Duochun Wang

Detection of Vibrio cholerae O1 and O139 in environmental water samples by an immunofluorescent-aggregation assay.

ABSTRACT Environmental waters are an important reservoir for Vibrio cholerae, and effective surveillance of the pathogen can help to warn of and prevent infection with this potentially fatal pathogen. An immunofluorescent-aggregation (IFAG) assay to detect V. cholerae O1 and O139 was established and evaluated with estuarine water samples. The practical application of this assay was compared with the conventional culture method and real-time PCR. The IFAG method had a sensitivity of 103 CFU/ml for detection of V. cholerae O1 and O139 strains in a suspension containing 10 different species of enterobacterial strains (total, 105 CFU/ml). Ten fluorescent bacterial aggregate colonies were randomly picked and tested positive in serum agglutination tests for the V. cholerae O1 and O139 strains, showing a high specificity. The enrichment broths of 146 samples of estuarine water were tested, and the percentage positive by the IFAG assay was 19.9% (29/146), which was significantly higher than that of the conventional culture method (10.3%, 15/146; P < 0.01) but lower than that of real-time PCR (29.5%, 43/146; P < 0.01). The coincidence rates of real-time PCR and IFAG detection were decreased with the reduction of the V. cholerae concentration. The IFAG method, with a high specificity and a relatively high sensitivity, may be used for detection and isolation of V. cholerae in environmental water samples.

Distribution of Virulence-Associated Genes and Genetic Relationships in Non-O1/O139 Vibrio cholerae Aquatic Isolates from China

ABSTRACT Non-O1/O139 Vibrio cholerae is naturally present in aquatic ecosystems and has been linked with cholera-like diarrhea and local outbreaks. The distribution of virulence-associated genes and genetic relationships among aquatic isolates from China are largely unknown. In this study, 295 aquatic isolates of V. cholerae non-O1/O139 serogroups from different regions in China were investigated. Only one isolate was positive for ctxB and harbored a rare genotype; 10 (3.4%) isolates carried several types of rstR sequences, eight of which carried rare types of toxin-coregulated pili (tcpA). Furthermore, 16 (5.4%) isolates carried incomplete (with partial open reading frames [ORFs]) vibrio seventh pandemic island I (VSP-I) or VSP-II clusters, which were further classified as 11 novel types. PCR-based analyses revealed remarkable variations in the distribution of putative virulence genes, including mshA (95.6%), hlyA (95.3%), rtxC (89.8%), rtxA (82.7%), IS1004 (52.9%), chxA (30.2%), SXT (15.3%), type III secretion system (18.0%), and NAG-ST (3.7%) genes. There was no correlation between the prevalence of putative virulence genes and that of CTX prophage or TCP genes, whereas there were correlations among the putative virulence genes. Further multilocus sequence typing (MLST) placed selected isolates (n = 70) into 69 unique sequence types (STs), which were different from those of the toxigenic O1 and O139 counterparts, and each isolate occupied a different position in the MLST tree. The V. cholerae non-O1/O139 aquatic isolates predominant in China have high genotypic diversity; these strains constitute a reservoir of potential virulence genes, which may contribute to evolution of pathogenic isolates.

Molecular characteristics and antimicrobial resistance in invasive and noninvasive Group B Streptococcus between 2008 and 2015 in China

Group B streptococcus (GBS) is an increasing pathogen threat to newborns and adults with immunodepressive diseases. Here, a total of 193 GBS, including 51 invasive and 142 noninvasive isolates, were collected from the patients with infections in 7 tertiary hospitals from 5 cities in China during the year 2008 to 2015. The strains of GBS were characterized by classical and molecular techniques for capsular polysaccharide serotyping, genes for pilus island (PI) and α-like protein (alp), and antibiotic resistance profiling. Of 193 isolates, the predominant serotypes were III (45.6%) and Ia (18.7%). All strains carried at least 1 PI gene. The combination of PI-2b and PI-1 was present in 46.1% isolates, followed by PI-2a alone (80, 41.5%) and PI-2b alone (23, 11.9%). The most prevalent alp gene was rib (87, 45.1%), followed by α-C (47, 24.4%), ε (33, 17.1%), alp2/3 (7, 3.6%) and alp4 (2, 1.0%), respectively. The clonal relationships between strains were investigated using multilocus sequence typing. The strains were distinguished into 26 individual sequence typing, and further clustered into 6 clonal complexes. A significant association was noted between the distributions of alp genes, serotyping and PI profiles, such as serotype III-rib-PI+PI-2a, Ib-α-C, and Ia-ε-PI-2a. No penicillin-resistant strains were detected, and 74.1%, 64.2%, and 68.9% were resistant to erythromycin, clindamycin, and tetracycline, respectively. The infective GBS isolates in China demonstrated epidemical features.

The first case of bacteraemia due to non-O1/non-O139 Vibrio cholerae in a type 2 diabetes mellitus patient in mainland China

Bacteraemia due to non-O1/non-O139 Vibrio cholerae is rarely documented in mainland China. We report such a case in a 70-year-old male with type 2 diabetes mellitus. The clinical features, phenotypic analyses, and presence of a panel of known virulence genes in the isolated strain are described. To the best of our knowledge, this is the first reported case of bacteraemia due to this strain in a T2DM patient without other coexisting underlying diseases in mainland China.

A Molecular Surveillance Reveals the Prevalence of Vibrio cholerae O139 Isolates in China from 1993 to 2012

ABSTRACT Vibrio cholerae serogroup O139 was first identified in 1992 in India and Bangladesh, in association with major epidemics of cholera in both countries; cases were noted shortly thereafter in China. We characterized 211 V. cholerae O139 isolates that were isolated at multiple sites in China between 1993 and 2012 from patients (n = 92) and the environment (n = 119). Among clinical isolates, 88 (95.7%) of 92 were toxigenic, compared with 47 (39.5%) of 119 environmental isolates. Toxigenic isolates carried the El Tor CTX prophage and toxin-coregulated pilus A gene (tcpA), as well as the Vibrio seventh pandemic island I (VSP-I) and VSP-II. Among a subset of 42 toxigenic isolates screened by multilocus sequence typing (MLST), all were in the same sequence type as a clinical isolate (MO45) from the original Indian outbreak. Nontoxigenic isolates, in contrast, generally lacked VSP-I and -II, and fell within13 additional sequence types in two clonal complexes distinct from the toxigenic isolates. In further pulsed-field gel electrophoresis (PFGE) (with NotI digestion) studies, toxigenic isolates formed 60 pulsotypes clustered in one group, while the nontoxigenic isolates formed 43 pulsotypes which clustered into 3 different groups. Our data suggest that toxigenic O139 isolates from widely divergent geographic locations, while showing some diversity, have maintained a relatively tight clonal structure across a 20-year time span. Nontoxigenic isolates, in contrast, exhibited greater diversity, with multiple clonal lineages, than did their toxigenic counterparts.

High Prevalence of Macrolide-resistance and Molecular Characterization of Streptococcus pyogenes Isolates Circulating in China from 2009 to 2016

Streptococcus pyogenes, or group A Streptococcus, is a pathogen responsible for a wide range of clinical manifestations, from mild skin and soft tissue infections and pharyngitis to severe diseases. Its epidemiological characteristics should be comprehensively under surveillance for regulating the national prevention and treatment practice. Herein, a total of 140 S. pyogenes, including 38 invasive and 102 noninvasive isolates, were collected from infected patients in 10 tertiary general hospitals from 7 cities/provinces in China during the years 2009–2016. All strains were characterized by classical and molecular techniques for its emm types/subtypes, virulent factors and antibiotic resistance profiling. Of 140 isolates, 15 distinct emm types and 31 subtypes were detected, dominated by emm12 (60 isolates, 42.9%), emm1(43, 30.7%), and emm89 (10, 7.1%), and 8 new emm variant subtypes were identified. All strains, invasive or not, harbored the superantigenic genes, speB and slo. The other virulence genes, smeZ, speF, and speC accounted for 96.4, 91.4, and 87.1% of collected isolates, respectively. Further multilocus sequence typing (MLST) placed all strains into 22 individual sequence types (STs), including 4 newly-identified STs (11, 7.9%). All isolates were phenotypically susceptible to penicillin, ampicillin, cefotaxime, and vancomycin, whereas 131(93.5%), 132(94.2%), and 121(86.4%) were resistant to erythromycin, clindamycin, and tetracycline, respectively. Our study highlights high genotypic diversity and high prevalence of macrolide resistance of S. pyogenes among clinical isolates circulating in China.

Molecular characterization and antibiotic resistance of clinical Streptococcus dysgalactiae subsp. equisimilis in Beijing, China.

Streptococcus dysgalactiae subsp. equisimilis (SDSE) is presently considered as a human pathogen associated with clinical infection. We characterized 56 SDSE isolates collected from two tertiary hospitals in Beijing, China. Sixteen distinct emm types/subtypes were detected, dominated by stG245.0 (32.1%), stG652.0 (10.7%), stG6.1 (10.7%) and stG485.0 (10.7%), and a novel stG840.0 variant type was identified. All isolates possessed virulence genes of sagA and scpA, and most carried slo (98.2%), ska (98.2%) and speG(dys) (35.7%). By multilocus sequence typing (MLST) analysis, 17 individual sequence types (STs) were distinguished, including 7 newly-identified STs (26.8% of isolates), of which ST127 (30.4%), ST7 (12.5%) and ST44 (10.7%) dominated. Meanwhile, pulsed-field gel electrophoresis (PFGE) analysis revealed 33 pattern types (PTs), which were further combined into 16 pattern clusters (PCs), and 59.3% of isolates were distributed into 2 dominant PCs. Notably, emm types had both close relationship and consistency with STs and PFGE PCs. Furthermore, of 56 SDSE isolates, the predominant antibiotic resistances were erythromycin (71.4%), clindamycin (71.4%) and tetracycline (60.7%). Correspondingly, the prevalent resistance genes of macrolide and tetracycline were erm(B) (78.6%) and tet(M) (73.2%). In addition, multiple point mutations of parC, one of fluoroquinolone resistance genes, were observed (accounting for 75%), and were divided into 12 types, with parC 07 as the predominant type. Our data suggested the wide molecular diversity and distinctive regional features of SDSE from clinical infection in Beijing, China.

High prevalence and diversity of pre‐CTXΦ alleles in the environmental Vibrio cholerae O1 and O139 strains in the Zhujiang River estuary

Toxigenic conversion of environmental Vibrio cholerae strains through lysogenic infection by the phage CTXΦ is an important step in the emergence of new pathogenic clones. The precursor form of the CTXΦ phage, pre-CTXΦ, does not carry the cholera toxin gene. During our investigation, we frequently found pre-CTXΦ prophages in non-toxigenic isolates in the serogroups of O1 and O139 strains in the Zhujiang estuary. We observed high amounts of sequence variation of rstR and gIII(CTX) in the pre-CTXΦ alleles as well as in the tcpA sequences within the strains. In addition, a new pre-CTXΦ allele, with a novel rstR sequence type and hybrid RS2, was identified. Our findings show that active, complicated gene recombination and horizontal transfer of pre-CTXΦs occurs within V. cholerae environmental strains, which creates a complex intermediate pool for the generation of toxigenic clones in the estuarine environment.

Development and evaluation of an up-converting phosphor technology-based lateral flow assay for the rapid, simultaneous detection of Vibrio cholerae serogroups O1 and O139

Vibrio cholerae serogroups O1 and O139 are etiological agents of cholera, a serious and acute diarrheal disease, and rapid detection of V. cholerae is a key method for preventing and controlling cholera epidemics. Here, a point of care testing (POCT) method called Vch-UPT-LF, which is an up-converting phosphor technology-based lateral flow (UPT-LF) assay with a dual-target detection mode, was developed to detect V. cholerae O1 and O139 simultaneously from one sample loading. Although applying an independent reaction pair made both detection results for the two Vch-UPT-LF detection channels more stable, the sensitivity slightly declined from 104 to 105 colony-forming units (CFU) mL−1 compared with that of the single-target assay, while the quantification ranges covering four orders of magnitude were maintained. The strip showed excellent specificity for seven Vibrio species that are highly related genetically, and nine food-borne species whose transmission routes are similar to those of V. cholerae. The legitimate arrangement of the two adjacent test lines lessened the mutual impact of the quantitation results between the two targets, and the quantification values did not differ by more than one order of magnitude when the samples contained high concentrations of both V. cholerae O1 and O139. Under pre-incubation conditions, 1×101 CFU mL−1 of V. cholerae O1 or O139 could be detected in fewer than 7 h, while the Vch-UPT-LF assay exhibited sensitivity as high as a real-time fluorescent polymerase chain reaction with fewer false-positive results. Therefore, successful development of Vch-UPT-LF as a dual-target assay for quantitative detection makes this assay a good candidate POCT method for the detection and surveillance of epidemic cholera.

Influence of climate factors on Vibrio cholerae dynamics in the Pearl River estuary, South China

Abstract Current research has seldom focused on the quantitative relationships between Vibrio cholerae (V. cholerae) and climate factors owing to the complexities and high cost of field observation in the aquatic environment. This study has focused on the relationships between V. cholerae and climate factors based on linear regression method and data partition method. Data gathered from 2008 to 2009 in the Pearl River estuary, South China, were adopted. Positive rate of V. cholerae was correlated closely with monthly climate factors of water temperature and air temperature, respectively in 2009. Quarterly data analysis from 2008 to 2009 showed that there existed seasonal characteristic for V. cholerae. Positive rate of V. cholerae was correlated positively with quarterly climate factors of land surface temperature, pH, water temperature, air temperature and rainfall, respectively and negatively with quarterly air pressure. Partition data analysis in 2009 showed that there existed geography region characteristic for V. cholerae. V. cholerae dynamics was closely correlated to climate factors in the downstream area. However, it was more greatly affected by human geography factors in the urban area. Positive annual rate of V. cholerae was higher in the downstream area than in the urban area both in 2008 and 2009. At last, a cellular automaton model was used to simulate V. cholerae diffusion downstream, and the distribution of V. cholerae obtained from this model was similar to that obtained from the field observations.