl or presence of sodium deoxycholate (NaDC), corroborating with the RNA-Seq data. Seven differentially expressed genes are associated with NaDC resistance, including the adenine-specific DNA-methyltransferase gene (dam), multidrug efflux system subunit gene mdtA, hyperosmotically inducible periplasmic protein gene osmY and oxidation-reduction related genes while two differentially expressed genes (osmY and pspD) are likely to be related to resistance to osmotic pressure. A number of differentially expressed genes were virulence associated including four genes encoding T3SS effectors from the chromosome and ehxD from pO157. Through complementation of Xuzhou21m with a plasmid construct carrying the pO157_Sal hha homolog we further showed that the pO157_Sal hha represses the expression of T3SS effectors. These findings demonstrated that the plasmid pO157_Sal affects the transcription of the chromosomal and pO157 plasmid genes and contributes to the enhanced ability to resist stress. We conclude that pO157_Sal plays an important role in regulating global gene expression and affects the virulence and adaptation of E. coli O157:H7.

A novel Escherichia coli O157:H7 clone causing a major hemolytic uremic syndrome outbreak in China.

An Escherichia coli O157:H7 outbreak in China in 1999 caused 177 deaths due to hemolytic uremic syndrome. Sixteen outbreak associated isolates were found to belong to a new clone, sequence type 96 (ST96), based on multilocus sequence typing of 15 housekeeping genes. Whole genome sequencing of an outbreak isolate, Xuzhou21, showed that the isolate is phylogenetically closely related to the Japan 1996 outbreak isolate Sakai, both of which share the most recent common ancestor with the US outbreak isolate EDL933. The levels of IL-6 and IL-8 of peripheral blood mononuclear cells induced by Xuzhou21 and Sakai were significantly higher than that induced by EDL933. Xuzhou21 also induced a significantly higher level of IL-8 than Sakai while both induced similar levels of IL-6. The expression level of Shiga toxin 2 in Xuzhou21 induced by mitomycin C was 68.6 times of that under non-inducing conditions, twice of that induced in Sakai (32.7 times) and 15 times higher than that induced in EDL933 (4.5 times). Our study shows that ST96 is a novel clone and provided significant new insights into the evolution of virulence of E. coli O157:H7.