Vibrio metschnikovii as an emergent pathogen: analyses of phylogeny and O-antigen and identification of possible virulence characteristics.

Vibrio metschnikovii is an emergent pathogen that causes human infections which may be fatal. However, the phylogenetic characteristics and pathogenicity determinants of V. metschnikovii are poorly understood. Here, the whole-genome features of 103 V. metschnikovii strains isolated from different sources are described. On phylogenetic analysis V. metschnikovii populations could be divided into two major lineages, defined as lineage 1 (L1) and 2 (L2), of which L1 was more likely to be associated with human activity. Meanwhile, we defined 29 V. metschnikovii O-genotypes (VMOg, named VMOg1-VMOg29) by analysis of the O-antigen biosynthesis gene clusters (O-AGCs). Most VMOgs (VMOg1 to VMOg28) were assembled by the Wzx/Wzy pathway, while only VMOg29 used the ABC transporter pathway. Based on the sequence variation of the wzx and wzt genes, an in silico O-genotyping system for V. metschnikovii was developed. Furthermore, nineteen virulence-associated factors involving 161 genes were identified within the V. metschnikovii genomes, including genes encoding motility, adherence, toxins, and secretion systems. In particular, V. metschnikovii was found to promote a high level of cytotoxicity through the synergistic action of the lateral flagella and T6SS. The lateral flagellar-associated flhA gene played an important role in the adhesion and colonization of V. metschnikovii during the early stages of infection. Overall, this study provides an enhanced understanding of the genomic evolution, O-AGCs diversity, and potential pathogenic features of V. metschnikovii.

Genomic Epidemiology of Vibrio cholerae O139, Zhejiang Province, China, 1994-2018.

Cholera caused by Vibrio cholerae O139 was first reported in Bangladesh and India in 1992. To determine the genomic epidemiology and origins of O139 in China, we sequenced 104 O139 isolates collected from Zhejiang Province, China, during 1994-2018 and compared them with 57 O139 genomes from other countries in Asia. Most Zhejiang isolates fell into 3 clusters (C1-C3), which probably originated in India (C1) and Thailand (C2 and C3) during the early 1990s. Different clusters harbored different antimicrobial resistance genes and IncA/C plasmids. The integrative and conjugative elements carried by Zhejiang isolates were of a new type, differing from ICEVchInd4 and SXTMO10 by single-nucleotide polymorphisms and presence of genes. Quinolone resistance-conferring mutations S85L in parC and S83I in gyrA occurred in 71.2% of the Zhejiang isolates. The ctxB copy number differed among the 3 clusters. Our findings provided new insights for prevention and control of O139 cholera .

Population Structure and Multidrug Resistance of Non-O1/Non-O139 Vibrio cholerae in Freshwater Rivers in Zhejiang, China.

To understand the environmental reservoirs of Vibrio cholerae and their public health significance, we surveyed freshwater samples from rivers in two cities (Jiaxing [JX] and Jiande [JD]) in Zhejiang, China. A total of 26 sampling locations were selected, and river water was sampled 456 times from 2015 to 2016 yielding 200 V. cholerae isolates, all of which were non-O1/non-O139. The average isolation rate was 47.3% and 39.1% in JX and JD, respectively. Antibiotic resistance profiles of the V. cholerae isolates were examined with nonsusceptibility to cefazolin (68.70%, 79/115) being most common, followed by ampicillin (47.83%, 55/115) and imipenem (27.83%, 32/115). Forty-two isolates (36.52%, 42/115) were defined as multidrug resistant (MDR). The presence of virulence genes was also determined, and the majority of the isolates were positive for toxR (198/200, 99%) and hlyA (196/200, 98%) with few other virulence genes observed. The population structure of the V. cholerae non-O1/non-O139 sampled was examined using multilocus sequence typing (MLST) with 200 isolates assigned to 128 STs and 6 subpopulations. The non-O1/non-O139 V. cholerae population in JX was more varied than in JD. By clonal complexes (CCs), 31 CCs that contained isolates from this study were shared with other parts of China and/or other countries, suggesting widespread presence of some non-O1/non-O139 clones. Drug resistance profiles differed between subpopulations. The findings suggest that non-O1/non-O139 V. cholerae in the freshwater environment is a potential source of human infections. Routine surveillance of non-O1/non-O139 V. cholerae in freshwater rivers will be of importance to public health.

Comparison of xMAP Salmonella Serotyping Assay With Traditional Serotyping and Discordance Resolution by Whole Genome Sequencing.

Salmonella spp. are a major cause of foodborne illness throughout the world. Traditional serotyping by antisera agglutination has been used as a standard identification method for many years but newer nucleic acid-based tests have become available that may provide advantages in workflow and test turnaround time. In this study, we evaluated the Luminex® xMAP® Salmonella Serotyping Assay (SSA), a multiplex nucleic acid test capable of identifying 85% of the most common Salmonella serotypes, in comparison to the traditional serum agglutination test (SAT) on 4 standard strains and 255 isolates from human (224), environmental, and food (31) samples. Of the total of 259 isolates, 256 could be typed by the SSA. Of these, 197 (77.0%) were fully typed and 59 (23.0%) were partially typed. By SAT, 246 of the 259 isolates (95%) were successfully typed. Sixty isolates had discrepant results between SAT and SSA and were resolved using whole genome sequencing (WGS). By SAT, 80.0% (48/60) of the isolates were consistent with WGS while by SSA 91.7% (55/60) were partially consistent with WGS. By serovar, all 30 serovars except one tested were fully or partially typable. The workflow comparison showed that SSA provided advantages over SAT with a hands-on time (HOT) of 3.5 min and total turnaround time (TAT) of 6 h, as compared to 1 h HOT and 2-6 days TAT for SAT. Overall, this study showed that molecular serotyping is promising as a rapid method for Salmonella serotyping with good accuracy for typing most common Salmonella serovars circulating in China.

Molecular characteristics of Clostridium difficile in children with acute gastroenteritis from Zhejiang.

BACKGROUND: Clostridium difficile infection (CDI) has an increasing pediatric prevalence worldwide. However, molecular characteristics of C. difficile in Chinese children with acute gastroenteritis have not been reported. METHODS: A five-year cross-sectional study was conducted in a tertiary children's hospital in Zhejiang. Consecutive stool specimens from outpatient children with acute gastroenteritis were cultured for C. difficile, and isolates then were analyzed for toxin genes, multi-locus sequence type and antimicrobial resistance. Diarrhea-related viruses were detected, and demographic data were collected. RESULTS: A total of 115 CDI cases (14.3%), and 69 co-infected cases with both viruses and toxigenic C. difficile, were found in the 804 stool samples. The 186 C. difficile isolates included 6 of toxin A-positive/toxin B-positive/binary toxin-positive (A+B+CDT+), 139 of A+B+CDT-, 3 of A-B+CDT+, 36 of A-B+CDT- and 2 of A-B-CDT-. Sequence types 26 (17.7%), 35 (11.3%), 39 (12.4%), 54 (16.7%), and 152 (11.3%) were major genotypes with significant differences among different antimicrobial resistances (Fisher's exact test, P < 0.001). The A-B+ isolates had significantly higher resistance, compared to erythromycin, rifampin, moxifloxacin, and gatifloxacin, than that of the A+B+ (χ2 = 7.78 to 29.26, P < 0.01). The positive CDI rate in infants (16.2%) was significantly higher than that of children over 1 year old (10.8%) (χ2 = 4.39, P = 0.036). CONCLUSIONS: CDI has been revealed as a major cause of acute gastroenteritis in children with various genotypes. The role of toxigenic C. difficile and risk factors of CDI should be emphatically considered in subsequent diarrhea surveillance in children from China.

High-Level Resistance of Toxigenic Clostridioides difficile Genotype to Macrolide-Lincosamide- Streptogramin B in Community Acquired Patients in Eastern China.

BACKGROUND: Clostridioides difficile resistant to macrolide-lincosamide-streptogramin B (MLSB) has not been reported in China. METHODS: In a cross-sectional study in two tertiary hospitals, C. difficile isolates from stool specimens from community-onset, hospital-associated diarrheal patients were analyzed for toxin genes, genotype, and antibiotic resistance, and the patients' clinical charts were reviewed. RESULTS: A total of 190 (15.2%) isolates (102 A+B+ and 88 A-B+) from 1250 community acquired (CA) patients were recovered and all were susceptible to vancomycin and metronidazole. High-level resistance (minimum inhibitory concentration > 128 mg/L) to erythromycin and clindamycin was recorded in 77.9% and 88.4% of the tested isolates, respectively. Furthermore, 89.3% (159/178) of the isolates resistant to MLSB carried the erythromycin resistance methylase gene (ermB). The statistically significant factors associated with C. difficile infection (CDI) induced by A-B+ isolates with MLSB resistance included a severity score of >2 (odds ratio [95% confidence interval], 7.43 [2.31-23.87]) and platelet count (cells × 109 cells/L) < 100 [5.19 (1.58-17.04)]. The proportion of A-B+ increased with enhanced CDI severity (x2 = 21.62, P < 0.001), which was significantly higher than that of ermB-positive A+B+ in severity score of 4 (x2 = 8.61, P = 0.003). The average severity score of ermB-positive isolates was significantly higher than that of ermB-negative isolates in A-B+ (Z = -2.41, P = 0.016). CONCLUSION: The ermB-positive A-B+ C. difficile with MLSB resistance is described for the first time as a potential epidemic clone inducing severe CDI in CA diarrheal patients in Eastern China.

Different molecular characteristics and antimicrobial resistance profiles of Clostridium difficile in the Asia-Pacific region.

Molecular epidemiology of Clostridium difficile infection (CDI) has been extensively studied in North America and Europe; however, limited data on CDI are available in the Asia-Pacific region. A multicentre retrospective study was conducted in this region. C. difficile isolates were subjected to multilocus sequence typing (ST) and antimicrobial susceptibility testing. Totally, 394 isolates were collected from Hangzhou, Hong Kong, China; Busan, South Korea; Fukuoka, Japan; Singapore; Perth, Sydney, Australia; New York, the United States. C. difficile isolates included 337 toxin A-positive/B-positive/binary toxin-negative (A+B+CDT-), 48 A-B+CDT-, and nine A+B+CDT+. Distribution of dominant STs varied geographically with ST17 in Fukuoka (18.6%), Busan (56.0%), ST2 in Sydney (20.4%), Perth (25.8%). The antimicrobial resistance patterns were significantly different among the eight sites (χ2 = 325.64, p < 0.001). Five major clonal complexes correlated with unique antimicrobial resistances. Healthcare-associated (HA) CDI was mainly from older patients with more frequent antimicrobial use and higher A-B+ positive rates. Higher resistance to gatifloxacin, tetracycline, and erythromycin were observed in HA-CDI patients (χ2 = 4.76-7.89, p = 0.005-0.029). In conclusion, multiple C. difficile genotypes with varied antimicrobial resistance patterns have been circulating in the Asia-Pacific region. A-B+ isolates from older patients with prior antimicrobial use were correlated with HA-CDI.

Evaluation of an UltraFast LabChip V280 assay for detection of toxigenic Clostridium difficile.

In this study, we compared the performance of an UltraFast LabChip (UL) V280 system for Clostridium difficile detection in stool with that of Xpert C. difficile/Epi and VIDAS CDAB. Among 176 stool specimens, UL V280 detected toxigenic C. difficile in 22 (22/176, 12.5%) with a sensitivity, specificity, positive predictive value, negative predictive value (NPV) of 100.0%, 99.4%, 99.5% and 100.0%, respectively, which were higher than 95.2%, 97.4%, 83.3%, and 99.3% of Xpert C. difficile/Epi (P > 0.05). Notably, the sensitivity and NPV of ULV280 were significantly higher than those of VIDAS CDAB 52.4% (P < 0.001, odds ratio [OR] = 20.0, 95% confidence interval [CI] = 2.26-176.81) and 93.8% (P = 0.002, OR = 10.27, 95% CI = 1.30-81.17). UL V280 turnaround time (35 min) and cost (6.24 Dollars [$]) per specimen were less than those for Xpert C. difficile/Epi (47 min, 59.26 $) and VIDAS CDAB (65 min, 11.70 $). UL V280 possessed an analytical sensitivity limit of 2500 CFU/ml, 95% [CI] = (Ct: 30.76-34.90), and no cross-reactions with other pathogens were found. The study demonstrates that UL V280 based on a microfluidic chip is a rapid, accurate, easy, and cost-effective diagnostic test for toxigenic C. difficile in stool.

Coinfection with 2 Clostridium difficile ribotypes in China: A case report.

RATIONALE: Clostridium difficile infections (CDIs) have been reported in China, but detailed clinical symptoms of coinfection by 2 C difficile ribotypes have not been documented. PATIENTS CONCERNS: An 83-year-old male with a 10-day history of diarrhea and urinary tract infection was admitted to the hospital. The patient had received ofloxacin for several days, but his clinical response was poor. Laboratory workup revealed high white blood cell (WBC), serum creatinine (Scr), and C-reactive protein (CRP) levels. Based on these abnormal lab results, rapid detection of glutamate dehydrogenase and toxin A and B was performed. DIAGNOSIS: Severe CDI. INTERVENTIONS: Oral vancomycin was administered for 8 days. OUTCOMES: Diarrhea symptoms improved and C difficile culture was negative after oral vancomycin administration for 8 days. Clostridium difficile was isolated from 3 consecutive stool samples at 2-day intervals because the patient was admitted to the hospital. Polymerase chain reaction ribotyping revealed ribotype (RT) 017 in the first 2 samples and RT 001 in the third sample. RT 017 caused significantly higher increases in the levels of WBC, Scr, and CRP than RT 001. LESSONS: It is necessary to improve clinicians' awareness of CDI and reduce the severity of CDI caused by RT 017 in China.

Simultaneous detection and characterization of toxigenic Clostridium difficile directly from clinical stool specimens.

We employed a multiplex polymerase chain reaction (PCR) coupled with capillary electrophoresis (mPCR-CE) targeting six Clostridium difficile genes, including tpi, tcdA, tcdB, cdtA, cdtB, and a deletion in tcdC for simultaneous detection and characterization of toxigenic C. difficile directly from fecal specimens. The mPCR-CE had a limit of detection of 10 colony-forming units per reaction with no cross-reactions with other related bacterial genes. Clinical validation was performed on 354 consecutively collected stool specimens from patients with suspected C. difficile infection and 45 isolates. The results were compared with a reference standard combined with BD MAX Cdiff, real-time cell analysis assay (RTCA), and mPCR-CE. The toxigenic C. difficile species were detected in 36 isolates and 45 stool specimens by the mPCR-CE, which provided a positive rate of 20.3% (81/399). The mPCR-CE had a specificity of 97.2% and a sensitivity of 96.0%, which was higher than RTCA (x2 = 5.67, P = 0.017) but lower than BD MAX Cdiff (P = 0.245). Among the 45 strains, 44 (97.8%) were determined as nonribotype 027 by the mPCR-CE, which was fully agreed with PCR ribotyping. Even though ribotypes 017 (n = 8, 17.8%), 001 (n = 6, 13.3%), and 012 (n = 7, 15.6%) were predominant in this region, ribotype 027 was an important genotype monitored routinely. The mPCR-CE provided an alternative diagnosis tool for the simultaneous detection of toxigenic C. difficile in stool and potentially differentiated between RT027 and non-RT027.

Clostridium difficile colonization in preoperative colorectal cancer patients.

The entire process of Clostridium difficile colonization to infection develops in large intestine. However, the real colonization pattern of C. difficile in preoperative colorectal cancer patients has not been studied. In this study, 33 C. difficile strains (16.1%) were isolated from stool samples of 205 preoperative colorectal cancer patients. C. difficile colonization rates in lymph node metastasis patients (22.3%) were significantly higher than lymph node negative patients (10.8%) (OR=2.314, 95%CI=1.023-5.235, P =0.025). Meanwhile, patients positive for stool occult blood had lower C. difficile colonization rates than negative patients (11.5% vs. 24.0%, OR=0.300, 95%CI=0.131-0.685, P =0.019). A total of 16 sequence types were revealed by multilocus sequence typing. Minimum spanning tree and time-space cluster analysis indicated that all C. difficile isolates were epidemiologically unrelated. Antibiotic susceptibility testing showed all isolates were susceptible to vancomycin and metronidazole. The results suggested that the prevalence of C. difficile colonization is high in preoperative colorectal cancer patients, and the colonization is not acquired in the hospital. Since lymph node metastasis colorectal cancer patients inevitably require adjuvant chemotherapy and C. difficile infection may halt the ongoing treatment, the call for sustained monitoring of C. difficile in those patients is apparently urgent.

Molecular Epidemiology of Clostridium difficile Infection in Hospitalized Patients in Eastern China.

Few studies on risk factors for and transmission of Clostridium difficile infection (CDI) in China have been reported. A cross-sectional study was conducted for 3 years in eastern China. Consecutive stool specimens from hospitalized patients with diarrhea were cultured for C. difficile. C. difficile isolates from these patients then were analyzed for toxin genes, genotypes, and antimicrobial resistance. A severity score for the CDI in each patient was determined by a blinded review of the medical record, and these scores ranged from 1 to 6. A total of 397 out of 3,953 patients (10.0%) with diarrhea were found to have CDI. Severity of CDI was mild to moderate, and the average (± standard deviation) severity score was 2.61 ± 1.01. C. difficile was isolated from stool specimens in 432 (10.9%) of all the patients who had diarrhea. C. difficile genotypes were determined by multilocus sequence analysis and PCR ribotyping; sequence type 37 (ST37)/ribotype 017 (RT017) (n = 68, 16.5%) was the dominant genotype. Eleven patients (16.2%) with this genotype had a CDI severity score of 5. Overall, three RTs and four STs were predominant; these genotypes were associated with significantly different antimicrobial resistance patterns in comparison to all genotypes (χ2 = 79.56 to 97.76; P < 0.001). Independent risk factors associated with CDI included age greater than 55 years (odds ratio [95% confidence interval], 26.80 [18.76 to 38.29]), previous hospitalization (12.42 [8.85 to 17.43]), previous antimicrobial treatment within 8 weeks (150.56 [73.11 to 310.06]), hospital stay more than 3 days before sampling (2.34 [1.71 to 3.22]), undergoing chemotherapy (3.31 [2.22 to 4.92]), and undergoing abdominal surgery (4.82 [3.54 to 6.55]). CDI is clearly a problem in eastern China and has a prevalence of 10.0% in hospitalized patients. Among risk factors for CDI, the advanced age threshold was younger for Chinese patients than that reported for patients in developed countries.

US Gulf-like toxigenic O1 Vibrio cholerae causing sporadic cholera outbreaks in China.

OBJECTIVES: Cholera is potentially a life threatening disease caused by toxigenic Vibrio cholerae. Here we report the identification and characterisation of 76 non-7th pandemic clone O1 V. cholerae isolates including 65 clinical isolates from diarrhoeal patients from 2005 to 2014 in Zhejiang Province, China. METHODS: We used multilocus sequence typing (MLST) to characterise 65 V. cholerae isolates. Pulse-Field Gel Electrophoresis (PFGE) was performed on a subset of the isolates and whole-genome sequencing was done on 13 isolates. RESULTS: MLST separated 65 isolates into 19 sequence types (STs). Thirty three isolates belonged to ST75 which also contains the US Gulf Coast clone. PFGE separated the 33 isolates into 16 pulsotypes. Whole genome sequencing of 10 ST75 isolates showed that the US Gulf Coast clone and the Chinese ST75 isolates can be separated into two distinct lineages, ST75a and ST75b. All Zhejiang ST75 isolates were ST75b. CONCLUSION: PFGE and genome sequencing confirmed the linked cases and identified small outbreaks caused by ST75b. The emergence and potential spread of ST75b may pose significant threat to public health. Epidemiological surveillance is required to further understand its epidemic potential.

Genome Sequence and Analysis of Peptoclostridium difficile Strain ZJCDC-S82.

Peptoclostridium difficile (Clostridium difficile) is the major pathogen associated with infectious diarrhea in humans. Concomitant with the increased incidence of C. difficile infection worldwide, there is an increasing concern regarding this infection type. This study reports a draft assembly and detailed sequence analysis of C. difficile strain ZJCDC-S82. The de novo assembled genome was 4.19 Mb in size, which includes 4,013 protein-coding genes, 41 rRNA genes, and 84 tRNA genes. Along with the nuclear genome, we also assembled sequencing information for a single plasmid consisting of 11,930 nucleotides. Comparative genomic analysis of C. difficile ZJCDC-S82 and two other previously published strains, such as M120 and CD630, showed extensive similarity. Phylogenetic analysis revealed that genetic diversity among C. difficile strains was not influenced by geographic location. Evolutionary analysis suggested that four genes encoding surface proteins exhibited positive selection in C. difficile ZJCDC-S82. Codon usage analysis indicated that C. difficile ZJCDC-S82 had high codon usage bias toward A/U-ended codons. Furthermore, codon usage patterns in C. difficile ZJCDC-S82 were predominantly affected by mutation pressure. Our results provide detailed information pertaining to the C. difficile genome associated with a strain from mainland China. This analysis will facilitate the understanding of genomic diversity and evolution of C. difficile strains in this region.

Real-time cell analysis for monitoring cholera toxin-induced human intestinal epithelial cell response.

The pathogenic mechanism of Vibrio cholerae manifests as diarrhea and causes life-threatening dehydration. Here, we observe the human intestinal epithelial cells (HIEC) response to Cholera toxin (CT) by a real-time cell analysis (RTCA) platform, and disclose the difference from CT-induced cytotoxicity and others in HIEC. An HIEC cell of 1.0 × 10(5) cells/mL was characterized as the suitable concentration for each well. For experimentation, the assay requires an inoculation of CT dissolved in Dulbecco's phosphate-buffered saline with 0.1 % gelatin for a period of 18-25 h. The dimensionless impedance cell index curve presented characteristic dose- and time-dependent drop responses at the first stage, and the CT-induced cytotoxicity was the most remarkable following exposure for 18-25 h (P = 0.0002). Following the obvious cytotoxic reaction, the CI curve gradually increased over time until the original CI value, indicating that self-recovery occurred. The CT-induced CI curve for HIEC was different from that induced by other toxins, including diphtheria and Clostridium difficile toxin. Collectively, these results suggest that the CT-induced cytotoxicity in HIEC was absolutely different from that induced by C. difficile and other toxins because of the different pathogeneses that were correlated with the specific CI curve generated by the RTCA system. In summary, our data show that the assay described here is a convenient and rapid high-throughput tool for real-time monitoring of host cellular responses to CT on the basis of the characteristic CI curve.

Molecular analysis of non-O1/non-O139 Vibrio cholerae isolated from hospitalised patients in China.

BACKGROUND: Cholera is still a significant public health issue in developing countries. The aetiological agent is Vibrio cholerae and only two serogroups, O1 and O139, are known to cause pandemic or epidemic cholera. In contrast, non-O1/non-O139 V. cholerae has only been reported to cause sporadic cholera-like illness and localised outbreaks. The aim of this study was to determine the genetic diversity of non-O1/non-O139 V. cholerae isolates from hospitalised diarrhoeal patients in Zhejiang Province, China. RESULTS: In an active surveillance of enteric pathogens in hospitalised diarrhoeal patients, nine non-O1/non-O139 V. cholerae isolates were identified from 746 diarrhoeal stool samples at a rate of 1.2%. These isolates and an additional 31 isolates from sporadic cases and three outbreaks were analysed using pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). PFGE divided the isolates into 25 PFGE types while MLST divided them into 15 sequence types (STs). A single ST, ST80, was predominant which persisted over several years in different cities and caused two outbreaks in recent years. Antibiotic resistance varied with the majority of the isolates resistant to sulphamethoxazole/trimethoprim and nearly all isolates either resistant or intermediate to erythromycin and rifampicin. None of the isolates carried the cholera toxin genes or toxin co-regulated pilus genes but the majority carried a type III secretion system as the key virulence factor. CONCLUSIONS: Non-O1/non-O139 V. cholerae is an important contributor to diarrhoeal infections in China. Resistance to commonly used antibiotics limits treatment options. Continuous surveillance of non-O1/non-O139 V. cholerae is important for control and prevention of diarrhoeal infections.

Rapid molecular identification of Listeria species by use of real-time PCR and high-resolution melting analysis.

Identification of Listeria species via a molecular method is critical for food safety and clinical diagnosis. In this study, an assay integrating real-time quantitative PCR (Q-PCR) with high-resolution melting (HRM) curve analysis was developed and assessed for rapid identification of six Listeria species. The ssrA gene, which encodes a transfer-messenger RNA (tmRNA) is conserved and common to all bacterial phyla, contains a variable domain in Listeria spp. Therefore, Q-PCR and a HRM profile were applied to characterize this gene. Fifty-three Listeria species and 45 non-Listeria species were detected using one primer set, with an accuracy of 100% in reference to conventional methods. There was a 93.3% correction rate to 30 artificially contaminated samples. Thus, Q-PCR with melting profiling analysis proved able to identify Listeria species accurately. Consequently, this study demonstrates that the assay we developed is a functional tool for rapidly identifying six Listeria species, and has the potential for discriminating novel species food safety and epidemiological research.

[Development of multiplex real time PCR methodology for better identification and discrimination of Vibrio cholerae and O139 serotype].

OBJECTIVE: To develop a rapid, sensitive and specific assay method, based on multiplex real time PCR for identifying Vibrio cholerae and distinguishing Vibrio cholerae O139 serotype from Vibrio cholerae. METHODS: Cholera toxin A subunit gene (ctxA) and glycosyltransferase gene (LPSgt) were chosen as targets according to Vibrio cholerae and Vibrio cholerae O139 serotype, and then the primers and TaqMan-MGB probe were designed. The 5'end of probes was labeled with FAM and VIC fluoresceins respectively while the 3'end of probes was labeled with MGB. The PCR reaction was optimized systematically. Then the specificity, sensitivity and reproducibility of multiplex real time PCR were estimated. Finally, multiplex real time PCR was applied to detect the clinical specimens. RESULTS: Vibrio cholerae was identified by multiplex real time PCR accurately and quickly, which could distinguish Vibrio cholerae O139 serotype from Vibrio cholerae. Vibrio cholerae was identified positive for primer pairs and probes from ctxA gene, and Vibrio cholerae O139 serotype tested was positive for LPSgt gene. Meanwhile, none of other bacteria was identified. Sensitivity of the test was 2 × 10(2) cfu/ml. When this assay was applied directly to identify 45 clinical specimens, results showed that 10 were positive to Vibrio cholerae, in which 4 clinical specimens were positive to Vibrio cholerae O139 serotype. All the results were the same to the one that had been obtained from the conventional assays. CONCLUSION: Our results showed that the multiplex real time PCR was a reliable, accurate and feasible method for identifying Vibrio cholerae that carrying toxin gene and distinguishing Vibrio cholerae O139 serotype from Vibrio cholerae. This method could be applied to the cholera surveillance, prevention and control system for identifying and distinguishing Vibrio cholerae in the labs.

[Detection of co-infection with Lyme spirochetes and Spotted fever group rickettsiae in a group of Haemaphysalis longicornis].

OBJECTIVE: The present study was conducted to investigate the infection of Lyme disease, Spotted fever, Ehrlichiosis (anaplasmosisin) in wild animals and ticks in the mountain areas of Zhejiang province. METHODS: Nested polymerase chain reaction was used to amplify specific DNA sequences of Lyme spirochetes, Spotted fever group rickettsiae, Ehrlichia(anaplasma) from samples of mice and ticks. RESULTS: 14 positive samples were identified from 121 mice and 105 groups of ticks. Among mice samples, one positive 5S-23S rDNA intergenic spacer of Borrelia burgdorferi and two 5' fragments of Ehrlichia (anaplasma) 16S rDNA were obtained. 11 positive results were detected from tick samples including three 5S-23S rDNA intergenic spacer regions of Borrelia burgdorferi and eight 5' fragments of Spotted fever group rickettsiae outer member protein A gene. One group of adult ticks, Haemaphysalis longicornis, which had been collected from eastern mountain area were detected to have co-infected with Lyme spirochetes and Spotted fever group rickettsiae. The positive sequences of 5S-23S rDNA intergenic spacer and ompA gene were tested and analyzed as Lyme spirochetes while rickettsia which was closely related to Borrelia valaisiana and R. massiliae. CONCLUSION: This was the first report about co-infection of Lyme spirochetes and Spotted fever group rickettsiae found in the same group of adult Haemaphysalis longicornis. It is very important to strengthen the surveillance program on tick-borne infectious disease and their pathogenic in vectors, wild animals and targeted high risk groups and to differentiate the clinical manifestation and diagnosis to extend the knowledge of tick-borne infectious diseases in Zhejiang.