Dynamic PB2-E627K substitution of influenza H7N9 virus indicates the in vivo genetic tuning and rapid host adaptation.

Avian-origin influenza viruses overcome the bottleneck of the interspecies barrier and infect humans through the evolution of variants toward more efficient replication in mammals. The dynamic adaptation of the genetic substitutions and the correlation with the virulence of avian-origin influenza virus in patients remain largely elusive. Here, based on the one-health approach, we retrieved the original virus-positive samples from patients with H7N9 and their surrounding poultry/environment. The specimens were directly deep sequenced, and the subsequent big data were integrated with the clinical manifestations. Unlike poultry/environment-derived samples with the consistent dominance of avian signature 627E of H7N9 polymerase basic protein 2 (PB2), patient specimens had diverse ratios of mammalian signature 627K, indicating the rapid dynamics of H7N9 adaptation in patients during the infection process. In contrast, both human- and poultry/environment-related viruses had constant dominance of avian signature PB2-701D. The intrahost dynamic adaptation was confirmed by the gradual replacement of 627E by 627K in H7N9 in the longitudinally collected specimens from one patient. These results suggest that host adaptation for better virus replication to new hosts, termed "genetic tuning," actually occurred in H7N9-infected patients in vivo. Notably, our findings also demonstrate the correlation between rapid host adaptation of H7N9 PB2-E627K and the fatal outcome and disease severity in humans. The feature of H7N9 genetic tuning in vivo and its correlation with the disease severity emphasize the importance of testing for the evolution of this avian-origin virus during the course of infection.

Reverse transcription recombinase-aided amplification assay for rapid detection of the influenza A(H1N1)pdm09 H275Y mutation that confers oseltamivir resistance.

The emergence of the influenza A(H1N1)pdm09 virus with the NA-H275Y mutation, which confers oseltamivir resistance, must be monitored, especially in patients undergoing neuraminidase inhibitor treatment. In this study, we developed a reverse transcription recombinase-aided amplification assay that has high sensitivity (detection limit: 1.0 × 101 copies/µL) and specificity for detecting the oseltamivir-resistant H275Y mutation; the assay is performed within 30 min at a constant temperature of 39° Celsius using an isothermal device. This method is suitable for the clinical application of targeted testing, thereby providing technical support for precision medicine in individual drug applications for patients with severe infection or immunosuppression.

Epidemiological and Genomic Characterization of Campylobacter jejuni Isolates from a Foodborne Outbreak at Hangzhou, China.

BACKGROUND: Foodborne outbreaks caused by Campylobacter jejuni have become a significant public health problem worldwide. Applying genomic sequencing as a routine part of foodborne outbreak investigation remains in its infancy in China. We applied both traditional PFGE profiling and genomic investigation to understand the cause of a foodborne outbreak in Hangzhou in December 2018. METHOD: A total of 43 fecal samples, including 27 sick patients and 16 canteen employees from a high school in Hangzhou city in Zhejiang province, were recruited. Routine real-time fluorescent PCR assays were used for scanning the potential infectious agents, including viral pathogens (norovirus, rotavirus, adenovirus, and astrovirus), and bacterial pathogens (Salmonella, Shigella, Campylobacter jejuni, Vibrio parahaemolyticus and Vibrio cholerae). Bacterial selection medium was used to isolate and identify the positive bacteria identified by molecular test. Pulsed field gel electrophoresis (PFGE), and next generation sequencing (NGS) were applied to fifteen recovered C. jejuni isolates to further understand the case linkage of this particular outbreak. Additionally, we retrieved reference genomes from the NCBI database and performed a comparative genomics analysis with the examined genomes produced in this study. RESULTS: The analyzed samples were found to be negative for the queried viruses. Additionally, Salmonella, Shigella, Vibrio parahaemolyticus and Vibrio cholera were not detected. Fifteen C. jejuni strains were identified by the real-time PCR assay and bacterial selection medium. These C. jejuni strains were classified into two genetic profiles defined by the PFGE. Out of fifteen C. jejuni strains, fourteen have a unified consistent genotype belonging to ST2988, and the other strain belongs to ST8149, with a 66.7% similarity in comparison with the rest of the strains. Moreover, all fifteen strains harbored blaOXA-61 and tet(O), in addition to a chromosomal mutation in gyrA (T86I). The examined fourteen strains of ST2988 from CC354 clone group have very minimal genetic difference (3~66 SNPs), demonstrated by the phylogenomic investigation. CONCLUSION: Both genomic investigation and PFGE profiling confirmed that C. jejuni ST2988, a new derivative from CC354, was responsible for the foodborne outbreak Illustrated in this study.

The occurrence and distribution characteristics of Cronobacter in diverse cereal kernels, flour, and flour-based products.

Cronobacter was positive in cereals at a relatively high rate. In the present study, we investigated the occurrence and characteristics of this pathogen systematically in diverse cereals. All sampled food (N = 467) contained Cronobacter with a high positive rate of 54.0%. The enumeration experiment showed the concentration ranged from 0.3 to more than 110 MPN/100 g, and 87.9% of 127 samples were less than 10 MPN/100 g. There were significant differences (p < 0.05) in positive rates for Cronobacter among cereal kernels (40.2%), cereal flour (66.7%), cereal products made from raw cereal flour (87.6%), and cereal products made from flour (ready-to-eat) (17.4%). The dominant Cronobacter species was C. sakazakii and C. dublinensis, followed by C. malonaticus and C. turicensis. Two interesting clusters with more than 90% similarities were identified by pulsed-field gel electrophoresis (PFGE). The C1 cluster (four isolates) indicated these strains were derived from a common source and persisted in the food production environment for an extended time. The C2 cluster (six isolates) indicated the pathogen could be transmitted via cereal processing. Our research provided baseline data for Cronobacter in diverse cereals and was helpful for understanding Cronobacter transmission. The results also indicate that additional control measures should be developed to reduce the risk of infection by these opportunistic pathogenic bacteria.

Potential reservoirs and routes of Cronobacter transmission during cereal growing, processing and consumption.

Cronobacter are opportunistic bacterial pathogens of both infants and adults. We investigated the incidence and distribution of Cronobacter in 1245 samples of cereal and related environments. 39.1% (101/258) rice-related and 46.9% (98/209) wheat-related samples tested positive for Cronobacter, and the positive rate differed notably according to processing method. Cronobacter was found in rice and wheat plants at the tillering, filling and mature stages. Soil, water and swab samples from nearby milling plants were assayed, and results revealed that 6.3% (7/122) of water from paddy fields, 49.1% (28/57) and 62.1% (41/67) of swab samples from rice and wheat flour milling plants were Cronobacter positive. Pulsed-field gel electrophoresis (PFGE) subtyping indicated that some strains had a common profile, which suggested their persistence in the environment, potential transmission routes and cross-contamination in processing. Finally, we surveyed 18 families to evaluate potential risks. None of the families who primarily ate rice cooked with water tested positive for Cronobacter, though of 66.7% families (6/9) whose food staples were produced from wheat flour tested positive. Taken together, our results are important for understanding Cronobacter transmission and will aid in the development of additional control measures to reduce the risk of infection by these opportunistic pathogenic bacteria.

Associations between genetic mutations in different SARS-CoV-2 strains and negative conversion time of viral RNA among imported cases in Hangzhou: A cross-sectional study.

PURPOSE: Previous studies on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have focused on factors that influence the achievement of negative conversion of viral RNA. This study aimed to investigate the effects of the genetic mutations in different SARS-CoV-2 strains on the negative conversion time (NCT) among imported cases in Hangzhou, Zhejiang Province, China, in order to provide valuable insights for developing targeted epidemic prevention guidelines. METHODS: This retrospective study involved 146 imported SARS-CoV-2 cases in Hangzhou from 8 April 2021 to 11 June 2022. We compared the SARS-CoV-2-specific indicators, clinical indexes, and NCT among the wild-type (WT), Delta, and Omicron groups. Spearman correlation analysis was used to identify the correlations of NCT with mutation types/frequencies. RESULTS: The mean age of the imported cases was 35.3 (SD: 12.3) years, with 71.92 % males and 28.08 % females. The mean cycle threshold (Ct) values of open reading frame 1ab (ORF1ab) and nucleocapsid (N) RNA were 25.17 (SD: 6.44) and 23.4 (SD: 6.76), respectively. The mutations of SARS-CoV-2 strains were mainly located in N, membrane (M), spike (S), ORF1a, ORF1b, ORF3a, ORF6, and ORF9b genes among the WT, Delta, and Omicron groups. NCT was significantly prolonged in the WT and Delta groups compared to the Omicron group. T lymphocyte, white blood cell, eosinophil, and basophil counts were dramatically higher in the WT group than the Delta group. White blood cell, red blood cell, and basophil counts were significantly lower in the Delta group than the Omicron group. Spearman correlation analysis revealed a significant correlation between the NCT of viral RNA and mutation types of viral genes of WT and Omicron strains. Additionally, NCT was markedly negatively correlated with the frequencies of five mutations in Omicron strains (ORF1b:P1223L, ORF1b:R1315C, ORF1b:T2163I, ORF3a:T223I, and ORF6:D61L). CONCLUSIONS: This study indicates that five mutations in Omicron strains (ORF1b:P1223L/R1315C/T2163I, ORF3a:T223I and ORF6:D61L) shortened NCT in imported SARS-CoV-2 cases.

Population genomics implies potential public health risk of two non-toxigenic Vibrio cholerae lineages.

Diarrheal cases caused by non-toxigenic Vibrio cholerae have been reported globally. Lineages L3b and L9, characterized as ctxAB-negative and tcpA-positive (CNTP), pose the highest risk and have caused long-term epidemics in different regions worldwide. From 2001 to 2018, two waves (2001-2012 and 2013-2018) of epidemic caused by non-toxigenic V. cholerae occurred in the developed city of Hangzhou, China. In this study, through the integrated analysis of 207 genomes of Hangzhou isolates from these two waves (119 and 88) and 1573 publicly available genomes, we showed that L3b and L9 lineages together caused the second wave as had happened in the first wave, but the dominant lineage shifted from L3b (first wave: 69%) to L9 (second wave: 50%). We further found that the genotype of a key virulence gene, tcpF, in the L9 lineage during the second wave shifted to type I, which may have enhanced bacterial colonization in humans and potentially promoted the pathogenic lineage shift. Moreover, we found that 21% of L3b and L9 isolates had changed to predicted cholera toxin producers, providing evidence that gain of complete CTXφ-carrying ctxAB genes, rather than ctxAB gain in pre-CTXφ-carrying isolates, led to the transition. Taken together, our findings highlight the possible public health risk associated with L3b and L9 lineages due to their potential to cause long-term epidemics and turn into high-virulent cholera toxin producers, which necessitates a more comprehensive and unbiased sampling in further disease control efforts.

An integrated nationwide genomics study reveals transmission modes of typhoid fever in China.

IMPORTANCE: Typhoid fever is a life-threatening disease caused by Salmonella enterica serovar Typhi, resulting in a significant disease burden across developing countries. Historically, China was very much close to the global epicenter of typhoid, but the role of typhoid transmission within China and among epicenter remains overlooked in previous investigations. By using newly produced genomics on a national scale, we clarify the complex local and global transmission history of such a notorious disease agent in China spanning the most recent five decades, which largely undermines the global public health network.

Human infection with avian-origin H5N6 influenza a virus after exposure to slaughtered poultry.

Exposure to poultry in live poultry markets is strongly associated with human infection with avian influenza virus. To effectively prevent the transmission of viruses from live poultry to humans, people have been forced to change their living habits from purchasing live poultry for consumption to purchasing freshly slaughtered poultry after the permanent closure of live poultry markets in China. In this study, we reported a case of human infection by the H5N6 virus in Hangzhou after exposure to a freshly slaughtered chicken, defying the traditional hypothesis that human infection requires a history of exposure to live poultry and indicating a novel route of infection. Rapid genomic characterization of H5N6 influenza A variants from the patient and the associated environment suggested that these viral variants were of avian origin, belonged to clade 2.3.4.4b H5 and were adapting to the human host after infection. Comparative analysis of the local H5N6 genomes showed that viral contamination in the associated environment and the poultry market was complex. Considering this case of H5N6 infection, conducting surveillance for any possible new avian influenza virus reassortment spillover to humans or other animal species is critical, and awareness of the risk of exposure to possible viral variants from infected slaughtered poultry or the associated environment must be seriously improved.Highlights We reported the first case of human infection with avian-origin influenza A (H5N6) virus in Zhejiang Province, southeastern China.Rapid genomic characterization of H5N6 influenza A variants from a patient and the associated environment suggested that these viral variants were of avian origin and were adapting to the human host after infection.Comparative analysis of the H5N6 genomes showed that viral contamination in the associated environment and poultry market was complex.Considering this case of H5N6 infection, the risk of exposure to possible viral variants from infected slaughtered poultry or the associated environment must be seriously considered.

A nontyphoidal Salmonella serovar domestication accompanying enhanced niche adaptation.

Invasive nontyphoidal Salmonella (iNTS) causes extraintestinal infections with ~15% case fatality in many countries. However, the mechanism by which iNTS emerged in China remains unaddressed. We conducted clinical investigations of iNTS infection with recurrent treatment failure, caused by underreported Salmonella enterica serovar Livingstone (SL). Genomic epidemiology demonstrated five clades in the SL population and suggested that the international animal feed trade was a likely vehicle for their introduction into China, as evidenced by multiple independent transmission incidents. Importantly, isolates from Clade-5-I-a/b, predominant in China, showed an invasive nature in mice, chicken and zebrafish infection models. The antimicrobial susceptibility testing revealed most isolates (> 96%) in China are multidrug-resistant (MDR). Overall, we offer exploiting genomics in uncovering international transmission led by the animal feed trade and highlight an emerging hypervirulent clade with increased resistance to frontline antibiotics.

[Molecular characteristics and antibiotic resistances of Vibrio cholerae O1 isolates in Hangzhou in 2009].

OBJECTIVE: To study the molecular characteristics and antibiotic resistances of Vibrio cholerae (V. cholerae) O1 isolates in Hangzhou in 2009. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Biofilm formation and prevalence of adhesion genes among Staphylococcus aureus isolates from different food sources.

To assess biofilm formation ability and identify differences in the prevalence of genes involved in biofilm formation among Staphylococcus aureus strains isolated from different food samples, the ability of biofilm formation among 97 S. aureus strains was evaluated using a colorimetric microtiter plate assay. Thirteen genes encoding microbial surface components recognizing adhesive matrix molecules, and the intracellular adhesion genes were detected by PCR using specific primers. Approximately 72% of the isolates produced biofilms. Among these isolates, 54.64% were weak biofilm producers, while 14.43% and 3.09% produced moderate and strong biofilms, respectively. The icaADBC, clfA/B, cidA, and fib genes were detected in all the S. aureus strains, whereas the bap gene was not present in any of the strains. The occurrence of other adhesin genes varied greatly between biofilm-producing and nonbiofilm-producing strains. However, a significant difference was observed between these two groups with respect to the fnbpB, cna, ebps, and sdrC genes. No obvious evidence was found to support the link between PFGE strain typing and the capacity for biofilm formation. Considerable variation in biofilm formation ability was observed among S. aureus strains isolated from food samples. The prevalence of adhesin-encoding genes also varied greatly within strains. This study highlights the importance of biofilm formation and the adhesins of S. aureus strains in food samples.

Rapid genomic characterization of SARS-CoV-2 viruses from clinical specimens using nanopore sequencing.

The novel SARS-CoV-2 outbreak has swiftly spread worldwide. The rapid genome sequencing of SARS-CoV-2 strains has become a helpful tool for better understanding the genomic characteristics and origin of the virus. To obtain virus whole-genome sequences directly from clinical specimens, we performed nanopore sequencing using a modified ARTIC protocol in a portable nanopore sequencer and validated a routine 8-h workflow and a 5-h rapid pipeline. We conducted some optimization to improve the genome sequencing workflow. The sensitivity of the workflow was also tested by serially diluting RNA from clinical samples. The optimized pipeline was finally applied to obtain the whole genomes of 29 clinical specimens collected in Hangzhou from January to March 2020. In the 29 obtained complete genomes of SARS-CoV-2, 33 variations were identified and analyzed. The genomic variations and phylogenetic analysis hinted at multiple sources and different transmission patterns during the COVID-19 epidemic in Hangzhou, China. In conclusion, the genomic characteristics and origin of the virus can be quickly determined by nanopore sequencing following our workflows.

Genomic epidemiology of Vibrio cholerae reveals the regional and global spread of two epidemic non-toxigenic lineages.

Non-toxigenic Vibrio cholerae isolates have been found associated with diarrheal disease globally, however, the global picture of non-toxigenic infections is largely unknown. Among non-toxigenic V. cholerae, ctxAB negative, tcpA positive (CNTP) isolates have the highest risk of disease. From 2001 to 2012, 71 infectious diarrhea cases were reported in Hangzhou, China, caused by CNTP serogroup O1 isolates. We sequenced 119 V. cholerae genomes isolated from patients, carriers and the environment in Hangzhou between 2001 and 2012, and compared them with 850 publicly available global isolates. We found that CNTP isolates from Hangzhou belonged to two distinctive lineages, named L3b and L9. Both lineages caused disease over a long time period with usually mild or moderate clinical symptoms. Within Hangzhou, the spread route of the L3b lineage was apparently from rural to urban areas, with aquatic food products being the most likely medium. Both lineages had been previously reported as causing local endemic disease in Latin America, but here we show that global spread of them has occurred, with the most likely origin of L3b lineage being in Central Asia. The L3b lineage has spread to China on at least three occasions. Other spread events, including from China to Thailand and to Latin America were also observed. We fill the missing links in the global spread of the two non-toxigenic serogroup O1 V. cholerae lineages that can cause human infection. The results are important for the design of future disease control strategies: surveillance of V. cholerae should not be limited to ctxAB positive strains.

Persistent Asymptomatic Human Infections by Salmonella enterica Serovar Newport in China.

Salmonella enterica serovar Newport (S Newport) infections are gradually on the rise in China from the last decade. For humans' infections, S Newport has been ranked among the top five serovars responsible for persistent infections, globally. A total of 290 S. Newport strains with their relevant clinical metadata were analyzed, and the strains were subjected to whole-genome sequence analysis. Among these, 62.4% (n = 181) were from diarrheic patients and 28.9% (n = 84) were from asymptomatic individuals (including adults and youngsters) while 8.6% (n = 25) were from cases of persistent diarrhea in infants (28%, n = 7) and toddlers (72%, n = 18). The association between the sequence types (STs) and the variations in the clinical presentation was statistically significant (P = 0.0432), with ST46 causing diarrhea or representing asymptomatic patients and ST31 or ST68 causing persistent diarrhea. Genomic analysis revealed that the highest proportion of the isolates (98.5%, n = 279), primarily from patients with or without diarrhea rather than from asymptomatic individuals, carried antimicrobial resistance determinants corresponding to the aminoglycosides and beta-lactams, highlighting the need for cautionary usage of antimicrobials in such patients. These findings also suggest that cases of nontyphoidal Salmonella infection with symptoms of acute diarrhea or persistent diarrhea caused by S Newport should be handled with caution, due to the high chance of development of an antimicrobial resistance phenotype that might lead to therapeutic failures. Together, S Newport ST31 and ST46, which have the highest frequency of carriage of multidrug resistance, are potentially responsible for antimicrobial-resistant diarrhea/persistent diarrhea in infants and children, while adult humans are more likely to be (asymptomatic) carriers of the S Newport strains.IMPORTANCE Human infections caused by Salmonella Newport generally lead to gastrointestinal diseases. These infections are normally self-limiting; however, in certain cases, broad-spectrum antimicrobials are prescribed for the treatment. The Chinese National Foodborne Disease Surveillance Network has reported a gradual increase in the incidence of multidrug-resistant S Newport infections in humans. After careful evaluation of the dynamic relationship among the clinical findings, the age group, and the genomic sequence data, it was found that young patients represented the major group with persistent diarrhea, whereas adults were either asymptomatic or diarrheic. Furthermore, all these strains contained multiple acquired antimicrobial resistance determinants, which limited the use of antimicrobials for human patients of all age groups. This analysis of the laboratory-confirmed cases, coupled with genetic analysis of the corresponding pathogen, revealed that antimicrobial treatment of persistent infections by S Newport in infants and toddlers, and in asymptomatic or diarrheic adults, may not be successful. If the antimicrobials must be prescribed at all, they must be used with caution because of the presence of multiple acquired antimicrobial resistance determinants in such strains.

Characterization of fluoroquinolone-resistant Shigella flexneri in Hangzhou area of China.

OBJECTIVES: The aim of this study was to characterize fluoroquinolone-resistant Shigella and determine whether the qnr and aac(6')-Ib-cr genes could contribute to sporadic shigellosis at the clinic in the Hangzhou area of China. METHODS: A total of 202 strains of Shigella (79 Shigella sonnei and 123 Shigella flexneri ) isolated from sporadic cases of shigellosis from 1998 to 2007 were analysed for their antimicrobial susceptibility. The gyrA, gyrB, parC, parE, qnr and aac(6')-Ib-cr genes and the profiles and incompatibility of plasmids were characterized. Chromosomal DNA fingerprinting was determined by XbaI-based digestion and PFGE. RESULTS: All strains of S. sonnei were susceptible to fluoroquinolones (ciprofloxacin and levofloxacin) while 15 out of 123 strains of S. flexneri were resistant. All of the 15 resistant strains displayed common mutations in the gyrA and parC genes and formed eight distinct groups with unique molecular characteristics. Notably, 10 isolates showed mutations at codon 87 of gyrA, and the other 5 were qnrS-positive. Two strains were positive for the aac(6')-Ib-cr gene. Importantly, this is the first report of qnrS- and aac(6')-Ib-cr-positive Shigella in China, the qnrS-positive S. flexneri serotypes 1a, 2a and 4c and the aac(6')-Ib-cr-positive S. flexneri serotypes 2a and 4c worldwide. CONCLUSIONS: The common mutations at position 83 of gyrA and position 80 of parC were crucial for resistance to nalidixic acid in S. flexneri. The mutation at position 87 of gyrA or the presence of the qnrS gene is necessary for high-level resistance to fluoroquinolones in Shigella isolates from China.

[Multiplex PCR assay for dissemination and diversity of extended-spectrum beta-lactamase genes in Shigella isolates].

OBJECTIVE: 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. METHODS: 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. RESULTS: 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%. CONCLUSION: 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.

Multiple Lineages of Dengue Virus Serotype 2 Cosmopolitan Genotype Caused a Local Dengue Outbreak in Hangzhou, Zhejiang Province, China, in 2017.

During July to November 2017, a large dengue outbreak involving 1,138 indigenous cases occurred in Hangzhou, Zhejiang Province, China. All patients were clinically diagnosed as mild dengue. Epidemiology investigation and phylogenetic analysis of circulating viruses revealed that at least three lineages of dengue virus serotype 2 (DENV-2) Cosmopolitan genotype initiated the outbreak during a short time. The analysis of the time to most recent common ancestor estimated that the putative ancestor of these DENV-2 lineages might rise no later than March, 2017, suggesting independent introductions of these lineages into Hangzhou. We presumed that group travelers visiting dengue-endemic areas gave rise to multiple introductions of these lineages during so short a time. Co-circulating of multiple DENV-2 lineages, emerging of disease in urban areas, hot and humid weather in Hangzhou adequate for mosquito breeding, and limited dengue diagnosis abilities of local hospitals, were the reasons causing the large local outbreak in Hangzhou.

Vibrio cholerae O139 multiple-drug resistance mediated by Yersinia pestis pIP1202-like conjugative plasmids.

A conjugative plasmid, pMRV150, which mediated multiple-drug resistance (MDR) to at least six antibiotics, including ampicillin, streptomycin, gentamicin, tetracycline, chloramphenicol, and trimethoprim-sulfamethoxazole, was identified in a Vibrio cholerae O139 isolate from Hangzhou, eastern China, in 2004. According to partial pMRV150 DNA sequences covering 15 backbone regions, the plasmid is most similar to pIP1202, an IncA/C plasmid in an MDR Yersinia pestis isolate from a Madagascar bubonic plague patient, at an identity of 99.99% (22,180/22,183 nucleotides). pMRV150-like plasmids were found in only 7.69% (1/13) of the O139 isolates tested during the early period of the O139 epidemic in Hangzhou (1994, 1996, and 1997); then the frequency increased gradually from 60.00% (3/5) during 1998 and 1999 to 92.16% (47/51) during 2000 to 2006. Most (42/51) of the O139 isolates bearing pMRV150-like plasmids were resistant to five to six antibiotics, whereas the plasmid-negative isolates were resistant only to one to three antibiotics. In 12 plasmid-bearing O139 isolates tested, the pMRV150-like plasmids ranged from approximately 140 kb to 170 kb and remained at approximately 1 or 2 copies per cell. High (4.50 x 10(-2) and 3.08 x 10(-2)) and low (0.88 x 10(-8) to 3.29 x 10(-5)) plasmid transfer frequencies, as well as no plasmid transfer (under the detection limit), from these O139 isolates to the Escherichia coli recipient were observed. The emergence of pMRV150-like or pIP1202-like plasmids in many bacterial pathogens and nonpathogens occupying diverse niches with global geographical distribution indicates an increasing risk to public health worldwide. Careful tracking of these plasmids in the microbial ecosystem is warranted.

[PFGE of Shigella flexneri 4c isolates from food-poisoning outbreaks and sporadic diarrhea patients].

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.