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MPLA derivatives are expected to be a promising platform for the development of new adjuvants used for a RBD-hFc based SARS-CoV-2 vaccine.

Salmonella is widely known as one of the most common foodborne pathogens, and antibiotics remain effective in clinical therapies against its infections. To guide better clinical antibiotic treatment, we analyzed the antimicrobial resistance (AMR) profiles of a nation-wide collection of 36,822 Salmonella isolates derived from sporadic diarrhea cases in China from 2014 to 2021. A panel of 15 antibiotics, including 10 critically important and 5 highly important antimicrobial agents for human medicine based on the WHO CIA List, was selected for AMR surveillance. Salmonella enterica serovar Typhimurium, Enteritidis, I 4,[5],12:i:-, London, and Stanley were turns to be the top five serotypes from human diarrhea cases in China. Antimicrobial susceptibility testing revealed that the majority of the isolates (87.2%) were resistant to at least one antimicrobial agent, and 66.5% were multidrug resistant (MDR). Salmonella isolates were found to be highly resistant to ampicillin (73.4%) while sensitive to imipenem (98.73%). Over the eight years, the isolates were demonstrated generally an increase in resistance to ampicillin, chloramphenicol, trimethoprim-sulfamethoxazole, and azithromycin, but displayed varied resistance profiles in terms of serotypes. Stanley (0.66–27.18%) and Agona (0.78–45.30%) had lower resistance rates compared to Typhimurium (1.11–85.6%), Enteritidis (1.55–91.29%), and I 4,[5],12:i:- (1.02–94.28%). In conclusion, our results provide systematic data on the resistance characteristics of Salmonella isolates from human diarrhea cases in China. Furthermore, this data identifies priorities for the clinical treatments of antibiotics.

Currently circulating SARS-CoV-2 Omicron variants feature highly mutated spike proteins with extraordinary abilities in evading acute-infection-induced germline antibodies isolated earlier in the pandemic. We identified that memory B cells from Delta variant breakthrough-infection patients expressed antibodies with more extensive somatic hypermutations (SHMs) allowing isolation of a number of broadly neutralizing antibodies with activities against heterologous variants of concerns (VOCs) including Omicron variant. Structural studies identified that SHM introduced altered amino acids and highly unusual HCDR2 insertions respectively in two representative broadly neutralizing antibodies - YB9-258 and YB13-292. Previously, insertion/deletion were rarely reported for antiviral antibodies except for those induced by HIV-1 chronic infections. Identified SHMs involved heavily in epitope recognition, they broadened neutralization breadth by rendering antibodies resistant to VOC mutations highly detrimental to previously isolated antibodies targeting similar epitopes. These data provide molecular mechanisms for enhanced immunity to heterologous SARS-CoV-2 variants after repeated antigen exposures with implications for future vaccination strategy.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been redetected after discharge in some coronavirus disease 2019 (COVID-19) patients. The reason for the recurrent positivity of the test and the potential public health concern due to this occurrence are still unknown. Here, we analyzed the viral data and clinical manifestations of 289 domestic Chinese COVID-19 patients and found that 21 individuals (7.3%) were readmitted for hospitalization after detection of SARS-CoV-2 after discharge. First, we experimentally confirmed that the virus was involved in the initial infection and was not a secondary infection. In positive retests, the virus was usually found in anal samples (15 of 21, 71.4%). Through analysis of the intracellular viral subgenomic messenger RNA (sgmRNA), we verified that positive retest patients had active viral replication in their gastrointestinal tracts (3 of 16 patients, 18.7%) but not in their respiratory tracts. Then, we found that viral persistence was not associated with high viral titers, delayed viral clearance, old age, or more severe clinical symptoms during the first hospitalization. In contrast, viral rebound was associated with significantly lower levels of and slower generation of viral receptor-binding domain (RBD)-specific IgA and IgG antibodies. Our study demonstrated that the positive retest patients failed to create a robust protective humoral immune response, which might result in SARS-CoV-2 persistence in the gastrointestinal tract and possibly in active viral shedding. Further exploration of the mechanism underlying the rebound in SARS-CoV-2 in this population will be crucial for preventing virus spread and developing effective vaccines.

ObjectivesTo investigate the prevalence and transmission of mcr-3 among Salmonella enterica serotype Typhimurium and 1,4,[5],12:i:−.MethodsA total of 4724 clinical Salmonella isolates were screened for the presence of mcr-3 in China during 2014–19. The clonal relationship of the mcr-3-positive isolates and their plasmid contents and complete sequence were also characterized based on WGS data from the Illumina and MinION platforms.ResultsWe identified 10 mcr-3-positive isolates, and all were MDR, mostly resistant to colistin, cefotaxime, ciprofloxacin, doxycycline and florfenicol. mcr-3 was co-present with blaCTX-M-55-qnrS1 on hybrid ST3-IncC-FII conjugatable plasmids (n = 6) and an ST3-IncC non-conjugatable plasmid (n = 1) and embedded into a pCHL5009T-like IncFII plasmid on the Salmonella chromosome (n = 3). Four distinctive genetic contexts surrounded mcr-3 and all but one were closely related to each other and to the corresponding region of IncFII plasmid pCHL5009T. IS15DI was most likely the vehicle for integration of mcr-3-carrying IncFII plasmids into ST3-IncC plasmids and the chromosome and for shaping the MDR regions. In addition, a phylogenetic tree based on the core genome revealed a unique Salmonella lineage (≤665 SNPs) that contained these 10 mcr-3-positive isolates and another 38 (33 from patients) mcr-3-positive Salmonella from five countries. In particular, most of the 51 mcr-3-positive isolates belonged to ST34 and harboured diverse antibiotic resistance genes (ARGs), including mcr-3-blaCTX-M-55-qnrS1, and possessed similar ARG profiles.ConclusionsOur findings revealed global clonal spread of MDR ST34 Salmonella from clinical isolates co-harbouring mcr-3 with blaCTX-M-55 and qnrS1 and a flexibility of mcr-3 co-transmittance with other ARGs mediated by mobile genetic elements.

Gastroenteritis caused by non-O1/non-O139We obtained the whole genome sequence of 66Two outbreaks caused by non-O1/non-O139Our analysis indicated the risk of non-O1/non-O139

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes the COVID-19 pandemic, is rapidly evolving. Due to the limited efficacy of vaccination in prevention of SARS-CoV-2 transmission and continuous emergence of variants of concern (VOC), including the currently most prevalent Delta variant, orally bioavailable and broadly efficacious antiviral drugs are urgently needed. Previously we showed that adenosine analogue 69-0 (also known as GS-441524), possesses potent anti-SARS-CoV-2 activity. Herein, we report that esterification of the 5’-hydroxyl moieties of 69-0 markedly improved the antiviral potency. The 5’-hydroxyl-isobutyryl prodrug, ATV006, showed excellent oral bioavailability in rats and cynomolgus monkeys and potent antiviral efficacy against different VOCs of SARS-CoV-2 in cell culture and three mouse models. Oral administration of ATV006 significantly reduced viral loads, alleviated lung damage and rescued mice from death in the K18-hACE2 mouse model challenged with the Delta variant. Moreover, ATV006 showed broad antiviral efficacy against different mammal-infecting coronaviruses. These indicate that ATV006 represents a promising oral drug candidate against SARS-CoV-2 VOCs and other coronaviruses.

SummaryThe COVID-19 pandemic and the SARS-CoV-2 with its variants have posed unprecedented challenges worldwide. Existing vaccines have limited effectiveness against the SARS-CoV-2 variants. Therefore, novel vaccines to match current mutated viral lineages with long-term protective immunity are urgently in demand. In the current study, we for the first time designed a recombinant Adeno-Associated Virus 5 (rAAV5)-based vaccine named as rAAV-COVID-19 vaccine (Covacinplus) by using RBD-plus of spike protein with both the single-stranded and the self-complementary AAV5 delivering vectors (ssAAV5 and scAAAV5), which provides excellent protection from SARS-CoV-2 infection. A single dose vaccination induced the strong immune response against SARS-CoV-2. The induced neutralizing antibodies (NAs) titers were maintained at a high peak level of over 1:1024 even after more than one year of injection and accompanied with functional T-cells responses in mice. Importantly, both ssAAV- and scAAV-based RBD-plus vaccines exhibited high levels of serum NAs against current circulating variants including variants Alpha, Beta, Gamma and Delta. SARS-CoV-2 virus challenge test showed that ssAAV5-RBD-plus vaccine protected both young and old age mice from SARS-CoV-2 infection in the upper and the lower respiratory tracts. Moreover, whole genome sequencing demonstrated that AAV vector DNA sequences were not found in the genome of the vaccinated mice after one year vaccination, demonstrating excellent safety of the vaccine. Taken together, this study suggests that rAAV5-based vaccine is powerful against SARS-CoV-2 and its variants with long-term protective immunity and excellent safety, which has great potential for development into prophylactic vaccination in human to end this global pandemic.

Herein, we report that a recombinant fusion protein, containing a 457 amino acid SARS-CoV-2 receptor binding domain (RBD, residues 319-541) and a mouse IgG1 Fc domain, could induce highly potent neutralizing antibodies and stimulate humoral and cellular immunity in mice. The antibodies also effectively suppressed SARS-CoV-2 RBD binding to soluble ACE2, indicating that RBD-mFc may be further developed as a safe and effective SARS-CoV-2 vaccine.

Background: A novel coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is causing pneumonia-associated respiratory disease pandemic across the world. Until now, the viral prevalence patterns, radiological characters, and antibody responses at initial stage of SARS-CoV-2 infection have been reported. The prolonged viral RNA shedding and coexistence with viral specific IgG antibody in discharged patients have also been reported recently. To date, however, correlations among viral specific RNA shedding, radiological pattern shift, and serum neutralizing antibody response covering the whole acute- and convalescent- phase in patients infected with SARS-CoV-2 are unknown. Focusing on a family cluster, present study is aim to give a bird view on the correlation of neutralizing antibody responses with viral clearance and lung disease prognosis from acute to convalescent phases. Methods: We conducted a surveillance study on a familial cluster of COVID-19 patients from Jan 17 to Apr 27 (99 days), 2020, in Guangzhou, Guangdong province, China. Epidemiological study was performed to clarify the virus transmission inside a family. NPAs, stool, and serum samples were collected and conducted RT-PCR to test the presence of SARS-CoV-2. Serum was used to perform neutralizing antibody tests to determine dynamic levels of neutralizing antibody. Radiological images as well as high-resolution CT scans for each patients were continually recorded to monitor outcomes of lung recovery. Findings: In this family cluster, three members (P2, P3, P4) in Guangzhou were infected by SARS-CoV-2 due to a close contact with P1 (index), who came back Guangzhou from Wuhan on Jan, 17, 2020, while another two members (H1 and H2) were unsusceptible and negative to SARS-CoV-2 test. The patients (P1, P2, P3) presented varying degrees of clinical symptoms on admission including recurrent fever, cough, and dyspnea, while P4 was asymptomatic. CT images showed ground-glass opacities in lungs of P1-3. The median length from illness onset to discharge for four-confirmed cases was 21.3 (4.7) days (range from 15 to 26). However, all patients returned positive for SARS-CoV-2 in follow-up investigation. The mean time for RNA shedding was 36.5 (3.2) days in P1, P2, and P4. The neutralizing antibody of P1, P2, and P4 was detectable during illness day 16 to 22. Then, the level was apparently ascent to a relative high titer and remained stable during 34-40, 31-41, and 35 days of illness onset for P1, P2, and P4, respectively, while declined gradually with time. The last positive time for SARS-CoV-2 test for P1, P2, and P4 was 40, 35, and 34 days of illness onset, respectively. Follow-up chest CT showed good prognosis in lung disease of P1 and P2, and CT scan was normal for P4. P3 was an exceptional case in this family cluster, who experienced the longest RNA shedding time (up to 68 days) as well as lung disease. In particular, radiological worsen was observed at day 34 of illness onset, high-resolution CT image revealed a glass-ground opacity in middle segment of right lung since day 45 to 99 of illness onset. The neutralizing antibody level in P3 was low during the entire study and significantly lower than that in other patients. Interpretation: The efficiency of SARS-CoV-2 virus clearance and lung disease prognosis were closely related to the levels of neutralizing antibody in COVID-19 patients. Viral clearance was observed along with high level of neutralizing antibody in P1, P2, and P4. However, the level of neutralizing antibody in P3 was not high enough to clear the virus completely, leading a prolong RNA shedding and lung disease. We proposed that high level of neutralizing antibody was essential for viral clearance and lung recovery, and virus was thoroughly eliminated during the period when neutralizing antibody reached high level in serum. Funding Statement: This work was supported by grants from the Guangzhou Science and Technology Program (201904010012), Guangdong Provincial Science and Technology Program (2018B020207006), Guangdong Provincial Novel Coronavirus Scientific and Technological Project (2020111107001). Declaration of Interests: The authors declare no competing interests. Ethics Approval Statement: The study was approved by the Institutional Review Board of the Guangdong Second Provincial General Hospital. All patients involved in this study signed the clinical research informed consent.

Noroviruses (NoVs) are the leading cause of acute gastroenteritis (AGE) outbreaks. Since 2014, novel genetic variants of NoV have been continuously identified and have caused a sharp increase in the number of AGE outbreaks. The specific geographical distribution and expanding genetic diversity of NoV has posed a challenge to conventional surveillance. Here, we describe the long-term dynamic correlation between NoV distribution in sewage and in the local population through the molecular surveillance of NoV in Guangdong, 2013-2018. The relative viral loads of the GI and GII genotypes in sewage were calculated through RT-PCR. A high-throughput sequencing method and operational taxonomic unit (OTU) clustering pipeline were developed to illustrate the abundances of different genotypes and genetic variants in sewage. Our results showed that the NoV viral loads and the emergence of new variants in sewage were closely associated with NoV outbreak risks in the population. Compared with the outbreaks surveillance, the dominance of the newly emerged variants, GII.P17-GII.17 and GII.P16-GII.2, could be detected one or two months ahead in sewage of a hub city. In addition, the dynamics of pre-epidemic variants, which were rarely detected in clinics, could be captured through sewage surveillance, thus improving our understanding of the origin and evolution of these novel epidemic variants. Our data highlight that sewage surveillance could provide nearly real-time and high-throughput data on NoV circulation in the community. With the advances in sequencing techniques, the sewage surveillance system could also be extended to other related infectious diseases.

Objectives To understand persistence of the virus in body fluids and immune response of infected host to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), an agent of coronavirus disease 2019 (COVID-19). Methods We determined the kinetics of viral load in several body fluids through real time reverse transcription polymerase chain reaction (rRT-PCR), serum antibodies of IgA, IgG and IgM by enzyme linked immunosorbent assay (ELISA), and neutralizing antibodies by microneutralization assay in 35 COVID-19 cases from two hospitals in Guangdong, China. Results We found higher viral loads and prolonged shedding of virus RNA in severe cases of COVID-19 in nasopharyngeal (1.3×106 vs 6.4×104, p, Graphical abstract Image 1

SUMMARYVibrio cholerae O139 emerged as a causative agent of epidemic cholera in 1992 in India and Bangladesh, and was subsequently reported in China in 1993. The genetic relatedness and molecular characteristics of V. cholerae O139 in Guangdong Province, located in the southern coastal area of China, remains undetermined. In this study, we investigated 136 clinical V. cholerae O139 isolates from 1993 to 2013 in Guangdong. By conventional PCR, 123 (90·4%) isolates were positive for ctxB, ace and zot. Sequencing of the positive amplicons indicated 113 (91·7%) isolates possessed the El Tor allele of ctxB (genotype 3); seven carried the classical ctxB type (genotype 1) and three harboured a novel ctxB type (genotype 5). With respect to tcpA, 123 (90·4%) isolates were positive for the El Tor allele. In addition, pulsed-field gel electrophoresis (with NotI digestion) differentiated the isolates into clusters A and B. Cluster A contained seven of the non-toxigenic isolates from 1998 to 2000; another six non-toxigenic isolates (from 1998 and 2007) and all of the toxigenic isolates formed cluster B. Our results suggest that over a 20-year period, the predominant O139 clinical isolates have maintained a relatively tight clonal structure, although some genetic variance and shift has occurred. Our data highlight the persistence of toxigenic V. cholerae O139 in clinical settings in the southern coastal area of China.

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.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.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.The PCR system is specific and sensitive for the identification of SPA, SPB, SPC and ST.

Salmonella enterica serovar Weltevreden is the most common non-typhoid Salmonella found in South and Southeast Asia. It causes zoonoses worldwide through the consumption of contaminated foods and seafood, and is considered as an important food-borne pathogen in China, especially in the Southern coastal area. We compared the whole genomes of 44 S. Weltevreden strains isolated from human stool and contaminated food samples from Southern Coastal China, in order to investigate their phylogenetic relationships and establish their genetic relatedness to known international strains. ResFinder analysis of the draft genomes of isolated strains detected antimicrobial resistance (AMR) genes in only eight isolates, equivalent to minimum inhibitory concentration assay, and only a few isolates showed resistance to tetracycline, ciprofloxacin or ampicillin. In silico MLST analysis revealed that 43 out of 44 S. Weltevreden strains belonged to sequence type 365 (CC205), the most common sequence type of the serovars. Phylogenetic analysis of the 44 domestic and 26 international isolates suggested that the population of S. Weltevreden could be segregated into six phylogenetic clusters. Cluster I included two strains from food and strains of the "Island Cluster", indicating potential inter-transmission between different countries and regions through foods. The predominant S. Weltevreden isolates obtained from the samples from Southern coastal China were found to be phylogenetically related to strains from Southern East Asia, and formed clusters II-VI. The study has demonstrated that WGS-based analysis may be used to improve our understanding of the epidemiology of this bacterium as part of a food-borne disease surveillance program. The methods used are also more widely applicable to other geographical regions and areas and could therefore be useful for improving our understanding of the international spread of S. Weltevreden on a global scale.

Toxigenic conversion of environmental Vibrio 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 gIII(CTX) 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.

Vibrio parahaemolyticus has been the most common food-borne pathogen in southern China, especially the O3:K6 pandemic clone and its serovariants. Recently, the serotype O4:K8 became more and more prevalent in southern China, which was different from the O3:K6 pandemic clone. Thus, the aim of the present work was to elucidate the molecular characteristics of the O4:K8. Some O3:K6 pandemic clone and its serovariants isolated in the same period were selected for comparative analysis, which were still dominant clone locally. The whole genome sequencing (WGS) was applied to characterize 20 strains of V. parahaemolyticus isolated from food-borne diarrheal cases and belonging to the serotype O4:K8, O3:K6 and O1:KUT (untypable), prevalent serotypes in recent southern China. The results showed that all these isolates were positive for the thermostable direct hemolysin gene (tdh), while negative for the TDH-related hemolysin gene (trh). We compared the V. parahaemolyticus strains to those of 31 strains isolated overseas and were available from NCBI genome database. A WGS-SNPs phylogenetic analysis of all the genomes revealed that the strains formed an important genetic lineage, which was genetically distinct from the O3:K6, O1:KUT and other internationals strains. Comparative genome analysis also revealed that all the O4:K8 strains carried the entire T3SS-1 and VpaI-7 (T3SS-2) regions, the most important virulent elements of the O3:K6 pandemic clone. However, all the O4:K8 strains lacked the entire VpaI-1 and VpaI-4 regions and carried only few ORFs of the VpaI-5 and VpaI-6, which were considered to be unique among post-1995 strains belonging to the O3:K6 pandemic clone. Our data showed that the O4:K8 strains possessed the virulence factors similar to the O3:K6 pandemic clone, which may have enabled them to become prevalent in southern China. Our study also revealed that WGS-bases analysis may help improve understanding epidemiology of this bacterium in food-borne disease surveillance.

We report on inapparent infections in adult/commercial tilapia in major tilapia fish farms in Guangdong. A total of 146 suspected isolates were confirmed to be S. agalactiae using an API 20 Strep system and specific PCR amplification. All isolates were identified as serotype Ia using multiplex serotyping PCR. An MLST assay showed single alleles of adhP (10), atr (2), glcK (2), glnA (1), pheS (1), sdhA (3) and tkt (2) and this profile was designated ‘unique ST 7’. The analysis of virulence genes resulted in 10 clusters, of which dltr-bca-sodA-spb1-cfb-bac (62, 42.47%) was the predominant virulence gene profile. The PFGE analysis of S. agalactiae yielded 6 distinct PFGE types (A, B, C, D, F and G), of which Pattern C (103) was the predominant type, accounting for approximately 70.55% (103/146) of the total S. agalactiae strains. Therefore, unlike what has been found in juvenile tilapia, in which PFGE pattern D/F is the major prevalent pattern, we found that pattern C was the major prevalent pattern in inapparent infected adult/commercial tilapia in Guangdong, China. In conclusion, we close a gap in the current understanding of S. agalactiae epidemiology and propose that researchers should be alert for inapparent S. agalactiae infections in adult/commercial tilapia to prevent a potential threat to food safety.

To investigate the serotypes, antimicrobial resistance, virulence genes and molecular characteristics of food-borne Vibrio parahaemolyticus isolated in Guangdong province in 2014.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.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.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.

Salmonella is one of the most common foodborne pathogens in humans. Laboratory-based surveillance for non-typhoidal Salmonella infection was conducted in Guangdong Province, China to improve understanding about the disease burden and detection of dispersed outbreaks. Salmonella isolated from patients with diarrhea were sent from 16 sentinel hospitals to local public health laboratories for confirmation, serotyping, antimicrobial susceptibility testing, and pulsed-field gel electrophoresis (PFGE). PFGE patterns were analyzed to identify clusters representing potential outbreaks. Between September 2009 and October 2010, 352 (4%) Salmonella isolates were obtained from 9167 stool specimens. Salmonella enterica serotype Typhimurium (45%) and Salmonella enterica serotype Enteritidis (13%) were the most common serotypes, and multidrug resistance was high, especially in Salmonella Typhimurium isolates. PFGE patterns of obtained Salmonella isolates were found to be diverse, but a unique PFGE pattern comprising 53 Salmonella Typhimurium isolates were found to occur almost exclusively in infants. Epidemiologic studies are ongoing to determine whether a common exposure is the source of the Salmonella Typhimurium strain frequently isolated from infants.

To enhance the understanding of epidemiological impact of environmental Vibrio cholerae O139 strains, we characterized 10 clinical and 20 environmental isolates collected from human clinical samples and Pear River estuary during 2006 to 2008. Isolates were tested by PCR for eight virulence genes: cholera toxin (ctxA), zonula occludens toxin (zot), accessory cholera enterotoxin (ace), hemolysin (hlyA), NAG-specific heat-stable toxin (st), toxin-coregulated pilus (tcpA), outer membrane protein (ompU), and regulatory protein genes (tcpI). Genetic relatedness was assessed by pulsed-field gel electrophoresis (PFGE), and antibiotic susceptibility was determined using disk diffusion. Seven of eight virulence markers were detected in six clinical isolates and one environmental isolate. One clinical and one environmental isolate were positive for six virulence markers. 60% clinical isolates showed multi-drug resistance to tetracycline (TET), Nalidixic acid (NAL), chloramphenicol (CHL), and ampicillin (AMP), 70% were resistant to Trimethoprim + Sulfamethoxazole (SXT), while only 35% environmental strains were resistant to SXT. PFGE analysis revealed that the isolates in this study were formed three clusters. Cluster III was more related to strains from diarrheal patients than the strains in other clusters. Different from the clinical strains, most environmental strains lacked CTX and TCP gene clusters. Most environmental strains possess a single resistance profile, while most clinical isolates show multidrug resistant. PFGE analysis indicated the cluster III has more possibility to become a potential pathogenic clonal cluster.

Aims To investigate whether the Luminex xMAP® Salmonella Serotyping Assay (xMAP SSA) is applicable to serotype Salmonella from humans in southern China. Methods and Results Two hundred and five Salmonella isolates from diarrhoea patients were serotyped by xMAP SSA in parallel with the traditional serotyping. Forty serotypes were identified among 205 isolates; the most prevalent serotypes identified were Salmonella Enteritidis, Salmonella Stanley, Salmonella I 4,5,12:i:-, and Salmonella Typhimurium. One hundred and ninety-five (95·1%, 195/205) isolates were serotyped completely by xMAP SSA, while 10 stereotypes were partially detected as they were not included in the assay. The xMAP SSA correctly identified 192 (98·5%, 192/195) isolates. Five nonmotile and three monophasic strains, which possessed flagellar antigen genes that weren't expressed, were completely serotyped by xMAP SSA; however, these isolates were left undetected by the traditional method. Conclusion The xMAP SSA used in the study is a microsphere-based, molecular assay that could rapidly and accurately serotype Salmonella. It is suitable to identify the serovars of Salmonella in southern China. Significance and Impact of the Study The xMAP SSA, with high-throughput characteristics, provides an accurate and rapid serotyping system that dramatically strengthens the capability of clinical and public health laboratories for Salmonella serotyping.

China's Guangdong Province is located along the same latitude as Kolkata, India and Dhaka, Bangladesh, and is also considered a source of epidemic cholera. However, molecular description and the genetic relationships between Vibrio cholerae O1 El Tor isolates in Guangdong remain unclear. In this study, 381 clinical V. cholerae O1 isolates recovered from cholera cases presenting in Guangdong between 1961 and 2013 were investigated by PCR, amplicon sequencing and pulsed-field gel electrophoresis (PFGE). During this time frame, four distinct epidemic periods (1-4) were observed based on the different dominant serotype leading its epidemic, correspond to years; or time periods from/to 1961-1969, 1978-1989, 1990-2000, 2001-2013, respectively. Molecular analysis of representative isolates indicated that a single dominating clone was associated with each epidemic stage. All isolates from periods 1 and 2 carried the typical El Tor ctxB; this allele was displaced by classical ctxB beginning in 1993. However all isolates carried the El Tor-specific toxin-coregulated pili subunit A (tcpA). Isolates were grouped into five clusters on the basis of Not I enzyme digested PFGE, and the first four clusters were associated with specific periods, cluster I (period 1), II (period 3), III (period 2) and IV (period 4), respectively. While cluster V consisted of isolates from all four epidemic periods, but was most heterogeneous in appearance. Our data indicate genetic variations that shape the relationship among emerging isolates of V. cholerae O1 in Guangdong Province contribute to the 7th global pandemic.

Background Non-typhoidal Salmonella is a common cause of infectious diarrhea in humans. Antimicrobial-resistant Salmonella has become a global concern. Methods Using laboratory-based surveillance system for Salmonella from September 2009 to December 2012 in Guangdong Province of China. The clinical information and samples of diarrhea patients were collected, according to the surveillance case definition. The lab tests were followed by standardized protocols, including sample isolation, isolates confirmation, serotyping, and antimicrobial susceptibility testing (AST). Results A total of 1,826 Salmonella isolates were identified from40,572 patients in 28 hospitals in11 prefectures. The isolates ratio was highest in autumn (38.8%, 708/1826) and lowest in winter (6.4%, 117/1826). Children aged 5 years. Resistance to at least one antimicrobial was found in 72% (1321/1,826) of the isolates. Resistance to at least three antimicrobials was found in 46% (850/1,826) of the isolates. Resistance to all 12 antimicrobials screened was observed in 8 isolates (0.44%, 8/1,826). The resistant prevalence to quinolones including nalidixic acid and ciprofloxacin was 61.9% (1131/1826), of which ciprofloxacin resistance rate was 8.05% (147/1826). The prevalence resistance to all three cephalosporin antimicrobials (cefepime, cefotaxime, and caftazidime) in

To study the serotypes, antimicrobial resistance, virulence genes and molecular characteristics of Vibrio parahaemolyticus isolated from outbreaks and sporadic cases in Guangdong, 2013.36 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).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.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.

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.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.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.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.

To analyze the etiologic characteristics of O1/O139 Vibrio cholerae in Guangdong province in 2009-2013.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.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.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.

Background Salmonella enterica serovar Typhimurium is the most important serovar associated with human salmonellosis worldwide. Here we aimed to explore the molecular epidemiology and genetic characteristics of this serovar in Guangdong, China. Methodology We evaluated the molecular epidemiology and genetic characteristics of 294 endemic Salmonella Typhimurium clinical isolates which were collected from 1977 to 2011 in Guangdong, China, and compared them with a global set of isolates of this serovar using epidemiological data and Multilocus Sequence Typing (MLST) analysis. Principal Finding The 294 isolates were assigned to 13 Sequencing types (STs) by MLST, of which ST34 and ST19 were the most common in Guangdong. All the STs were further assigned to two eBurst Groups, eBG1 and eBG138. The eBG1 was the major group endemic in Guangdong. Nucleotide and amino acid variability were comparable for all seven MLST loci. Tajima’s D test suggested positive selection in hisD and thrA genes (p0.05). In addition, The Tajima’s D test within each eBG using the global set of isolates showed positive selection in eBG1 and eBG138 (p0.05). We also analyzed the phylogenetic structure of Salmonella Typhimurium from worldwide sources and found that certain STs are geographically restricted. ACSSuT was the predominant multidrug resistance pattern for this serovar. The resistant profiles ACSSuTTmNaG, ACSSuTTmNa and ACSuTTmNaG seem to be specific for ST34, and ASSuTNa for ST19. Conclusion Here we presented a genotypic characterization of Salmonella Typhimurium isolates using MLST and found two major STs are endemic in Guangdong. Our analyses indicate that genetic selection may have shaped the Salmonella Typhimurium populations. However, further evaluation with additional isolates from various sources will be essential to reveal the scope of the epidemiological characteristics of Salmonella Typhimurium in Guangdong, China.

Vibrio parahaemolyticus infection has been considered the leading cause of bacterial illnesses mainly associated with seafood consumption in Guangdong province in China. In this study, epidemiological and etiological characteristics of 36 V. parahaemolyticus outbreaks that occurred from 2008 to 2010 in Guangdong province were analyzed; 284 strains involved were characterized by serotyping; virulence genes and 66 strains from four outbreaks therein were subtyped by pulsed-field gel electrophoresis (PFGE). Epidemiological evidence showed that 36% (13/36) of outbreaks were caused by food contamination, of which 84.6 % (11/13) were related to salted food, including viscera of pigs and cattle, meat, and vegetable salad. It was also indicated that 88.9% (32/36) of V. parahaemolyticus outbreaks appeared from June to September, 44.4% (16/36) of which occurred in canteens as well as 41.7% (15/36) in restaurants. As for the etiology, 31% (11/36) of outbreaks were caused by single serovar of strains, while 69% (25/36) were caused by multiserovars; O3:K6, O4:K8, O1:Kut, and O2:K3 were the dominant serovars. Among the 284 strains, 98.8% (254/257) of strains from patients were tdh-present and trh-absent, whereas 37.0% (10/27) from food were tdh-present. Cluster analysis of PFGE patterns demonstrated that strains in the same outbreak with identical serovar seemed to be diversified, whereas strains with various serovars could be closely related genetically. Moreover, cross-contamination between salted food and seafood was first confirmed by molecular subtyping in Guangdong, revealing that salted food might be a vital risk factor associated with V. parahaemolyticus outbreaks.

To the Editor: Brucellosis is one of the most prevalent zoonotic diseases in the world. It is principally an animal disease, but globally, >500,000 human cases are reported each year (1). Transmission to humans occurs primarily through contact with infected animals and consumption of contaminated food (2,3). Persons with occupational exposure are at highest risk for brucellosis, in particular those performing husbandry activities, butchering, and livestock trading (4,5). Although brucellosis has been eradicated from many industrialized countries, new foci of disease continually appear, particularly in parts of Asia (6–8). In China, 160,214 brucellosis cases were reported during 2005–2010; 90% of them occurred in 6 northern agricultural provinces: Neimenggu, Shanxi, Heilongjiang, Hebei, Jilin, and Shaanxi. Livestock, such as goats, cattle, and pigs, are the main infectious source. However, factors such as the rapid movement of people from northern to southern China, increased livestock trading, and lack of livestock quarantine mean that infected livestock or their products readily traverse provincial borders and transmit disease to persons who have no direct contact with livestock. With an illness rate of 400). The male:female ratio among these patients was 66:46. The age ranges were similar by sex; male patients were 18–71 (median 47) years of age, and female patients were 20–70 (median 43) years of age. The first 3 cases of brucellosis in 2005 were reported in Shenzhen in Guangdong Province. One case was culture confirmed by clinical laboratory, and the isolate was identified as Brucella melitensis biovar 3 by SAT and phage biotyping. The other 2 cases were in dairy farm workers; their infections were laboratory confirmed by SAT but could not be identified by biovar. Since 2005, more cities in Guangdong have reported brucellosis cases (Figure, Appendix). The Pearl River Delta region reported 100 cases: 48 in Guangzhou, 27 in Shenzhen, 7 in Zhongshan, 6 in Foshan, 6 in Jiangmen, 4 in Zhuhai, and 2 in Dongguan. Only 12 cases were reported from undeveloped rural areas in Guangdong: 5 in Zhaoqing, 2 in Yangjiang, and 1 each in Huizhou, Qingyuan, Meizhou, Maoming, and Yunfu. Figure Reported cases of brucellosis, by city, Guandong Province, China, 2005–2010. A total of 42 Brucella isolates were cultured during 2005–2009, and all were identified as B. melitensis biovar 3. However, of 19 Brucella isolates cultured during 2010, a total of 13 were identified as B. melitensis biovar 3, 4 as B. melitensis biovar 1, and 2 as B. suis biovar 3. These results indicate a shift in species and biovar for Brucella spp. circulating in China. We conducted a retrospective epidemiologic investigation of the 112 brucellosis cases reported during 2005–2010 to identify the vehicles and sources of infection. Among the cases identified, 33 (29.46%) patients had occupational exposure history: 13 were pig or goat butchers, 12 dairy farmers, 5 animal market workers in charge of leading the animals to and from transportation, and 3 mutton and pork sellers in wet markets. The remaining 79 (70.54%) cases were in patients who denied having contact with living animals. Among these patients were retired persons, housekeeping matrons, teachers, doctors, white collar workers, and the unemployed. However, 17 of these patients recalled having purchased or handled goat placenta to be prepared for home consumption or having eaten goat products through barbequing or hot pot. The other 62 could not remember if they had contacted with livestock or their products. These findings indicate that nonoccupational exposure may pose a risk for brucellosis infection among persons who handle fresh meat and meat products for home cooking. In conclusion, Guangdong Province has become an emerging foci for brucellosis in China. The species and biovars of Brucella spp. circulating in this region are changing, and many persons are infected by nonoccupational exposure. Measures need to be taken by central and provincial governments to address these issues and prevent epidemics of brucellosis in humans.

Toxigenic conversion of environmental Vibrio 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 gIII(CTX) 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.

Although international clinical guidelines generally recommend performing bacterial stool culture in patients with acute diarrhea and fever and discourage routine antibiotic prescribing, clinical practice varies. Understanding practice patterns can help health officials assess the sensitivity of laboratory-based enteric infection surveillance systems and the need to improve antibiotic prescribing practices. We surveyed physicians in Guangdong province, China, to measure their practices for patients with acute diarrhea. A standardized questionnaire was used to interview physicians working in hospitals participating in a Salmonella surveillance system in Guangdong, China. The questionnaire asked physicians about their routine practice for patients with diarrhea, including how they managed the last patient they had seen with acute diarrhea. We calculated the odds ratio and 95% confidence interval for factors associated with ordering a stool culture and for prescribing antibiotics. We received surveys from 237 physicians across 22 hospitals in Guangdong. For the last patient with diarrhea whom they had evaluated, 134 (57%) reported ordering a stool culture. The most common reasons for not ordering a stool culture included that it takes too long to receive the result, that the patient is not willing to pay for the test, and that the patient's illness was too mild to warrant testing. Most physicians prescribed at least one medication for the last patient with diarrhea whom they had evaluated. Of the 237 physicians surveyed, 153 (65%) prescribed antibiotics, 135 (57%) probiotics, and 115 (49%), a gastric mucosal protective drug. In conclusion, physicians in Guangdong, China, reported high rates of ordering bacterial stool cultures from patients with diarrhea, possibly associated with their hospital's participation in a special surveillance project. The high rate of antibiotic prescribing suggests that efforts to promote judicious antibiotic use, such as physician education, are needed.

From September 2008 to December 2010, 112 Haemophilus parasuis strains were isolated from 536 pigs with clinical signs of Glässer’s disease in South China, for a frequency of 21%. The 112 strains were subjected to serovar analysis by gel diffusion (GD) and indirect hemagglutination (IHA) tests and to genotype analysis by means of pulsed-field gel electrophoresis (PFGE). With a combination of the GD and IHA results, serovars 5 and 4 were found to be the most prevalent, at 23% and 17%, respectively, followed by serovars 2 (8%), 15 (7%), 13 (6%), and 12 (5%); 20% of the strains were nontypeable. The 112 strains were genetically diverse, with 85 genotypes identified (discriminatory index 0.992). The 89 typeable isolates belonged to 15 H. parasuis serovars displaying 63 different PFGE profiles. The 23 nontypeable strains displayed 22 different PFGE profiles. These findings confirmed that 15 serovars and diverse genotypes of H. parasuis were widely distributed in southern China.

Environmental waters are an important reservoir for Vibrio cholerae , and effective surveillance of the pathogen can help to warn of and prevent infection with this potentially fatal pathogen. An immunofluorescent-aggregation (IFAG) assay to detect V. cholerae O1 and O139 was established and evaluated with estuarine water samples. The practical application of this assay was compared with the conventional culture method and real-time PCR. The IFAG method had a sensitivity of 10 3 CFU/ml for detection of V. cholerae O1 and O139 strains in a suspension containing 10 different species of enterobacterial strains (total, 10 5 CFU/ml). Ten fluorescent bacterial aggregate colonies were randomly picked and tested positive in serum agglutination tests for the V. cholerae O1 and O139 strains, showing a high specificity. The enrichment broths of 146 samples of estuarine water were tested, and the percentage positive by the IFAG assay was 19.9% (29/146), which was significantly higher than that of the conventional culture method (10.3%, 15/146; P < 0.01) but lower than that of real-time PCR (29.5%, 43/146; P < 0.01). The coincidence rates of real-time PCR and IFAG detection were decreased with the reduction of the V. cholerae concentration. The IFAG method, with a high specificity and a relatively high sensitivity, may be used for detection and isolation of V. cholerae in environmental water samples.