Drug resistance and genomic characteristics of Salmonella enterica serovar London from clinical and food sources in Hangzhou City from 2017 to 2021 / 中华预防医学杂志

Objective: To analyze the drug resistance and genomic characteristics of Salmonella enterica serovar London isolated from clinical and food sources in Hangzhou City from 2017 to 2021. Methods: A total of 91 Salmonella enterica serovar London strains isolated from Hangzhou City from 2017 to 2021 were analyzed for drug susceptibility, pulsed field gel electrophoresis (PFGE) typing and whole genome sequencing. Multilocus sequence typing (MLST), core genome multilocus sequence typing (cgMLST) and detection of drug resistance genes were performed by using the sequencing data. Phylogenetic analysis was conducted to compare the 91 genomes from Hangzhou City with 347 genomes from public databases. Results: No significant difference in the drug resistance rate was observed between clinical strains and food strains to 18 drugs in Hangzhou City(all P>0.05), and the multidrug resistance (MDR) rate was 75.8% (69/91). Most strains were resistant to 7 drug classes simultaneously. One strain was resistant to Polymyxin E as well as positive for mcr-1.1, and 50.5% (46/91) of the strains were resistant to Azithromycin and were positive for mph(A). All 91 Salmonella enterica serovar London strains were ST155, which were subdivided into 44 molecular types by PFGE and 82 types by cgMLST. Phylogenetic analysis showed that most strains from Hangzhou City (83/91) were clustered together, and a small number of human isolates from Europe, North America and pork isolates from Hubei and Shenzhen were mixed in the cluster. Other strains from Hangzhou City (8/91) were closely related to strains from Europe, America and Southeast Asia. Strains isolated from pork were the most closely related to clinical strains. Conclusion: The epidemic of Salmonella enterica serovar London in Hangzhou City is mainly caused by the spread of ST155 strains, which is mainly transmitted locally. At the same time, cross-region transmission to Europe, North America, Southeast Asia, and other provinces and cities in China may also occur. There is no significant difference in the drug resistance rate between clinical strains and food strains, and a high level of MDR is found in the strains. Clinical infection of Salmonella enterica serovar London may be closely related to pork consumption in Hangzhou City.

Molecular characteristics and antibiotic resistances of Vibrio cholerae O1 isolates in Hangzhou in 2009 / 中华预防医学杂志

<p><b>OBJECTIVE</b>To study the molecular characteristics and antibiotic resistances of Vibrio cholerae (V. cholerae) O1 isolates in Hangzhou in 2009.</p><p><b>METHODS</b>The virulence genes ctxA and tcpA of the thirty V. cholerae O1 isolates from 7 counties and districts of Hangzhou were detected by PCR. Pulsed-field gel electrophoresis (PFGE) was performed for molecular typing and similarity analysis. Antibiotic resistances of these isolates were measured by the Kirby-Bauer method.</p><p><b>RESULTS</b>Virulence gene analysis showed that 80.00% (24/30) of the genotype in V. cholerae isolates was ctxA- and tcpA+, 13.33% (4/30) was ctxA- and tcpA-, and 6.67% (2/30) was ctxA+ and tcpA+. Twenty-seven isolates tested were typed into 11 PFGE patterns (P1-P11). Twenty-three isolates with genotype ctxA- and tcpA+ were clustered into 7 PFGE patterns (P1-P7, termed P1-like cluster) with the similarity to be equal or greater than 91.4%, and 56.52%(13/23) of them belong to P1. 7 isolates with very high similarity (97.6%), belonging to P1 (6 isolates), and P2 (1 isolate), respectively, were collected from one foodborne disease outbreak. The resistant rates of the 24 isolates with genotype ctxA- and tcpA+ to ampicillin, tobramycin and amikacin were 20.83% (5/24), 4.17% (1/24) and 4.17% (1/24), respectively.</p><p><b>CONCLUSION</b>The genotype of the epidemic strains of V. cholerae O1 isolates in Hangzhou in 2009 with high similarity was ctxA- and tcpA+; The level of drug resistances of this kind of V. cholerae O1 isolates were not high.</p>

Multiplex PCR assay for dissemination and diversity of extended-spectrum beta-lactamase genes in Shigella isolates / 中华预防医学杂志

<p><b>OBJECTIVE</b>To develop a rapid and simple multiplex polymerase chain reaction (PCR) method which discriminates extended-spectrum beta-lactamases (ESBLs) genes in sporadic Shigella isolates from 1998 to 2007 in Hangzhou city, China.</p><p><b>METHODS</b>After ESBLs screening according to the Clinical and Laboratory Standards Institute (CLSI) method, CTX-M, TEM, SHV and OXA-1 encoding genes were detected by using a multiplex PCR method, and the results were verified by 8 single gene PCR amplification.</p><p><b>RESULTS</b>Seventeen isolates harbored ESBLs genes among 195 Shigella isolates (8.72%). Genes encoding CTX-M (17 strains), TEM (2 strains), OXA-1 (10 strains) and SHV (0 strains) were discriminated with multiplex PCR analysis, which coincided with eight single gene PCR analysis at 94.12%.</p><p><b>CONCLUSION</b>Multiplex PCR should be a suitable tool for initial rapid screening and discriminating ESBLs genes in Shigella isolates. With similar trend of national surveillance data, the proportion of sporadic Shigella isolates harbouring ESBLs genes might probably be on increase.</p>

Multiplex real-time PCR detecting Salmonella, Shigella and diarrheagenic Escherichia coli / 中华预防医学杂志

<p><b>OBJECTIVE</b>To develop a multiplex real-time PCR for the detection of Salmonella invasion protein A gene (invA), enterotoxigenic Escherichia coli (ETEC) heat-labile I enterotoxin gene (elt), and Shigella or enteroinvasive E. coli (EIEC) invasive plasmid antigen H gene (ipaH).</p><p><b>METHODS</b>Under the optimized reaction conditions of the multiplex real-time PCR, invA, elt, and ipaH were determined in 10-fold series of dilution of DNA extracted from Salmonella enterica serovar Typhimurium, ETEC 44815 strain and Shigella F301 strain. The three genes were examined in 90 fecal samples from diarrhea patients using the multiplex real-time PCR. When PCR-positive samples were found, the target strains were isolated and identified.</p><p><b>RESULTS</b>The detectable concentration for this multiplex real-time PCR was 10 CFU/microl for Shigella F301 strain, 10(2) CFU/microl for S. enterica serovar Typhimurium and ETEC 44815 strain, respectively. Out of 90 fecal samples from diarrhea patients, thirteen were found positive for elt gene (14.4%), and five were found positive for ipaH gene (5.6%). Three E. coli strains positive for elt gene and four E. coli strains positive for ipaH gene were isolated successfully from the PCR-positive samples mentioned above. The detection of invA, elt and ipaH genes was completed in 10 h, which included an enrichment period of 6 h.</p><p><b>CONCLUSION</b>The multiplex real-time PCR assay can detect invA, elt, ipaH simultaneously in a single reaction, moreover, it can detect for virulence genes in strains of Salmonella, ETEC, and Shigella or EIEC and screen these pathogens in fecal specimens from patients with diarrhea with a high specificity.</p>

PFGE of Shigella flexneri 4c isolates from food-poisoning outbreaks and sporadic diarrhea patients / 中华预防医学杂志

<p><b>OBJECTIVE</b>To know the molecular characteristic of Shigella flexneri 4c isolates from patients in two food-poisoning outbreaks and one sporadic diarrhea case in Hangzhou, China.</p><p><b>METHODS</b>S. flexneri isolates from patients in two food-poisoning outbreaks (outbreak 1 and outbreak 2, n = 13 and n = 12, respectively) and one sporadic diarrhea patient (n = 1) in Hangzhou during 2003 and 2005 were serotyped. Antibiotic resistances of these isolates were measured by the Kirby-Bauer method. Invasive plasmid antigen gene ipaH was examined by PCR. Pulse field gel electrophoresis (PFGE) was performed for molecular typing.</p><p><b>RESULTS</b>In outbreak 1, all 13 isolates were S. flexneri 4c, of them 6 isolates tested were quite different in PFGE patterns with dice coefficient from 0.78 to 0.92. In outbreak 2, 10 isolates were S. flexneri 4c and 2 isolates were S. flexneri X, however their PFGE patterns were almost identical (dice coefficient > 0.8). Compared to the two outbreaks isolates, the sporadic isolate was demonstrated with a distinct PFGE pattern (dice coefficient < 0.8). The antibiotic resistance patterns with 14 kinds of antibiotics had a little difference among the isolates from outbreak 1, outbreak 2 and sporadic diarrhea patient, but the same pattern was found among 10 isolates of S. flexneri 4c and 2 isolates of S. flexneri X from outbreak 2.</p><p><b>CONCLUSIONS</b>PFGE might distinguish the isolates from these two outbreaks and the sporadic diarrhea patient. Some differences in PFGE patterns, serotypes and antibiotic resistance patterns might occur among S. flexneri 4c isolates during an outbreak.</p>

Detection of severe acute respiratory syndrome probable patients' virus RNA in Hangzhou by using a two loci and a modified nested real-time reverse transcription polymerase chain reaction / 中华预防医学杂志

<p><b>OBJECTIVE</b>To detect the RNA of severe acute respiratory syndrome virus (SARS-CoV) by using reverse transcription polymerase chain reaction (RT-PCR) targeted for a two loci and a modified nested real-time RT-PCR as to improving the reliability and sensitivity of tests.</p><p><b>METHODS</b>A nested RT-PCR was used for detecting one fragment of SARS-CoV RNA in oropharyngeal swabs from 3 SARS probable patients, 4 SARS suspect patients and other 27 patients with fever in Hangzhou, and the nested RT-PCR product from one SARS probable patient was sequenced. Meanwhile in these 3 SARS probable patients, other three RT-PCR methods, including a hemi-nested RT-PCR targeted for another fragment of SARS-CoV RNA, a real-time RT-PCR and a modified nested real-time RT-PCR, were employed to detect SARS-CoV RNA.</p><p><b>RESULTS</b>Two positives were found in the 3 SARS probable patients, and none positive in 4 SARS suspect patients and other 27 patients with fever, using the nested RT-PCR. The sequence of the nested RT-PCR product from one SARS probable patient was identified with the counterpart of SARS-CoV genomes published in public database. The results of the hemi-nested RT-PCR, the real-time RT-PCR and the modified nested real-time RT-PCR in the 3 SARS patients were consistent with the one of the nested RT-PCR. During detecting specimen with low copies of RNA, a weak positive signal was produced after about 35 cycles in the real-time RT-PCR, but a strong positive signal was found only after 10 cycles in the modified nested real-time RT-PCR.</p><p><b>CONCLUSION</b>It might improve the reliability of test by employing RT-PCR targeted for two or more fragments in SARS-CoV genome. The modified nested real-time RT-PCR might have higher sensitivity than the routine real-time RT-PCR.</p>

Development of Multiplex Real-time PCR for Detection of Toxigenic Vibrio cholerae and Virbio parahaemolyticus / 微生物学通报

A multiplex real-time PCR was developed to detect ctxA of Vibrio cholerae, gyrB and tdh of Vibrio parahaemolyticus simultaneously. The multiplex real-time PCR were evalidated by detection for the three genes in 47 toxigenic V. cholerae O1 and O139 strains (ctxA+; O1=3, O139=44), 25 non-toxigenic V. cholerae strains (ctxA-; O1=12, O139=6, non-O1 and non-O139=7), 116 V. parahaemolyticus strains with or without tdh (73 or 43) and 9 other bacteria strains. The specificity and sensitivity of the multiplex real-time PCR in detection for the ctxA and the tdh genes in the strains tested were both 100.0%, compared to the results by routine PCRs. In the detection for V. parahaemolyticus specific gyrB using the multiplex real-time PCR, all of 116 V. parahaemolyticus strains were positive, and 9 other strains and 72 V. cholerae strains were all negative. The multiplex real-time PCR is a sensitive, specific and quick assay not only for detecting virulence genes of V. cholerae and V. parahaemolyticus but also for identifying V. parahaemolyticus at species level. In addition, two real-time PCRs for detection of V. parahaemolyticus virulence genes trh1 and trh2 were also developed.