Epidemiologic characterization of 30 confirmed cases of human infection with avian influenza A(H7N9) virus in Hangzhou, China.

BACKGROUND: We examined the clinical and epidemiological characteristics of 30 cases of human infection with avian influenza A(H7N9) virus in Hangzhou and investigated their external environments to provide evidence for contact tracing and disease prevention and control. METHODS: The cases confirmed from April 1 through May 1, 2013 were studied. Field epidemiologic surveys were conducted to collect the clinical and epidemiologic data. Case-related and environmental specimens were collected for etiologic detection. RESULTS: Thirty cases of human infection with avian influenza A(H7N9) virus were confirmed in Hangzhou from April 1 through May 1, 2013, including one pregnant woman and three deaths. The median age of the patients was 62 years (range: 38-86 years). Twenty-three of the patients were men (76.67%). The median duration between disease onset and occurrence of respiratory failure and confirmed diagnosis was 5 and 6 days, respectively. Maximum medical observation of 666 close contacts of the patients revealed no irregularity. Of 314 external environmental specimens, the overall positive detection rate of H7N9 nucleic acid was 28.98%. Eight districts of Hangzhou city had positive detections in the external environments, the highest rate being in Yuhang District (78.13%). Statistical analysis of the specimen collection locations indicates a significant difference between the case-linked locations and the non-case locations (χ2 = 16.563, p < 0.05) in terms of H7N9 viral nucleic acid detection rate. No epidemiologic link has been found among the 30 cases. CONCLUSIONS: Most of the infected were retired individuals aged 60 years or older. Men made the majority. The cases are sporadic at present, with no evidence of human-to-human transmission. Exposures to poultry and live poultry markets may be important sources of infection.

Control of RNA stability by NrrF, an iron-regulated small RNA in Neisseria gonorrhoeae.

Regulation of gene expression by small noncoding RNAs (sRNAs) plays a critical role in bacterial response to physiological stresses. NrrF, a trans-acting sRNA in Neisseria meningitidis and Neisseria gonorrhoeae, has been shown in the meningococcus to control indirectly, in response to iron (Fe) availability, the transcription of genes encoding subunits of succinate dehydrogenase, a Fe-requiring enzyme. Given that in other organisms, sRNAs target multiple mRNAs to control gene expression, we used a global approach to examine the role of NrrF in controlling gonococcal transcription. Three strains, including N. gonorrhoeae FA1090, an nrrF deletion mutant, and a complemented derivative, were examined using a custom CombiMatrix microarray to assess the role of this sRNA in controlling gene expression in response to Fe availability. In the absence of NrrF, the mRNA half-lives for 12 genes under Fe-depleted growth conditions were longer than those in FA1090. The 12 genes controlled by NrrF encoded proteins with biological functions including energy metabolism, oxidative stress, antibiotic resistance, and amino acid synthesis, as well as hypothetical proteins and a regulatory protein whose functions are not fully understood.

Spontaneous mutation frequency and molecular mechanisms of Shigella flexneri fluoroquinolone resistance under antibiotic selective stress.

The incidence of fluoroquinolone-resistant Shigella strains has risen rapidly, presumably in response to ciprofloxacin antibiotic stress. Understanding the molecular mechanisms underlying this resistance phenotype is critical to developing novel and effective therapeutic strategies. In this study, the frequency of ciprofloxacin-induced mutation was measured in antibiotic resistance genes (gyrA, gyrB, parC, parE, marOR, and marA) of Shigella flexneri. The S. flexneri 2a strain 301 was cultured on Luria-Bertani agar plates containing one of seven different ciprofloxacin concentrations (range: 0.03125-2 µg mL(-1)). Resistant colonies were selected for gene-targeted sequencing analysis; the identified point mutations were subsequently confirmed by insertion into antibiotic cassette plasmids and growth under ciprofloxacin stress. The results demonstrated that the seven different ciprofloxacin concentrations produced dose-dependent frequencies of spontaneous mutations: 10(-8) (0.03125 and 0.0625 µg mL(-1)), 10(-9) (0.125 µg mL(-1)), and <10(-9) (0.25, 0.5, 1, 2 µg mL(-1)). PCR sequencing of the ten randomly selected resistant colonies (minimum inhibitory concentrations (MICs) of 0.125 µg mL(-1), n = 5 and 0.25 µg mL(-1), n = 5) revealed that all colonies had mutations in the gyrA gene at either codon 83 (Ser83 → Leu) or 87 (Asp87 → Tyr or → Gly), both of which were confirmed at MIC of 0.125 µg mL(-1). None of the spontaneous mutation colonies exhibited gyrB, parC, parE, marOR, or marA mutations. In conclusion, S. flexneri is normomutable under ciprofloxacin antibiotic stress and fluoroquinolone resistance by spontaneous mutation occurs at a low rate. Codon mutations gyrA 83 and/or gyrA 87 cause a 4-fold increase in the ciprofloxacin MIC, and may represent the natural mechanism of fluoroquinolone resistance.

Two adenovirus serotype 3 outbreaks associated with febrile respiratory disease and pharyngoconjunctival fever in children under 15 years of age in Hangzhou, China, during 2011.

Adenovirus serotype 3 and 7 outbreaks have occurred periodically in northern, eastern, and southern China since 1955, but there has been no report since the adenovirus serotype 7 outbreak first occurred in Hangzhou, China, in 1991. Here we explored the epidemiology and etiology of two adenovirus serotype 3 outbreaks in Hangzhou in 2011. One acute respiratory outbreak was found in Chun'an County, where a total of 371 cases were confirmed in 5 of 23 towns from 4 to 31 May 2011. The outbreak affected 18.57% (13/70) of schools and 14.49% (90/621) of classes. The incidence was 5.18% (371/7,163). The population was distributed among individuals ages 7 to 15 years. No parents or teachers were infected. Another pharyngoconjunctival fever outbreak was discovered in the Chenjinglun Swimming Center located in the Xihu District between 1 and 15 July 2011. A total of 134 cases were confirmed in 900 amateur swimmers, with an incidence of 14.89% (134/900). The ages ranged from 4 to 9 years. The two outbreaks had no severe complications or death. The viruses in 66.67% (10/15) of throat swabs from children with acute respiratory infections and 100% (10/10) of the swabs from children with pharyngoconjunctival fever were confirmed to be adenovirus serotype 3 with 100% homology by PCR. Of these samples, 60.0% (12/20) had a classical characteristic cytopathic effect, presented as grape-like clusters at 72 h after infection in HEp-2 cells. In conclusion, the acute respiratory infection and pharyngoconjunctival fever outbreak in Hangzhou were caused by the completely homologous type 3 adenovirus in subgenus B. Moreover, these outbreaks demonstrated rapid transmission rates, possibly due to close contact and droplet transmission.

[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.

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.

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.

Preparation of armored RNA as a control for multiplex real-time reverse transcription-PCR detection of influenza virus and severe acute respiratory syndrome coronavirus.

The common respiratory viruses, including influenza A, influenza B, and newly emerging severe acute respiratory syndrome (SARS) viruses, may cause similar clinical symptoms. Therefore, differential diagnosis of these virus pathogens is frequently required for single clinical samples. In addition, there is an urgent need for noninfectious and stable RNA standards and controls for multivirus detection. In this study, reverse transcription-PCR (RT-PCR) targeting of the RNAs of influenza A and influenza B viruses and SARS coronavirus was performed, and the resulting products were spliced into a fragment which was packaged into armored RNA for use as a noninfectious, quantifiable synthetic substitute. Furthermore, in the present study we developed a multiplex real-time RT-PCR assay in which the armored RNA was used as an external positive control and the three RNA viruses could be detected simultaneously in a single reaction mix. The detection limit of the multiplex real-time PCR was 10 copies/microl of armored RNA.

[Establishing a single-tube fluorescent bidirectional PCR method to detect the 609C/T polymorphism of NQO1 gene].

OBJECTIVE: To develop a single-tube fluorescent bidirectional PCR method to detect the 609C/T polymorphism of NAD(P)H: quinone oxidoreductase 1 (NQO1) gene. METHODS: Two primers of NQO1 gene C609T locus were designed. Using these primers, a SYBR Green I fluorescent bidirectional PCR, combined with melting curve analysis of the PCR products, were optimized to differentiate the 609C/T polymorphisms in 191 samples of human genomic DNA. The accuracy of the fluorescent bidirectional PCR was validated by the classical method of PCR-restriction fragment length polymorphism(RFLP) in 62 of these 191 samples. RESULTS: In the 62 samples, the genotypes determined by the fluorescent bidirectional PCR were 100% consistent with the ones by the PCR-RFLP. The frequencies of genotypes of homozygous wild-type (CC), heterozygous (CT), and homozygous mutant (TT) were 28%, 50%, and 22%, respectively, in the 191 samples. CONCLUSION: The single-tube fluorescent bidirectional PCR method established here provides a simple, rapid, accurate and inexpensive assay to determine the 609C/T polymorphism of NQO1 gene. The assay is suitable to detect the single nucleotide polymorphism in large-scale samples.

Complete Sequences and Characterization of Two Novel Plasmids Carrying aac(6')-Ib-cr and qnrS Gene in Shigella flexneri.

The complete sequences of two previously reported plasmids carrying plasmid-mediated quinolone resistance genes from Shigella flexneri in China have not been available. The present study using the p5-C3 assembly method revealed that (1) the plasmid pSF07201 with aac(6')-Ib-cr had 75,335 bp with antibiotic resistance genes CTX-M-3, TEM-1, and FosA3; (2) seven fragments of pSF07201 had more than 99% homology with the seven corresponding plasmids; (3) the other plasmid pSF07202 with qnrS had 47,669 bp with antibiotic resistance gene TEM-1 and 99.95% homology with a segment of pKF362122, which has the qnrS gene from location 162,490 to 163,146. A conjugation and electrotransformation experiment suggested that these two plasmids might horizontally transfer between and coexist in Escherichia coli J53 and S. flexneri 2a 301. Either the aac(6')-Ib-cr or qnrS gene contributed to, but only the coexistence of the two genes conferred to the resistance to ciprofloxacin in these two strains. To the best of our knowledge, this is the first report of the complete sequences of the aac(6')-Ib-cr- and qnrS-positive plasmids in Shigella isolates. Our findings indicate that two genes probably evolve through horizontal plasmid transfer between the different bacterial types.

[Characteristics of virulence gene in Vibrio parahaemolyticus strains isolated from clinical patients and environment in Hangzhou, China].

OBJECTIVES: To investigate the characteristics of virulence gene in Vibrio parahaemolyticus strains isolated from clinical patients and environment in Hangzhou, China. METHODS: Thermostable direct hemolysin gene (tdh) and thermostable direct hemolysin-related hemolysin gene (trh) were determined in a total of 174 strains of V. parahaemolyticus isolated from patients and environment (seafood) in Hangzhou area by PCR. RESULTS: The tdh was found in 92 out of 94 V. parahaemolyticus strains from food poisoning patients and in 33 out of 34 strains from sporadic diarrhea patients, and trh was not detected in all above clinical strains. Meanwhile the tdh was negative in all V. parahaemolyticus strains from environment, and the trh was also negative except one strain with urease activity. All strains with trh negative had no the activity of urease. CONCLUSIONS: The V. parahaemolyticus strains from food poisoning patients and sporadic diarrhea patients are tdh positive and trh negative. The V. parahaemolyticus strains with tdh negative and almost trh positive in environment might be a potential pathogen in Hangzhou.

[Preparation of armored RNA containing M gene of influenza H3N2].

OBJECTIVE: To prepare the armored RNA containing M gene of influenza H3N2. METHODS: The vector pAR-1 was constructed from expression vector pET30b in which the bacteriophage MS2 DNA fragment, containing the genes for maturase and coat protein and the pac site, was inserted. The M gene fragment of influenza A was inserted into the HindIII site downstream of the pac site on the pAR-1, which formed a new recombinant plasmid pAR-2. After the prokaryotic expression was carried out, armored RNA AR-2 containing M gene was obtained. AR-2 was purified, and then was quantified by real time RT-PCR. Moreover, the stability of AR-2 was checked. RESULTS: AR-2 was expressed successfully. AR-2 remained stable under various storage environments. Approximately 8.9 x 10(11) copies of AR-2 particles can be purified from one milliliter of culture. CONCLUSION: It showed that AR-2 was stable and RNase-resistant, which, as a virus surrogate, would be used as RT-PCR standards, controls and training or proficiency samples.

Molecular characteristics of class 1 and class 2 integrons and their relationships to antibiotic resistance in clinical isolates of Shigella sonnei and Shigella flexneri.

OBJECTIVES: To analyse the gene cassettes and determine the roles of class 1 and class 2 integrons in antibiotic-resistant strains of Shigella sonnei (n=31) and Shigella flexneri (n=33). METHODS: Various molecular techniques, including PCR and Southern-blotting analysis, were used to analyse various markers of class 1 and class 2 integrons in these 64 S. sonnei and S. flexneri isolates collected in Hangzhou, China. The gene cassette arrays in integrons were identified by DNA sequencing and/or restriction fragment length polymorphism. Two genomic DNA fragments, one containing intI1 from a S. flexneri isolate that contains intI1 but lacks 3'-conserved region and another containing intI2 from a S. sonnei isolate, were cloned into pUC19 vectors and sequenced. The links between integron gene cassette arrays and antibiotic resistance were analysed. RESULTS: Class 2 integrons were present in 80.6% (25/31) of the S. sonnei isolates and 87.9% (29/33) of the S. flexneri isolates. All of these integron 2-positive isolates contained constant gene cassette arrays of dfrA1+sat1+aadA1 which confer resistance to trimethoprim and streptomycin. It was demonstrated that the class 2 integron was located in the Tn7 region inside the attTn7 locus downstream of glmS in Shigella. Class 1 integrons were found in 9.4% (6/64) of Shigella spp. isolates. An atypical class 1 integron without a 3'-conserved segment on the Shigella chromosome, termed Shigella atypical class 1 integron (SAI), was present in 84.9% (28/33) of S. flexneri isolates. The SAI contained two gene cassettes, bla(OXA30) and aadA1; however, the SAI conferred resistance to ampicillin, but not to streptomycin, in Escherichia coli host. The bla(OXA30) and aadA1 cassettes of the SAI seemed to be always coordinately excised or integrated. CONCLUSIONS: Multiple and complex mechanisms involving mobile genetic elements in class 1 and class 2 integrons and antibiotic resistance have been developed in the evolution of Shigella strains.