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Objective:To observe the stability of severe acute respiratory syrdrome coronavirus （SARS-CoV-2） in cell cultures at different temperatures so as to provide basic data and scientific basis for the research and control of COVID-19 epidemic. Methods:The Vero E6 cells inoculated with SARS-CoV-2. According to TCID50， SARS-CoV-2 with different dilution （10-1， 10-3， 10-5， 10-6）were stored at 37 °C， 22.5 °C， and 4 °C for one to seven days， and then infectious titer was determined by micro cytopathogenic effect assay， observing cytopathic effect （CPE）， and real-time fluorescence quantitative testing. Results:SARS-CoV-2 was stable under 4 °C. The infectivity of high concentration （10-1 dilution） under 22.5 °C for seven days gradually decreased， while lower concentration completely lost infectivity after one day. The virus lost infectivity when stored at 37 °C for more than one day. Conclusion:SARS-CoV-2 is highly stable at 4 °C， sensitive to heat， and related to virus concentration.
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Objective:To determine the distribution and epidemic characteristics of Vibrio parahaemolyticus in Jinshan District of Shanghai from 2016 through 2018. Methods:Serotype analysis，examination of virulence genes， including thermolabilehemolysin（TLH），thermostable direct hemolysin（TDH），and TDH related hemolysin（TRH），and antimicrobial susceptibility test and pulsed field gel electrophoresis （PFGE） molecular typing were performed on 218 Vibrio parahaemolyticus isolated from diarrhea patients. Results:A total of 218 strains were divided into 8 groups and 21 serotypes. Of them， 147 strains were serotyped，with O3：K6 as the most common serotype （57.3%）. All tested strains were divided into 25 clusters based on the similarity of 85% and above，in which the dominant cluster was JSVP02，and the total similarity was 56.3%-100.0%. Two hundred and one strains （92.2%） carried tdh gene，13 strains （6.0%） carried trh gene，and 7 strains were negative for both tdh and trh. A total of 35 strains were completely sensitive to 17 kinds of antibiotics，while the remaining 183 strains showed different drug resistance. Conclusion:Vibrio parahaemolyticus isolated from diarrhea patients in Jinshan District from 2016 through 2018 is diverse. Majority of the strains have TDH gene and are resistant to the first generation cephalosporins such as cefazolin and penicillin ampicillin. Construction of regional PFGE molecular typing database will facilitate screening，identification and early warning of high-risk strains.
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Objective:To investigate the antimicrobial resistance characteristics of carbapenem-resistant Enterobacteriaceae （CRE） isolated from outpatients with diarrhea in Shanghai， and provide support for surveillance， prevention and control of CRE. Methods:A total of 800 fecal swabs of the outpatients with diarrhea were collected from 23 sentinel hospitals for diarrhea pathogen surveillance in Shanghai from January 2018 to December 2019. The drug-resistant strains were isolated using MacConkey plates containing 1 μg/μL meropenem. The collected strains were identified preliminarily by the VITEK-2 Compact system and VITEK mass spectrometry. The minimum inhibitory concentration （MIC） of the strain was determined by the broth microdilution method. The multi-locus sequence typing （MLST） method and pulsed-field gel electrophoresis （PFGE） were used to analyze the homology of drug-resistant strains. The transferability of the resistance gene was investigated by a junction experiment. High-throughput sequencing was used to characterize the isolates. Results:Seven non-repetitive CRE isolates were multi-drug resistant carbapenem-resistant Escherichia coli （CREC） strains that produce New Delhi metallo-β-lactamase （NDM） with resistance to several commonly used antibiotics in clinical therapy. The molecular typing results showed that the CRE strains had different sequence types， and diverse PFGE patterns. The stains were all positive for blaNDM genes， including blaNDM-5 and blaNDM-13， with blaNDM-5 as the main type. The carbapenem-resistant genes could be transferred to EC600 by conjugation. Conclusion:The intestinal carbapenem-resistant strains in this study are all NDM-producing Escherichia coli. The isolates carried blaNDM and other resistance genes. The MLST analysis showed that they belonged to different cloning types. Antimicrobial resistance genes could be horizontally transferred to EC600 by conjugation.
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[ Objective] To investigate the status quo of Campylobacter spp.infection in Shanghai and study its molecular characteristics and virulence and toxin genes. [ Methods ] Stool samples collected from diarrheal patients were cultured for bacterial pathogens using membrane filter method.The strains were identified by biochemical tests and PCR.PCR was applied to detect six virulence and toxin genes including flaA,cdtA,cdtB,cdtC,virB11,cadF.Pulsed-field gel electrophoresis ( PFGE) was carried out for subtyping. [Results] A total of 43 Campylobacter spp.(1.9%) were collected from 2 235 stool samples in Shanghai in 2014 including 41 Campylobacter jejuni isolates(95.3%) and 2 Campylobacter coli isolates(4.7%) .The data showed 100.0%(43/43) of the isolates were positive for flaA and cadF, and 93.0%(40/43) of the isolates positive for cdtA and cdtB.And 88.4%(38/43) of the isolates were posi-tive for cdtC.Only 7.0%(3/43) of the isolates were positive for virB11.Using PFGE, 43 Campylobacter jejuni and Campylobacter coli strains were subtyped into 6 clusters. [ Conclusion] The genes of flaA and cadF are ubiquitous on Campylobacter spp.isolates.The distribution of cdt gene cluster in Campylobacter spp.is high, while that of virB11 is low.The PFGE types of Campylobacter spp.isolated in Shanghai are of diversified and complicated features, which causes mainly sporatic diarrhea.
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Objective ] To study the molecular characteristics and antibiotic resistance of Salmonella diarrhea cases in sentinel hospitals through active surveillance system conducted by public health laboratory. [ Methods] Two sentinel hospitals were chosen for collection of stool specimens from food-borne infectious diarrhea cases and for Salmonella separation and detection immediately following serotyping and antimicrobial susceptible test ( AST ) on those isolates .Moreover , pulsed field gel electrophoresis ( PFGE) was used for the genetic homology analysis . [ Results] A total of 2 579 diarrhea specimens were collected and analyzed from 2010 to 2012, with 185 Salmonella isolates, covering 23 different serotypes (annual positive rates were 9.1%, 6.8%, 5.1%, with an average 7.2%).Salmonella enterica serovar Enteritidis(S.Enteritidis) and Typhimurium(S.Typhimurium) were the most common serotypes, of which 68.9% cases were seen in those aged 21 to 60 and 21.4% cases in those over 60 years old. 27.7%-96.9%S.Enteritidis and 2.6%-63.2% S.Typhimurium(P all <0.05) proved resistant to Nalidixic acid, Sultisoxazole, Streptomycin, Sulfamethoxydiazine, Gentamicinand Tetracycline. PFGE analysis on 22 S.Enteritidis strains showed 11 different clusters , while 20 S.Typhimurium strians showed 6. [Conclusion] S.Enteritidis and S.Typhimurium are the most common Salmonella serotypes, and molecular typing indicates the existence of clustering and sporadic outbreaks caused by dominance clones . We should be alert to early warnings on potential outbreaks of multiple-drug-resistant ( MDR ) S.Enteritidis.Active surveillance system based on public health laboratory should play an important role in the control of food-borne infectious diseases .
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To establish a new method for PFGE fingerprint analysis , which is easy-to-operate , inexpensive , software-low-dependent and readily-exchangeable . [ Methods ] A group of 44 vibrio parahaemolyticus was subtyped according to PulseNet standardized PFGE protocol .The fingerprint was analyzed with new gel-partition bands counting method and BioNumerics clustering separately and then the results by the two methods were compared with type number , cluster number and Simpson ’ s Index of Diversity ( DI) . [ Results] The 44 isolates could be typed into 32 types and 5 clusters were found by gel-partition bands counting method ( DI=97.6%) .BioNumerics could type the same isolates into 29 types and 4 clusters were found ( DI=95.5%) .The difference in the ability of finding clusters between the two methods was not statistically significant . [ Conclusion] Gel-partition bands counting method is based on the bands distribution among finger print and greatly reduces the number of visually observed spectrum , which is easy-to-operate, inexpensive , software-low-dependent and readily-exchangeable .
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Objective To describe the phenotype and molecular characteristics of Vibrio (V.) cholerae strains isolated in Shanghai,from 1962 to 2011.Methods K-B test was used to investigate the antibiotic resistance of V.cholerae strains.PCR was applied to detect seven virulence-related genes including cholera toxin (ctxA),zonula oecludens toxin (zot),accessory cholera enterotoxin (ace),hemolysin (hlyA),toxin-coregulated pilus (tcpA) outer membrane protein (ompU) and the regulatory protein genes (toxR).Genetic relation was assessed by pulsed-field gel electrophoresis (PFGE) and the patterns were clustered by BioNumerics software.Results V.cholerae strains isolated from 1962 to 1996 were sensitive to most of the antibiotics.However,the strains isolated from 2005 to 2011 were resistant to many antibiotics.V.cholerae O 139 group showed higher prevalence of resistance to several antibiotics compared with O l group,and the resistance rate of the O139 toxigenic isolates was higher than that of the non-toxigenic isolates.Most of the O1 strains isolated from 2005 to 2011 were non-toxigenic while O139 strains isolated from 2005 to 2011 were almost toxigenic.There were no strains ofctxA+ detected from the rivers from 2005 to 2011.Main gene type of the O1 strains detected from the aquatic products was hlyA+toxR+ompU+,while that of the O139 strains was hlyA+toxR+ompU+ ctxA + ace +zot + tcpA +.Using PFGE,222 V.cholerae strains were subtyped into 121 molecular types.O139 strains were divided to three clusters and O1 strains to five clusters.Conclusion The characteristics of V.cholerae strains isolated in Shanghai from 1962 to 2011 showed great changes,suggesting that more attention should be paid to the multiplication on antibiotic resistance of V.cholerae strains.
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<p><b>OBJECTIVE</b>To determine whether Acanthamoeba polyphaga could affect the survival and growth of Vibrio cholerae O139 in low temperature.</p><p><b>METHODS</b>V. cholerae O139 was co-cultured with the Acanthamoeba polyphaga to be examined on its intracellular growth and survival rate within cysts at low temperature, using methods as Gram-staining, electron microscope and passage culture.</p><p><b>RESULTS</b>V. cholerae O139 were observed to enter into the trophozoites and grow the within the vacuoles after 8 hour incubation with Acanthamoeba polyphaga. The germs survived in the vacuole and/or endo-layer of wall and could be re-isolated from the excystment of Acanthamoeba polyphaga. At 30 degrees C, V. cholerae O139 could survive for 120 days with the amoeba while less than 45 days in PAS. At 4 degrees C, the number of viable bacteria decreased and reached undetectable levels for both study and control groups after a 30-day incubation. V. cholerae O139 could be re-isolated from the 30-, 45-, 60- and 75-day's infected cysts after excystment. However the ability of excystment for 90-day's infected cysts decreased and V. cholerae O139 within the cyst could not be isolated again because the amoebae had lysed.</p><p><b>CONCLUSION</b>These findings indicated that V. cholerae O139 could grow within Acanthamoeba polyphaga and the survival time could be increased in the cysts at low temperature. It seemed that Acanthamoeba can provide an environmental reservoir for V. cholerae O139.</p>