Yanmei Xu

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.

Dynamics of fecal microbial communities in children with diarrhea of unknown etiology and genomic analysis of associated Streptococcus lutetiensis

BackgroundThe sequences of the 16S rRNA genes extracted from fecal samples provide insights into the dynamics of fecal microflora. This potentially gives valuable etiological information for patients whose conditions have been ascribed to unknown pathogens, which cannot be accomplished using routine culture methods. We studied 33 children with diarrhea who were admitted to the Children’s Hospital in Shanxi Province during 2006.ResultsNineteen of 33 children with diarrhea could not be etiologically diagnosed by routine culture and polymerase chain reaction methods. Eleven of 19 children with diarrhea of unknown etiology had Streptococcus as the most dominant fecal bacterial genus at admission. Eight of nine children whom three consecutive fecal samples were collected had Streptococcus as the dominant fecal bacterial genus, including three in the Streptococcus bovis group and three Streptococcus sp., which was reduced during and after recovery. We isolated strains that were possibly from the S. bovis group from feces sampled at admission, which were then identified as Streptococcus lutetiensis from one child and Streptococcus gallolyticus subsp. pasteurianus from two children. We sequenced the genome of S. lutetiensis and identified five antibiotic islands, two pathogenicity islands, and five unique genomic islands. The identified virulence genes included hemolytic toxin cylZ of Streptococcus agalactiae and sortase associated with colonization of pathogenic streptococci.ConclusionsWe identified S. lutetiensis and S. gallolyticus subsp. pasteurianus from children with diarrhea of unknown etiology, and found pathogenic islands and virulence genes in the genome of S. lutetiensis.

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.

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.

Rapid and Sensitive Detection of Shigella spp. and Salmonella spp. by Multiple Endonuclease Restriction Real-Time Loop-Mediated Isothermal Amplification Technique

Shigella and Salmonella are frequently isolated from various food samples and can cause human gastroenteritis. Here, a novel multiple endonuclease restriction real-time loop-mediated isothermal amplification technology (MERT-LAMP) were successfully established and validated for simultaneous detection of Shigella strains and Salmonella strains in only a single reaction. Two sets of MERT-LAMP primers for 2 kinds of pathogens were designed from ipaH gene of Shigella spp. and invA gene of Salmonella spp., respectively. Under the constant condition at 63°C, the positive results were yielded in as short as 12 min with the genomic DNA extracted from the 19 Shigella strains and 14 Salmonella strains, and the target pathogens present in a sample could be simultaneously identified based on distinct fluorescence curves in real-time format. Accordingly, the multiplex detection assay significantly reduced effort, materials and reagents used, and amplification and differentiation were conducted at the same time, obviating the use of postdetection procedures. The analytical sensitivity of MERT-LAMP was found to be 62.5 and 125 fg DNA/reaction with genomic templates of Shigella strains and Salmonella strains, which was consist with normal LAMP assay, and at least 10- and 100-fold more sensitive than that of qPCR and conventional PCR approaches. The limit of detection of MERT-LAMP for Shigella strains and Salmonella strains detection in artificially contaminated milk samples was 5.8 and 6.4 CFU per vessel. In conclusion, the MERT-LAMP methodology described here demonstrated a potential and valuable means for simultaneous screening of Shigella and Salmonella in a wide variety of samples.

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 β1 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-β1 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-β1 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.

Shiga Toxin-Producing Escherichia coli in Plateau Pika (Ochotona curzoniae) on the Qinghai-Tibetan Plateau, China

Shiga toxin-producing Escherichia coli (STEC) are an emerging group of zoonotic pathogens. Ruminants are the natural reservoir of STEC. In this study we determined the prevalence and characteristics of the STEC in plateau pika (Ochotona curzoniae) on the Qinghai-Tibetan Plateau, China. A total of 1116 pika samples, including 294 intestinal contents samples, 317 fecal samples, and 505 intestinal contents samples, were collected from May to August in the years 2012, 2013, and 2015, respectively. Twenty-one samples (1.88%) yielded at least one STEC isolate; in total, 22 STEC isolates were recovered. Thirteen different O serogroups and 14 serotypes were identified. One stx1 subtype (stx1a) and three stx2 subtypes (stx2a, stx2b, and stx2d) were present in the STEC isolates. Fifteen, fourteen, and three STEC isolates harbored the virulence genes ehxA, subA, and astA, respectively. Adherence-associated genes iha and saa were, respectively, present in 72.73 and 68.18% of the STEC isolates. Twenty antibiotics were active against all the STEC isolates; all strains were resistant to penicillin G, and some to cephalothin or streptomycin. The 22 STEC isolates were divided into 16 pulsed-field gel electrophoresis patterns and 12 sequence types. Plateau pikas may play a role in the ongoing circulation of STEC in the Qinghai-Tibetan plateau. This study provides the first report on STEC in plateau pikas and new information about STEC reservoirs in wildlife. Based on the serotypes, virulence gene profiles and multi-locus sequence typing (MLST) analysis, the majority of these pika STECs may pose a low public health risk.

Detection and dissemination of the colistin resistance gene, mcr-1, from isolates and faecal samples in China

Purpose. A recently identified colistin resistance gene, mcr‐1, has been reported in many countries. In this study, we established a new real‐time PCR method to detect it. Methodology. We used a real‐time PCR method to detect the mcr‐1 gene in a variety of isolates and faecal samples from 20 provinces and municipal cities in China. Results. Of the 2330 isolates (from 10 species) screened, 54 (2.3 %) isolates were positive for mcr‐1. All of the mcr‐1‐positive isolates that were identified belonged to Escherichia coli strains, among which 9, 1, and 44 were identified as enteropathogenic E. coli, enteroadherent E. coli, and non‐pathogenic E. coli, respectively. The majority of the mcr‐1‐positive isolates were obtained from farm animals from eight provinces and municipal cities across China. A total of 337 faecal samples, including 229 human and 108 pet animal faecal samples, were also screened for the mcr‐1 gene. Of the 337 samples analyzed, six and eight human and pet animal faecal samples were positive for the mcr‐1 gene, respectively. Conclusion. The data demonstrate that the mcr‐1 gene is highly prevalent in human and animal populations in China. This occurrence suggests that active surveillance of the mcr‐1 gene is imperative in curtailing its spread.

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 of eae -positive, lactose non-fermenting Escherichia albertii from retail raw meat in China

Escherichia albertii is a newly emerging enteric pathogen that has been associated with gastroenteritis in humans. Recently, E. albertii has also been detected in healthy and sick birds, animals, chicken meat and water. In the present study, the prevalence and characteristics of the eae-positive, lactose non-fermenting E. albertii strains in retail raw meat in China were evaluated. Thirty isolates of such strains of E. albertii were identified from 446 (6·73%) samples, including duck intestines (21·43%, 6/28), duck meat (9·52%, 2/21), chicken intestines (8·99%, 17/189), chicken meat (5·66%, 3/53), mutton meat (4·55%, 1/22) and pork meat (2·44%, 1/41). None was isolated from 92 samples of raw beef meat. Strains were identified as E. albertii by phenotypic properties, diagnostic PCR, sequence analysis of the 16S rRNA gene, and housekeeping genes. Five intimin subtypes were harboured by these strains. All strains possessed the II/III/V subtype group of the cdtB gene, with two strains carrying another copy of the I/IV subtype group. Pulsed-field gel electrophoresis showed high genetic diversity of E. albertii in raw meats. Our findings indicate that E. albertii can contaminate various raw meats, posing a potential threat to public health.