Research on the contamination, drug resistance and molecular typing of Vibrio parahaemolyticus isolated from aquatic products in Nanchang City market / 中国热带医学

@#Abstract: Objective　To understand the contamination status, drug resistance, virulence gene carrying status, and molecular typing characteristics of Vibrio parahaemolyticus (VP) in aquatic products sold in Nanchang City. Methods　A total of 170 commercial crayfishes, freshwater fish frogs and related smears samples were collected from various farmers' markets in Nanchang from March to September 2021. The strains of V. parahaemolyticus were detected and isolated from the samples. Antibiotic resistance test, virulence gene test, and pulsed-field gel electrophoresis (PFGE) molecular typing analysis were carried out. Results　Among the collected samples, V.parahaemolyticus was only isolated from crayfish and crayfish smear samples, with a total of 35 strains of VP isolated. No V.parahaemolyticus strain was isolated from other freshwater fish, frogs, and their smear samples. Among the 17 common antibiotics tested, only two trains showed resistance to ampicillin, and one strain to streptomycin, , and all were sensitive to other antibiotics; all 35 strains of V. parahaemolyticus carried the gene, but only one strain carried the heat-resistant related hemolysin gene trh, and no direct heat-resistant hemolysin gene tdh positive strain was found; PFGE pattern clustering showed that there was no strain with the same PFGE pattern, and there was no obvious dominant cluster among these strains, and their genetic relationship was relatively distant. Conclusions　The contamination of V. parahaemolyticus in small and medium-sized crayfish sold in the market in Nanchang City is relatively serious. The V. parahaemolyticus isolates in these polluted crayfish generally do not carry key virulence genes such as tdh, are sensitive to common antibiotics, and only have low-level resistance to ampicillin and streptomycin. PFGE pattern clustering showed that V. parahaemolyticus does not have no obvious dominant cluster, and these strains have rich genetic diversity, indicating that they may have different sources.

CqsA-introduced quorum sensing inhibits type VI secretion system 2 through an OpaR-dependent pathway in Vibrio parahaemolyticus.

The well-known food-borne pathogen Vibrio parahaemolyticus employs at least three quorum sensing signals to maintain its high environmental adaptability. V. parahaemolyticus CqsA, the synthase involved in 3-hydroxyundecan-4-one quorum sensing signal, introduces a quorum sensing network. The V. parahaemolyticus virulent factor type VI secretion system 2 (T6SS2), which is associated with adhesion to host cells, was previously reported to be regulated by a quorum sensing system. Herein, we set out to determine the role of CqsA-introduced quorum sensing (CIQS) in T6SS2-associated virulent regulation. Using a tandem mass tag (TMT)-based quantitative proteomics assay, 17 T6SS2 proteins were found having significantly higher abundances in the ΔcqsA strain than in the wild type strain. TMT proteomics assay results were confirmed by a parallel reaction-monitoring (PRM)-based proteomics assay. Two T6SS2 up-regulators, OpaR and CalR, were found under control of CIQS in the TMT proteomics assay, while OpaR was down-regulated and CalR was up-regulated by CIQS. Thus, it was hypothesized that CIQS would inhibit T6SS2 with an OpaR-dependent mechanism. Epistasis experiment with quantitative PCR was designed to analyze the role of OpaR in the process of CIQS inhibiting T6SS2 production. The mRNA levels of T6SS2 genes were up-regulated in the ΔcqsA strain while down-regulated in the ΔopaR strain and in the ΔcqsAΔopaR mutant, indicating that OpaR plays a predominant role in the regulation of T6SS2 by CIQS. Using a cell adhesion assay, we further found that the T6SS2-dependent adhesion activity of V. parahaemolyticus to Hela cells was also inhibited by CIQS and the inhibition was OpaR-dependent. In this study, we confirmed that V. parahaemolyticus CIQS inhibited T6SS2 through an OpaR-dependent pathway. It enriches the knowledge of how V. parahaemolyticus quorum sensing regulates its virulence.

Identification and Genomic Characterization of Escherichia albertii in Migratory Birds from Poyang Lake, China.

Escherichia albertii is an emerging zoonotic foodborne enteropathogen leading to human gastroenteritis outbreaks. Although E. albertii has been isolated from birds which have been considered as the potential reservoirs of this bacterium, its prevalence in migratory birds has rarely been described. In this study, E. albertii in migratory birds from Poyang Lake was investigated and characterized using whole genome sequencing. Eighty-one fecal samples from nine species of migratory birds were collected and 24/81 (29.6%) tested PCR-positive for E. albertii-specific genes. A total of 47 isolates was recovered from 18 out of 24 PCR-positive samples. All isolates carried eae and cdtB genes. These isolates were classified into eight E. albertii O-genotypes (EAOgs) (including three novel EAOgs) and three E. albertii H-genotypes (EAHgs). Whole genome phylogeny separated migratory bird-derived isolates into different lineages, some isolates in this study were phylogenetically closely grouped with poultry-derived or patient-derived strains. Our findings showed that migratory birds may serve as an important reservoir for heterogeneous E. albertii, thereby acting as potential transmission vehicles of E. albertii to humans.

The Impact of the Closure of the Live Poultry Market due to COVID-19 on the Avian Influenza Virus in Nanchang, Jiangxi Province, China.

This article aims to understand the changes in the detection rates of H5, H7, and H9 subtypes of avian influenza viruses (AIVs) in the live poultry markets (LPMs) in Nanchang City, Jiangxi Province, before and after the outbreak of the COVID-19. From 2019 to 2020, we monitored the LPM and collected specimens, using real-time reverse transcription polymerase chain reaction technology to detect the nucleic acid of type A AIV in the samples. The H5, H7, and H9 subtypes of influenza viruses were further classified for positive results. We analyzed 1,959 samples before and after the outbreak and found that the positive rates of avian influenza A virus (39.69%) and H9 subtype (30.66%) after the outbreak were significantly higher than before the outbreak (26.84% and 20.90%, respectively; P < 0.001). In various LPMs, the positive rate of H9 subtypes has increased significantly (P ≤ 0.001). Positive rates of the H9 subtype in duck, fecal, daub, and sewage samples, but not chicken samples, have increased to varying degrees. This study shows that additional measures are needed to strengthen the control of AIVs now that LPMs have reopened after the relaxing of COVID-19-related restrictions.

Pathogen change of avian influenza virus in the live poultry market before and after vaccination of poultry in southern China.

BACKGROUND: The fifth wave of H7N9 avian influenza virus caused a large number of human infections and a large number of poultry deaths in China. Since September 2017, mainland China has begun to vaccinate poultry with H5 + H7 avian influenza vaccine. We investigated the avian influenza virus infections in different types of live poultry markets and samples before and after genotype H5 + H7 vaccination in Nanchang, and analyzed the changes of the HA subtypes of AIVs. METHODS: From 2016 to 2019, we monitored different live poultry markets and collected specimens, using real-time reverse transcription polymerase chain reaction (RT-PCR) technology to detect the nucleic acid of type A avian influenza virus in the samples. The H5, H7 and H9 subtypes of influenza viruses were further classified for the positive results. The χ2 test was used to compare the differences in the separation rates of different avian influenza subtypes. RESULTS: We analyzed 5,196 samples collected before and after vaccination and found that the infection rate of AIV in wholesale market (21.73%) was lower than that in retail market (24.74%) (P < 0.05). Among all the samples, the positive rate of sewage samples (33.90%) was the highest (P < 0.001). After vaccination, the positive rate of H5 and H7 subtypes decreased, and the positive rate of H9 subtype and untypable HA type increased significantly (P < 0.001). The positive rates of H9 subtype in different types of LPMs and different types of samples increased significantly (P < 0.01), and the positive rates of untypable HA type increased significantly in all environmental samples (P < 0.05). CONCLUSIONS: Since vaccination, the positive rates of H5 and H7 subtypes have decreased, but the positive rates of H9 subtypes have increased to varying degrees in different testing locations and all samples. This results show that the government should establish more complete measures to achieve long-term control of the avian influenza virus.

Nanchangia anserum gen. nov., sp. nov., isolated from feces of greater white-fronted geese (Anser albifrons).

Two rod-shaped and Gram-stain-positive bacteria (strains C64T and C62) were isolated in 2020 from faeces of greater white-fronted geese (Anser albifrons) from Poyang Lake, PR China. Their optimal growth conditions were at 37 °C, pH 7.0 and with 0.5 % (w/v) NaCl. The two isolates showed a highest 16S rRNA gene sequence similarity to Bowdeniella nasicola DSM 19116T (92.1 %). Phylogenetic/phylogenomic analyses indicated that strains C64T and C62 clustered independently in the vicinity of the genera Varibaculum, Winkia and Mobiluncus within the family Actinomycetaceae, but could not be classified clearly as members of any of these known genera. The average amino acid identity values between our isolates and available genomes of members of the family Actinomycetaceae were around the genus threshold value (45-65 %). The major cellular fatty acids of the strains were C18 : 1ω9c and C16 : 0. The predominant polar lipids were phosphatidylinositol, phosphatidylglycerol, phosphatidylcholine, diacylglycerol, triacylglycerol and cardiolipin. The amino acid composition of peptidoglycan contained alanine, glutamic acid and glycine. The major respiratory menaquinones were MK-8(H4) and MK-9(H4). The whole cell sugars included galactose, arabinose and glucose. On the basis of the results of the 16S rRNA gene sequences comparison, whole-genome phylogenomic analysis, phenotypic and chemotaxonomic characteristics, we propose that strains C64T and C62 represent a novel species belonging to a novel genus within the family Actinomycetaceae, for which the name Nanchangia anserum gen. nov., sp. nov. is proposed. The type strain is Nanchangia anserum C64T (=CGMCC 1.18410T=GDMCC 1.1969T=KCTC 49511T=KACC 22143T).

Changes of avian influenza virus subtypes before and after vaccination in live poultry in Nanchang, China from 2016 to 2019.

We investigated fluctuations in the detection rates of avian influenza virus (AIV) subtypes H5, H7, and H9 in live poultry in Nanchang city, Chinese province Jiangxi, before and after the Chinese nationwide AIV vaccination campaign against highly pathogenic (HP) AIV subtype H5 and H7. Samples were tested for nucleic acid of type A avian influenza virus by real-time reverse transcription polymerase chain reaction technology. The H5, H7 and H9 subtypes of influenza viruses were further classified for the positive results. Based on the analysis of 2119 samples collected from February 2016 to December 2019, we found that AIV subtypes H5, H7, H9 showed a seasonal pattern, and the positive rate of avian influenza tended to reach its peak in the colder season. The detection rate of AIV subtypes H5, H7, H9 of chickens (39.26%) was significantly higher than that of ducks (5.78%) and pigeons (4.31%). After vaccination, the positive rates of the H5 subtype (0.27%) and the H7 subtype (0.00%) decreased significantly, while the positive rate of the H9 subtype (29.95%) increased significantly. The H9 subtype has become the dominant subtype detected in live poultry and occupies a dominant position in the live bird market. This study showed that the government of China should establish measures for the long-term control of avian influenza.

Impact of the Gut Microbiome on the Progression of Hepatitis B Virus Related Acute-on-Chronic Liver Failure.

The relationship between the progression of hepatitis B virus-related acute-on-chronic liver failure (HBV-ACLF) and the gut microbiota is poorly understood, and an HBV-ACLF-related microbiome has yet to be identified. In this study alterations in the fecal microbiome of 91 patients with HBV-ACLF (109 stool samples), including a cohort of nine patients at different stages of HBV-ACLF, were determined by high-throughput 16S rDNA sequencing. The operational taxonomic units and Shannon indexes indicated that the diversity and abundance of the gut microbiome significantly decreased with the progression of HBV-ACLF (p <0.05). The relative abundance of the Bacteroidetes phylum in the microbiome was significantly reduced, whereas the abundance of potentially pathogenic bacteria, such as Veilonella, Streptococcus, Enterococcus, and Klebsiella, was highly enriched in the HBV-ACLF group compared with the healthy control group. The abundance of Bacteroidetes was negatively correlated with the level of serum alpha fetoprotein, and the abundance of Veilonella was positively correlated with serum total bilirubin (TBIL). Furthermore, the abundance of Coprococcus was significantly negatively correlated with the level of serum TBIL and the international normalized ratio and positively correlated with prothrombin time activity. Our findings suggest that the gut microbiota plays an important role in the development of HBV-ACLF.

Corynebacterium anserum sp. nov., isolated from the faeces of greater white-fronted geese (Anser albifrons) at Poyang Lake, PR China.

Two Gram-stain-positive, facultatively aerobic, non-motile and rod- to coccoid-shaped bacterial strains, 23H37-10T and 4HC-13, were isolated from the faeces of greater white-fronted geese (Anser albifrons) at Poyang Lake, Jiangxi Province, PR China. Optimal growth was observed at 35-37 °C, pH 7.0-8.0 and with 0.5-1.5 % (w/v) NaCl. The 16S rRNA gene sequences of strains 23H37-10T and 4HC-13 were identical. Phylogenetic and phylogenomic analyses indicated that strains 23H37-10T and 4HC-13 formed an independent cluster within the genus Corynebacterium and showed 98.8, 97.4, 97.4 and 97.2 % 16S rRNA gene sequence similarity to Corynebacterium urogenitale LMM 1652T, Corynebacterium urealyticum DSM 7109T, Corynebacterium falsenii DSM 44353T and Corynebacterium jeikeium NCTC 11913T, respectively. Cells contained C18 :1 ω9c, C18 : 0 and C16 : 0 as the major cellular fatty acids and MK-9 (H2) as the predominant respiratory quinone. The polar lipids comprised diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, phosphatidyl inositol mannosides, two unidentified phospholipids, four unidentified glycolipids and one unidentified lipid. Strain 23H37-10T contained mycolic acids, with meso-diaminopimelic acid and arabinose as the major whole-cell hydrolysates. The genome G+C content of strains 23H37-10T and 4HC-13 was 55.2 mol%. The digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values between strains 23H37-10T and 4HC-13 were 94.4 and 99.6 %, respectively. Strains 23H37-10T and 4HC-13 had dDDH and ANI values of less than 70 and 96 % with all available genomes of the genus Corynebacterium, respectively. The differential genotypic inferences, together with phenotypic and biochemical characteristics, suggested that strains 23H37-10T and 4HC-13 represent a novel species within the genus Corynebacterium, for which the name Corynebacterium anserum sp. nov. is proposed. The type strain is 23H37-10T (=GDMCC 1.1737T=KACC 21672T).

Avian influenza H9N2 virus isolated from air samples in LPMs in Jiangxi, China.

BACKGROUND: Recently, avian influenza virus has caused repeated worldwide outbreaks in humans. Live Poultry Markets (LPMs) play an important role in the circulation and reassortment of novel Avian Influenza Virus (AIVs). Aerosol transmission is one of the most important pathways for influenza virus to spread among poultry, from poultry to mammals, and among mammals. METHODS: In this study, air samples were collected from LPMs in Nanchang city between April 2014 and March 2015 to investigate possible aerosol transmission of AIVs. Air samples were detected for Flu A by Real-Time Reverse Transcription-Polymerase Chain Reaction (RRT-PCR). If samples were positive for Flu A, they were inoculated into 9- to 10-day-old specific-pathogen-free embryonated eggs. If the result was positive, the whole genome of the virus was sequenced by MiSeq. Phylogenetic trees of all 8 segments were constructed using MEGA 6.05 software. RESULTS: To investigate the possible aerosol transmission of AIVs, 807 air samples were collected from LPMs in Nanchang city between April 2014 and March 2015. Based on RRT-PCR results, 275 samples (34.1%) were Flu A positive, and one virus was successfully isolated with embryonated eggs. The virus shared high nucleotide homology with H9N2 AIVs from South China. CONCLUSIONS: Our study provides further evidence that the air in LPMs can be contaminated by influenza viruses and their nucleic acids, and this should be considered when choosing and evaluating disinfection strategies in LPMs, such as regular air disinfection. Aerosolized viruses such as the H9N2 virus detected in this study can increase the risk of human infection when people are exposed in LPMs.

Clinical and epidemiological characteristics of a fatal case of avian influenza A H10N8 virus infection: a descriptive study.

BACKGROUND: Human infections with different avian influenza viruses--eg, H5N1, H9N2, and H7N9--have raised concerns about pandemic potential worldwide. We report the first human infection with a novel reassortant avian influenza A H10N8 virus. METHODS: We obtained and analysed clinical, epidemiological, and virological data from a patient from Nanchang City, China. Tracheal aspirate specimens were tested for influenza virus and other possible pathogens by RT-PCR, viral culture, and sequence analyses. A maximum likelihood phylogenetic tree was constructed. FINDINGS: A woman aged 73 years presented with fever and was admitted to hospital on Nov 30, 2013. She developed multiple organ failure and died 9 days after illness onset. A novel reassortant avian influenza A H10N8 virus was isolated from the tracheal aspirate specimen obtained from the patient 7 days after onset of illness. Sequence analyses revealed that all the genes of the virus were of avian origin, with six internal genes from avian influenza A H9N2 viruses. The aminoacid motif GlnSerGly at residues 226-228 of the haemagglutinin protein indicated avian-like receptor binding preference. A mixture of glutamic acid and lysine at residue 627 in PB2 protein--which is associated with mammalian adaptation--was detected in the original tracheal aspirate samples. The virus was sensitive to neuraminidase inhibitors. Sputum and blood cultures and deep sequencing analysis indicated no co-infection with bacteria or fungi. Epidemiological investigation established that the patient had visited a live poultry market 4 days before illness onset. INTERPRETATION: The novel reassortant H10N8 virus obtained is distinct from previously reported H10N8 viruses. The virus caused human infection and could have been associated with the death of a patient. FUNDING: Emergency Research Project on human infection with avian influenza H7N9 virus, the National Basic Research Program of China, and the National Mega-projects for Infectious Diseases.

Molecular epidemiology of human enterovirus 71 strains in the Nanchang region of China in 2011.

Hand, foot, and mouth disease (HFMD) is caused by enteroviruses, most commonly enterovirus 71 (EV71) and coxsackievirus A16 (CA16). In general, EV71 infection is more likely to induce severe complications and mortality than other enterovirus infections. The present study focuses on the molecular epidemiology of human EV71 strains in the Nanchang region of China in 2011. Overall, 651 specimens (throat or rectal swabs) were collected, and one-step reverse transcriptase-polymerase chain reaction was performed for analysis. Enteroviruses were detected in 62.2% (405/651) of the specimens. EV71, CA16, and other enteroviruses were found in 292, 73, and 40 specimens, respectively. Phylogenetic analysis of the VP1 region of the 8 EV71 strains found in the Nanchang region indicated that these strains belong to the C4 subgenotype. This study shows that the C4 subgenotype strain of EV71 was prevalent in the HFMD cases of Nanchang in 2011, and it reports the first incidence of adults being infected by EV71 in the Nanchang region. Thus, the surveillance of HFMD epidemiology and monitoring of HFMD severity should be continued.

[Clinical significance of the predominant bacterial strains on burn wound during early postburn stage].

OBJECTIVE: To investigate the clinical significance of the predominant bacterial colonization on burn wound in our department during recent years, so as to help select optimal antibiotics in burn patients with severe infections. METHODS: This bacterial investigation was carried out in 215 cases of severely burned patients. The bacterial culture and the drug susceptibility test were carried out. RESULTS: (1) One hundred and twenty-two strains of bacteria were cultured, in which 28 strains (23%) were Staphylococcus with negative coagulase, 27 (22%) S. aureus, 17 (14%) Pseudomonas aeruginosa, 11 (9%) Escherichia coli, 10 (8%) Enterobacter, 9 (7%), enterococci, 3 (2.5%) fungi, and 17 (14.5) other bacteria. (2) The resistance of S. aureus to ampicillin, oxacillin and amoxicillin/clavulanic acid was 81%, 38% and 31%, respectively. 11% and 16% of Pseudomonas aeruginosa resistant to Imipenem and Ceftazidime, respectively. (3) The sensitivity of G + cocci to vancomycin and norvancomycin, Chloramphenicol, Teicoplanin, Trimethoprim/Sulfamethoxaz, Rifampin was 100%, 100%, 100%, 94% and 88% respectively, and the Gram-negative bacilli to Meropenem, Imipenem, Amikacin, Cefepime, Cefoperazone/Sulbactam, Ceftazidime were 91%, 90%, 81%, 78%, 71% and 70%, respectively. Furthermore, the sensitivity of Pseudomonas aeruginosa to Cefoperazone/Sulbactam, Ceftazidime, Tobramycin, Meropenem, Amikacin, Ciprofloxacin, Amikacin, Cefepime were between 82% and 91%. MRSA was very sensitive to both vancomycin and norvancomycin. CONCLUSION: The results suggested that Staphylococcus with negative coagulase and S. aureus were the predominant bacteria and Pseudomonas aeruginosa ranked second. The resistance of these bacteria to antibiotics was on the increase. Moreover, colonization of enterococcus and fungi on burn wound increased recently, which were scarce before. This implied the importance of rational and correct use of antibiotics during early postburn stage.