Xiaolu Shi

Salmonella enterica Serovar Senftenberg Human Clinical Isolates Lacking SPI-1

ABSTRACT Nontyphoidal Salmonella species cause gastrointestinal disease worldwide. The prevailing theory of Salmonella enteropathogenesis is that bacterial invasion of the intestinal epithelium is essential for virulence and that this requires the virulence-associated genomic region Salmonella pathogenicity island 1 (SPI-1). Recent studies of Salmonella enterica infection models have demonstrated that enterocolitis and diarrhea in mice and cows can occur independently of SPI-1. In this study, we sought to confirm whether two S. enterica serovar Senftenberg clinical isolates lacked genes essential for SPI-1 function. Two clinical strains were isolated and identified as being S. enterica serovar Senftenberg from four stool samples from a food-borne disease outbreak affecting seven individuals in Shenzhen, Guangdong Province, China, using conventional methods, pulsed-field gel electrophoresis and multilocus sequence typing. The possibility of coinfection with other potential bacteria or usual viruses was excluded. Two isolates were analyzed for the presence of invA, sipA, ssaR, sifA, and sopE2 by PCR and Southern blotting and were then assayed for the presence of SPI-1 by PCR and long-range PCR for fhlA-hilA, hilA-spaP, and spaP-invH and Southern blot analysis. A long-range PCR fragment from fhlA to mutS covering the 5′ and 3′ flanks of SPI-1 was also amplified from the two clinical isolates and sequenced. In addition, the two clinical isolates were assayed for enteroinvasiveness in vitro. Murine infection models were also examined. Biochemical tests and serotyping confirmed that the two clinical isolates are S. enterica serovar Senftenberg. However, they lacked genes critical for SPI-1 function but contained SPI-2 genes and were attenuated for the invasion of cultured intestinal epithelial cells. In conclusion, clinical S. enterica serovar Senftenberg strains isolated from a food-borne disease outbreak lack the invasion-associated locus SPI-1, indicating that SPI-1 is not essential for human gastroenteritis.

Characterization of Staphylococcus aureus strains associated with food poisoning in Shenzhen, China.

ABSTRACT To characterize isolates of Staphylococcus aureus that were associated with staphylococcal food poisoning between 2006 and 2009 in Shenzhen, Southern China, a total of 52 Staphylococcus aureus isolates from 11 outbreaks were analyzed by using multilocus sequence typing (MLST), spa typing, and pulsed-field gel electrophoresis (PFGE). PCR analysis was used to analyze the staphylococcal enterotoxin (SE) genes sea to sei, and antimicrobial susceptibility testing was also performed. ST6 was the most dominant sequence type (ST), constituting 63.5% (34/52) of all of the isolates in 7 outbreaks. The next most common ST was ST943, which constituted 23.1% (12/52) of the isolates that were collected from 3 outbreaks. t701, t091, and t2360 were the most predominant spa types, constituting 67.3% (35/52) of the isolates that were collected from 11 outbreaks. Three PFGE types, (types A, B, and C) were the most frequently observed types, constituting 84.6% (44/52) of all of the isolates. The enterotoxin gene that we detected most frequently was sea (45/52; 86.5%). Four SE gene profiles were observed, including sea (n = 45), sec-seh (n = 3), seb (n = 2), and seg-sei (n = 2). With respect to antibiotic resistance, penicillin resistance was the most common (96.2%; 50/52), followed by resistance to tetracycline (28.8%; 15/52). Approximately 30.8% (16/52) of the isolates were resistant to at least two antibiotics, and 7.7% (4/52) of the isolates were resistant to three or more drugs. The two predominant S. aureus lineages, (i) PFGE types A and B with ST6 and (ii) PFGE type C with ST943, were identified in the outbreaks.

Vibrio parahaemolyticus, Southern Coastal Region of China, 2007–2012

We analyzed the prevalence and characteristics of Vibrio parahaemolyticus among patients with acute infectious diarrhea in the southern coastal region of China. V. parahaemolyticus was the leading cause of bacterial infectious diarrhea in this region during 2007–2012. Serotype O3:K6 strains were most common, followed by serotypes O4:K8 and O3:K29.

Simultaneous Detection of Five Enteric Viruses Associated with Gastroenteritis by Use of a PCR Assay: a Single Real-Time Multiplex Reaction and Its Clinical Application

ABSTRACT We developed a highly sensitive reverse transcription and multiplex real-time PCR (rtPCR) assay that can identify five viruses, including six genogroups, in a single reaction: norovirus genogroups I and II; sapovirus genogroups I, II, IV, and V; human rotavirus A; adenovirus serotypes 40 and 41; and human astrovirus. In comparison to monoplex rtPCR assays, the sensitivities and specificities of the multiplex rtPCR ranged from 75% to 100% and from 99% to 100%, respectively, evaluated on 812 clinical stool specimens.

Risk Factors for Vibrio parahaemolyticus Infection in a Southern Coastal Region of China

OBJECTIVES The objectives of the study were to identify dietary and medical risk factors for Vibrio parahaemolyticus (VP) infection in the coastal city Shenzhen in China. METHODS In April-October 2012, we conducted a case-control study in two hospitals in Shenzhen, China. Laboratory-confirmed VP cases (N = 83) were matched on age, sex, and other social factors to healthy controls (N = 249). Subjects were interviewed using a questionnaire on medical history; contact with seawater; clinical symptoms and outcome; travel history over the past week; and dietary history 3 days prior to onset. Laboratory tests were used to culture, serotype, and genotype VP strains. We used logistic regression to calculate the odds ratios for the association of VP infection with potential risk factors. RESULTS In multivariate analysis, VP infection was associated with having pre-existing chronic disease (adjusted odds ratio [aOR], 6.0; 95% confidence interval [CI], 1.5-23.7), eating undercooked seafood (aOR, 8.0; 95% CI, 1.3-50.4), eating undercooked meat (aOR, 29.1; 95% CI, 3.0-278.2), eating food from a street food vendor (aOR, 7.6; 95% CI, 3.3-17.6), and eating vegetable salad (aOR, 12.1; 95% CI, 5.2-28.2). CONCLUSIONS Eating raw (undercooked) seafood and meat is an important source of VP infection among the study population. Cross-contamination of VP in other food (e.g., vegetables and undercooked meat) likely plays a more important role. Intervention should be taken to lower the risks of cross-contamination with undercooked seafood/meat, especially targeted at people with low income, transient workers, and people with medical risk factors.

Comparative Screening of Digestion Tract Toxic Genes in Proteus mirabilis.

Proteus mirabilis is a common urinary tract pathogen, and may induce various inflammation symptoms. Its notorious ability to resist multiple antibiotics and to form urinary tract stones makes its treatment a long and painful process, which is further challenged by the frequent horizontal gene transferring events in P. mirabilis genomes. Three strains of P. mirabilis C02011/C04010/C04013 were isolated from a local outbreak of a food poisoning event in Shenzhen, China. Our hypothesis is that new genes may have been acquired horizontally to exert the digestion tract infection and toxicity. The functional characterization of these three genomes shows that each of them independently acquired dozens of virulent genes horizontally from the other microbial genomes. The representative strain C02011 induces the symptoms of both vomit and diarrhea, and has recently acquired a complete type IV secretion system and digestion tract toxic genes from the other bacteria.

Genomic and Phenotypic Analyses Reveal the Emergence of an Atypical Salmonella enterica Serovar Senftenberg Variant in China

ABSTRACT Human infections with Salmonella enterica subspecies enterica serovar Senftenberg are often associated with exposure to poultry flocks, farm environments, or contaminated food. The recent emergence of multidrug-resistant isolates has raised public health concerns. In this study, comparative genomics and phenotypic analysis were used to characterize 14 Salmonella Senftenberg clinical isolates recovered from multiple outbreaks in Shenzhen and Shanghai, China, between 2002 and 2011. Single-nucleotide polymorphism analyses identified two phylogenetically distinct clades of S. Senftenberg, designated SC1 and SC2, harboring variations in Salmonella pathogenicity island 1 (SPI-1) and SPI-2 and exhibiting distinct biochemical and phenotypic signatures. Although the two variants shared the same serotype, the SC2 isolates of sequence type 14 (ST14) harbored intact SPI-1 and -2 and hence were characterized by possessing efficient invasion capabilities. In contrast, the SC1 isolates had structural deletion patterns in both SPI-1 and -2 that correlated with an impaired capacity to invade cultured human cells and also the year of their isolation. These atypical SC1 isolates also lacked the capacity to produce hydrogen sulfide. These findings highlight the emergence of atypical Salmonella Senftenberg variants in China and provide genetic validation that variants lacking SPI-1 and regions of SPI-2, which leads to impaired invasion capacity, can still cause clinical disease. These data have identified an emerging public health concern and highlight the need to strengthen surveillance to detect the prevalence and transmission of nontyphoidal Salmonella species.

Genome Sequence of Proteus mirabilis Clinical Isolate C05028

ABSTRACT Genomic DNA of Proteus mirabilis C05028 was sequenced by an Illumina HiSeq platform and was assembled to 39 scaffolds with a total length of 3.8 Mb. Next, open reading frames (ORFs) were identified and were annotated by the KEGG, COG, and NR databases. Finally, we found special virulence factors only existing in P. mirabilis C05028.

The evaluation and application of multilocus variable number tandem repeat analysis (MLVA) for the molecular epidemiological study of Salmonella enterica subsp. enterica serovar Enteritidis infection

BackgroundSalmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis) is one of the most prevalent Salmonella serotypes that cause gastroenteritis worldwide and the most prevalent serotype causing Salmonella infections in China. A rapid molecular typing method with high throughput and good epidemiological discrimination is urgently needed for detecting the outbreaks and finding the source for effective control of S. Enteritidis infections.MethodsIn this study, 194 strains which included 47 from six outbreaks that were well-characterized epidemiologically were analyzed with pulse field gel electrophoresis (PFGE) and multilocus variable number tandem repeat analysis (MLVA). Seven VNTR loci published by the US Center for Disease Control and Prevention (CDC) were used to evaluate and develop MLVA scheme for S. Enteritidis molecular subtyping by comparing with PFGE, and then MLVA was applied to the suspected outbreaks detection. All S. Enteritidis isolates were analyzed with MLVA to establish a MLVA database in Shenzhen, Guangdong province, China to facilitate the detection of S. Enteritidis infection clusters.ResultsThere were 33 MLVA types and 29 PFGE patterns among 147 sporadic isolates. These two measures had Simpson indices of 0.7701 and 0.8043, respectively, which did not differ significantly. Epidemiological concordance was evaluated by typing 47 isolates from six epidemiologically well-characterized outbreaks and it did not differ for PFGE and MLVA. We applied the well established MLVA method to detect two S. Enteritidis foodborne outbreaks and find their sources successfully in 2014. A MLVA database of 491 S. Enteritidis strains isolated from 2004 to 2014 was established for the surveillance of clusters in the future.ConclusionsMLVA typing of S. Enteritidis would be an effective tool for early warning and epidemiological surveillance of S. Enteritidis infections.

Multicolor Melting Curve Analysis-Based Multilocus Melt Typing of Vibrio parahaemolyticus.

Vibrio parahaemolyticus is the leading cause of seafood-borne gastroenteritis outbreaks. To track the source of these diseases in a timely manner, a high throughput typing method is critical. We hereby describe a novel genotyping method for V. parahaemolyticus, termed multilocus melt typing (MLMT), based on multilocus sequence typing (MLST). MLMT utilizes melting curve analysis to interrogate the allelic types of a set of informative single nucleotide polymorphisms (SNPs) derived from the housekeeping genes used in MLST. For each SNP, one allelic type generates distinct Tm values, which are converted into a binary code. Multiple SNPs thus generate a series of binary codes, forming a melt type (MT) corresponding with a sequence type (ST) of MLST. Using a set of 12 SNPs, the MLMT scheme could resolve 218 V.parahaemolyticus isolates into 50 MTs corresponding with 56 STs. The discriminatory power of MLMT and MLST was similar with Simpson’s index of diversity of 0.638 and 0.646, respectively. The global (adjusted Rand index = 0.982) and directional congruence (adjusted Wallace coefficient, MT→ST = 0.965; ST→MT = 1.000) between the two typing approaches was high. The entire procedure of MLMT could be finished within 3 h with negligible hands on time in a real-time PCR machine. We conclude that MLMT provides a reliable and efficient approach for V. parahaemolyticus genotyping and might also find use in other pathogens.