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Toxigenic conversion of environmental V ibrio cholerae strains through lysogenic infection by the phage CTXΦ is an important step in the emergence of new pathogenic clones. The precursor form of the CTXΦ phage, pre- CTXΦ, does not carry the cholera toxin gene. During our investigation, we frequently found pre- CTXΦ prophages in non-toxigenic isolates in the serogroups of O1 and O139 strains in the Zhujiang estuary. We observed high amounts of sequence variation of rstR and gIIICTX in the pre- CTXΦ alleles as well as in the tcpA sequences within the strains. In addition, a new pre- CTXΦ allele, with a novel rstR sequence type and hybrid RS2, was identified. Our findings show that active, complicated gene recombination and horizontal transfer of pre- CTXΦs occurs within V . cholerae environmental strains, which creates a complex intermediate pool for the generation of toxigenic clones in the estuarine environment. [ABSTRACT FROM AUTHOR]

Continuous surveillance of enteroviruses (EVs) in urban domestic sewage can timely reflect the circulation of EVs in the environment and crowds, and play a predictive and early warning role in EV-related diseases. To better understand the long-term epidemiological trends of circulating EVs and EV-related diseases, we conducted a 9-year (2013 to 2021) surveillance study of non-polio EVs (NPEVs) in urban sewage in Guangzhou city, China. After concentrating and isolating the viruses from the sewage samples, NPEVs were detected and molecular typing was performed. Twenty-one different NPEV serotypes were identified. The most isolated EVs were echovirus 11 (E11), followed by coxsackievirus (CV) B5, E6, and CVB3. EV species B prevailed in sewage samples, but variations in the annual frequency of different serotypes were also observed in different seasons, due to spatial and temporal factors. E11 and E6 were detected continuously before 2017, and the number of isolates was relatively stable during the surveillance period. However, after their explosive growth in 2018 and 2019, their numbers suddenly decreased significantly. CVB3 and CVB5 had alternating trends; CVB5 was most frequently detected in 2013 to 2014 and 2017 to 2018, while CVB3 was most frequently detected in 2015 to 2016 and 2020 to 2021. Phylogenetic analysis showed that at least two different transmission chains of CVB3 and CVB5 were prevalent in Guangzhou City. Our results show that in the absence of a comprehensive and systematic EV-related disease surveillance system in China, environmental surveillance is a powerful and effective tool to strengthen and further investigate the invisible transmission of EVs in the population. IMPORTANCE This study surveilled urban sewage samples from north China for 9 years to monitor enteroviruses. Samples were collected, processed, and viral identification and molecular typing were performed. We detected 21 different non-polio enteroviruses (NPEVs) with yearly variations in prevalence and peak seasons. In addition, this study is very important for understanding the epidemiology of EVs during the COVID-19 pandemic, as the detection frequency and serotypes of EVs in sewage changed considerably around 2020. We believe that our study makes a significant contribution to the literature because our results strongly suggest that environmental surveillance is an exceptionally important tool, which can be employed to detect and monitor organisms of public health concern, which would otherwise be missed and under-reported by case-based surveillance systems alone., Competing Interests: The authors declare no conflict of interest.

Toll-like receptor 4 (TLR4) is a reliable target for the development of vaccine adjuvants. To identify novel TLR4 ligands with improved immunological properties for use as adjuvants for a RBD-hFc based SARS-CoV-2 vaccine, herein, natural E. coli monophosphoryl lipid A (MPLA) and nine of its derivatives were designed and synthesized. Immunological evaluation showed that compounds 1, 3, 5 and 7 exhibited comparative or better adjuvant activity than clinically used Al adjuvants, and are expected to be a promising platform for the development of new adjuvants used for a RBD-hFc based SARS-CoV-2 vaccine. Preliminary structure-activity relationship analysis of the MPLA derivatives showed that the replacement of the functional groups at the C-1, C-4' or C-6' position of E. coli MPLA has an effect on its biological activity. In addition, we found that the combination of MPLA and Al was feasible for immunotherapy and could further enhance immune responses, providing a new direction toward the immunological enhancement of RBD-hFc based SARS-CoV-2 vaccines., Competing Interests: The authors declare no competing financial interest., (This journal is © The Royal Society of Chemistry.)

Introduction: Gastroenteritis caused by non-O1/non-O139 Vibrio cholerae exhibited an increasing trend in recent years in China. Whole genome sequence (WGS) data could play an important role both in the identification of the outbreaks and in the determination of the serogroup. Here, we present the employment of WGS data in the investigation of two outbreaks caused by non-O1/non-O139 V. cholerae in Guangdong, China, 2020-2021., Methods: We obtained the whole genome sequence of 66 V. cholerae strains isolated in two outbreaks with next generation sequencing technology. We retrieved the publicly available WGS data of non-O1/non-O139 V. cholerae from public database. We used a pipeline integrated in China Pathogen Identification Net (PIN) to complete the phylogenetic analysis., Results: Two outbreaks caused by non-O1/non-O139 V. cholerae were identified using WGS data. These V. cholerae strains were determined as serogroup O5. Type 3 and 6 secretion systems were detected in these serogroup O5 strains. These serogroup O5 strains belonged to sequence type (ST) 88., Conclusions: Our analysis indicated the risk of non-O1/non-O139 V. cholerae leading to outbreaks of diarrheal diseases. The application of genomic data played an important role in the identification of the serogroup of non-O1/non-O139 V. cholerae in the lack of antiserum, which gave an example of the application of genome data in disease surveillance and public health emergency response., Competing Interests: No conflicts of interest. Figure 1Phylogenetic analysis and molecular characteristics of serogroup O5 V. cholerae isolated in two outbreaks (A) Phylogenetic tree of non-O1/non-O139 V. cholerae.(B) Phylogenetic tree of serogroup O5 V. cholerae. (C) Illustration of the O-PS biosynthesis gene locus in V. cholerae isolated in these two outbreaks. Notes: Maximum likelihood (ML) tree of 66 non-O1/non-O139 V. cholerae isolated in the 2 outbreaks, and the publicly available representative V. cholerae was constructed based on the SNVs in the core genome. Those isolated in this study were in red. This ML tree was constructed based on the SNVs in the non-repetitive and non-recombinant core-genome of 106 non-O1/non-O139 V. cholerae. O1 El Tor strain N16961 was used as reference. The ML tree was constructed on the SNVs identified in the non-repetitive, non-recombinant core-genome of the 66 most closely-related serogroup O5 V. cholerae. The presence and absence of virulence genes and mobile genetic elements usually detected in pandemic V. cholerae and antibiotic resistance genes were annotated on the right of the ML tree. Comparison between O-PS biosynthesis gene locus in serogroup O5 V. cholerae CO545 and that in a representative strain which was isolated in the outbreak in 2021. Abbreviations: MGEs=mobile genetic element; ARGs=antibiotic resistance genes; SNVs=single nucleiotide variation., (Copyright and License information: Editorial Office of CCDCW, Chinese Center for Disease Control and Prevention 2022.)

Objective: Establishment and application of Taq Man probe-based quadruple real-time PCR for detection of Salmonella paratyphi A/B/C and Salmonella typhi. Primers specific to Salmonella paratyphi A( SPAP), Salmonella paratyphi B( SPBP), Salmonella paratyphi C( SPCP), and Salmonella typhi( STP) were designed., Methods: A method of Taq Man probe-based quadruple real-time PCR was established according to the distinction of the 5'end of the probe mark of TET, ROX, FAM and HEX. 5 strains of SPA, 4 strains of SPB, 7 strains of SPC and 11 strains of ST were identified by amplification from SPAP, SPBP, SPCP and STP., Results: While other serotypes of salmonella and17 strains of non-salmonella got negative results of amplification. Amplification rate of SPAP, SPBP, SPCP, and STP were 84. 5%, 101. 8%, 92. 4% and 90. 9%, respectively. The linear correlation coefficient( R~2) were 0. 996, 0. 975, 0. 996 and 0. 984, respectively., Conclusion: The PCR system is specific and sensitive for the identification of SPA, SPB, SPC and ST.

Objective: To investigate the serotypes, antimicrobial resistance, virulence genes and molecular characteristics of food-borne Vibrio parahaemolyticus isolated in Guangdong province in 2014., Methods: Sixty V. parahaemolyticus strains were tested in this study. The serotyping and antibiotic resistance test were conducted, while the tdh and trh gens of the strains were detected with conventional PCR, and pulsed field gel electrophoresis (PFGE) and multiple locus sequence typing (MLST) were conducted too., Results: The 60 strains belonged to 13 serotypes, the major serotypes included O3: K6, O4: K8, O1: K36 and O4: KUT. The antibiotic resistance test indicated the isolates were highly resistant to ampicillin (100.0%), sulfonamides (43.3%) and cefalotin (28.3%). Up to 56.7%(34/60) of the strains were resistant to two or more antibiotics, and 2 strains showed resistance to three antibiotics. The virulence gene detection indicated that 63.3% (38/60) of the strains carried tdh⁺ trh⁻, while only 1 strain carried tdh⁺ trh⁺. The 60 strains digested by NotI belonged to 48 different PFGE patterns and 3 clusters. The cluster B included the strains isolated from sporadic food borne cases with serotype of O3: K6 and similarity of 62.6%-100.0%. The cluster C included O4: K8 strains with the PFGE pattern similarity of 56.7%-62.5%. The MLST indicated that the 60 strains had 26 sequence types (STs). The ST-3 was predominant, including 33 strains (serotypes O3: K6 and O1 :K36). The four O4: K8 strains formed another predominant colony, which was different from ST-3., Conclusion: The etiologic characteristics of V. parahaemolyticus varied, which might be one of the reasons for high incidence of food-borne V. parahaemolyticus infection in Guangdong. The molecular characteristics of O4: K8 strain were different from the other predominant serotypes. Close attention should be paid to the possible outbreak caused by O4: K8 strain in this area.

Objective: To study the serotypes, antimicrobial resistance, virulence genes and molecular characteristics of Vibrio parahaemolyticus isolated from outbreaks and sporadic cases in Guangdong, 2013., Methods: 36 Vibrio parahaemolyticus strains isolated from outbreaks and 43 strains from sporadic cases were sero-typed and tested for antimicrobial resistance. PCR was used to detect for tdh(thermostable direct hemolysin gene), trh (tdh(-) related hemolysin gene), GS-PCR and orf8 genes. All the samples were analyzed by pulsed-field gel electrophoresis (PFGE)., Results: 36 isolates from outbreaks were all identified as O3 : K6, and among the 43 sporadic isolates, O3 : K6 (23, 53.49%) was the dominant serotype. Vibrio parahaemolyticus isolates showed high resistance rate to ampicillin (96.20%) and cefalotin (40.50%), but were high sensitive to cotrimoxazole (100%) and chloramphenicol (100%). 83.33% (30/36) outbreak isolates were resistant to multi-drugs but only 37.21% (16/43) of the sporadic isolates showed so. Results from virulence gene detection suggested that all the 36 outbreak isolates belonged to tdh(+) trh(-) strains, while 86.05% (37/43) of the sporadic isolates were tdh(+)trh(-) and 11.63% (5/43)were tdh(-)trh(+) . Only one tdh(+)trh(+) strain was found. All the outbreak isolates contained GS-PCR and/or orf8 genes, whereas among the sporadic isolates only 51.16% (22/43) of them carrying the similar genes. Results from PFGE analysis suggested that 79 isolates were discriminated into 3 clusters and 32 different PFGE patterns with the similarity value between 59.8% and 100.0%. Outbreak isolates seemed to gather in the same cluster, while the sporadic isolates spreading in all the three clusters., Conclusion: O3 : K6 was the dominant serotype in Vibrio parahaemolyticus strains isolated in Guangdong, 2013. These strains showed high sensitivity to most antibiotics, but with multi-drug resistance. Positive rate of tdh gene was high, and most O3 : K6 strains contained GS-PCR and/or orf8 genes. PFGE analysis revealed genetic diversity was within the Vibrio parahaemolyticus strains in Guangdong.

Objective: To understand the distribution and the characteristics on molecular typing of Salmonella (S.) typhimurium isolates gathered from the surveillance program and to construct the standard S. typhimurium databank in the laboratory through surveillance network PulseNet, in Guangdong province to improve the capability of detection on laboratory-based foodborne outbreaks., Methods: With the application of standard pulse-field gel electrophoresis (PFGE) and multiple loci VNTR analysis (MLVA) including seven VNTRs loci protocols on PulseNet International Network, 275 isolates of S. typhimurium from ten cities in Guangdong province were typed and their patterns analyzed. The molecular typing databank was constructed by BioNumerics., Results: With S. typhimurium the most common serotypes, the average annual positive rate of Salmonella strains and S. typhimurium were 4.03% and 1.38% respectively. The positive rate and proportion presented a double-peak trend, appearing in May and September. The chromosomal DNA of S. typhimurium was digested with Xba I restricted endo-nucleotidase and 124 PFGE patterns were observed after pulse-field gel electrophoresis, with the discrimination index (D) as 0.928 6. The patterns including more than three S. typhimurium isolates and were further digested with the second enzyme Bln I to achieve 174 patterns, with the D value as 0.989 1. Under MLVA method, 143 variant patterns were obtained, with the D value reaching 0.966 5. Data showed that the discriminatory ability of the MLVA typing method in S. typhimurium was superior to PFGE-Xba I but equal to PFGE-Xba I-Bln I. In addition, when S. typhimurium strains were respectively analyzed by PFGE under double enzymes digestion and MLVA, the results appeared coincident and relative., Conclusion: The variant patterns showed by the two molecular typing methods indicating a genetic diversity existed among the clinical S. typhimurium isolates in Guangdong province. Databank of S. typhimurium was constructed and could be used in laboratory surveillance programs. Under the characterization of analyzing similarity and evolution among S. typhimurium isolates, MLVA was suitable for cluster analysis on early detection of outbreaks caused by S. typhimurium.

Objective: To analyze the etiologic characteristics of O1/O139 Vibrio cholerae in Guangdong province in 2009-2013., Methods: Isolates from cholera cases and from the environment surveillance points were investigated by serological typing, antibiotic susceptibility testings, toxic genes detection and molecular typing to analyze the similarities and differences of the identified species., Results: Totally, 190 isolations of O1/O139 V. cholerae were obtained from cholera cases (16 strains) and environmental samples (174 strains) in Guangdong province in 2009-2013. The sero-types would include Inaba (3 isolates), Ogawa (7 isolates) and O139 (6 isolates) in all the isolates from the cholera cases. Ten strains from the ctxA positive cases were detected by PCR. Two Ogawa strains carried incomplete CTXΦ phage. Results from the antibiotic susceptibility test indicated that 5 strains were absolutely sensitive to 11 antibiotic discs in vitro, while another 3 strains were simultaneously resistant to 4 antibiotic discs. Except for 2 stains, all the O139 strains from the environment were ctxA negative, detected by PCR. Incomplete CTXΦ phage was found in the Inaba (53 isolates), Ogawa (22 isolates) and O139 (2 isolates), respectively. Results from the antibiotic susceptibility test exhibited that 25 strains were resistant simultaneously to 4 and/or more antibiotic discs, especially the Inaba strains from the seafoods(13 isolates). 2 Inaba strains from seafood were simultaneously resistant to 7 antibiotic discs. Results from PFGE molecular typing indicated that the PFGE types digested by Not I expressed significant diversity. Inaba and O139 strains from cases were gathered in the same clusters, while the Ogawa strains from cases scattered in different clusters but no significant correlation among these strains were found. Our results suggested that a distant genetic relationship might exist between these two different sources strains., Conclusion: Complex and diverse as the virulence genes and genetic characteristics and with the grim situation of multi-drug resistant strains, all seemed important to strengthen the surveillance programs on the variation of strain types and antibiotics resistance of O1/O139 V. cholerae in Guangdong province.