Application of a core genome sequence typing (cgMLST) pipeline for surveillance of Clostridioides difficile in China.

Introduction: Clostridioides difficile (C. difficile) is a nosocomial bacterial pathogen that causes antibiotic-associated diarrhea mediated by cellular exotoxins secreted into the intestine during bacterial growth. Multilocus sequence typing (MLST) and PCR ribotyping are the main molecular typing for C. difficile. Whole genome sequencing (WGS) core genome multilocus sequence typing (cgMLST) was developed for genetic evolution and outbreak investigation of C. difficile with higher precision and accuracy. Methods: A total of 699 whole (complete and draft) genome sequences of distinct C. difficile strains were used in this study to identify core gene set (2469 core genes) and the cgMLST scheme for the phylogeny analysis of C. difficile. This cgMLST pipeline was then carried the Chinese Pathogen Identification Net (China PIN) for surveillance of C. difficile in China. Within the China PIN, 195 WGS of C. difficile and an outbreak of CDI with 12 WGS of C. difficile were used to evaluate the cgMLST pipeline. Results: The result displayed that mostly tested C. difficile isolates could be successfully divided into 5 classic clades and the outbreak event was also successfully identified. Discussion: The results are meaningful and offer a practicable pipeline for a national-wide surveillance of C. difficile in China.

Genetic Diversity, Antibiotic Resistance, and Pathogenicity of Aeromonas Species from Food Products in Shanghai, China.

Objective: Aeromonas has recently been recognized as an emerging human pathogen. Aeromonas-associated diarrhea is a phenomenon occurring worldwide. This study was designed to determine the prevalence, genetic diversity, antibiotic resistance, and pathogenicity of Aeromonas strains isolated from food products in Shanghai. Methods: Aeromonas isolates ( n = 79) collected from food samples were analyzed using concatenated gyrB- cpn60 sequencing. The antibiotic resistance of these isolates was determined using antimicrobial susceptibility testing. Pathogenicity was assessed using ß-hemolytic, extracellular protease, virulence gene detection, C. elegans liquid toxicity (LT), and cytotoxicity assays. Results: Eight different species were identified among the 79 isolates. The most prevalent Aeromonas species were A. veronii [62 (78.5%)], A. caviae [6 (7.6%)], A. dhakensis [3 (3.8%)], and A. salmonicida [3 (3.8%)]. The Aeromonas isolates were divided into 73 sequence types (STs), of which 65 were novel. The isolates were hemolytic (45.6%) and protease-positive (81.0%). The most prevalent virulence genes were act (73.4%), fla (69.6%), aexT (36.7%), and ascV (30.4%). The results of C. elegans LT and cytotoxicity assays revealed that A. dhakensis and A. hydrophila were more virulent than A. veronii, A. caviae, and A. bivalvium. Antibiotic resistance genes [ tetE, blaTEM, tetA, qnrS, aac(6)-Ib, mcr -1, and mcr-3] were detected in the isolates. The multidrug-resistance rate of the Aeromonas isolates was 11.4%, and 93.7% of the Aeromonas isolates were resistant to cefazolin. Conclusion: The taxonomy, antibiotic resistance, and pathogenicity of different Aeromonas species varied. The Aeromonas isolates A. dhakensis and A. hydrophila were highly pathogenic, indicating that food-derived Aeromonas isolates are potential risks for public health and food safety. The monitoring of food quality and safety will result in better prevention and treatment strategies to control diarrhea illnesses in China.

Comparative Study of the Genetic Diversity, Antimicrobial Resistance, and Pathogenicity of Aeromonas Isolates from Clinical Patients and Healthy Individuals.

OBJECTIVE: This study was performed to compare the genetic diversity, virulence, and antimicrobial resistance of Aeromonas strains isolated from patients and healthy individuals. METHODS: A total of 38 clinical strains and 19 strains from healthy individuals were isolated from the samples collected in Ma'anshan City, Anhui Province. Their taxonomy was investigated using concatenated gyrB- cpn60 sequences, and their resistance to 12 antibiotics was evaluated. The pathogenicity of these strains was examined through beta-hemolysis, protease activity, and virulence gene assays. RESULTS: The 57 Aeromonas strains were divided into 55 sequence types. Of these types, 21 were novel, suggesting that their genetic diversity was high. These Aeromonas isolates could be divided into 7 species, and the positive rates of beta-hemolysis and protease activity were 49.1% and 73.7%, respectively. The detection rate of clinical patients in terms of beta-hemolysis and protease activity was higher than that of healthy individuals. Among the four most common Aeromonas strains, A. dhakensis had the highest detection rate of virulence genes. The multidrug resistance rate of the clinical isolates was much higher than that of the strains isolated from healthy individuals. CONCLUSIONS: The taxonomy, virulence properties, and antibiotic resistance of Aeromonas isolates from patients differ from those of the isolates from healthy individuals.

Genetic Diversity, Antimicrobial Resistance, and Virulence Genes of Aeromonas Isolates from Clinical Patients, Tap Water Systems, and Food.

OBJECTIVE: This study aimed to evaluate the genetic diversity, virulence, and antimicrobial resistance of Aeromonas isolates from clinical patients, tap water systems, and food. METHODS: Ninety Aeromonas isolates were obtained from Ma'anshan, Anhui province, China, and subjected to multi-locus sequence typing (MLST) with six housekeeping genes. Their taxonomy was investigated using concatenated gyrB-cpn60 sequences, while their resistance to 12 antibiotics was evaluated. Ten putative virulence factors and several resistance genes were identified by PCR and sequencing. RESULTS: The 90 Aeromonas isolates were divided into 84 sequence types, 80 of which were novel, indicating high genetic diversity. The Aeromonas isolates were classified into eight different species. PCR assays identified virulence genes in the isolates, with the enterotoxin and hemolysin genes act, aerA, alt, and ast found in 47 (52.2%), 13 (14.4%), 22 (24.4%), and 12 (13.3%) of the isolates, respectively. The majority of the isolates (≥ 90%) were susceptible to aztreonam, imipenem, cefepime, chloramphenicol, gentamicin, tetracycline, and ciprofloxacin. However, several resistance genes were detected in the isolates, as well as a new mcr-3 variant. CONCLUSIONS: Sequence type, virulence properties, and antibiotic resistance vary in Aeromonas isolates from clinical patients, tap water systems, and food.

Two Cases of Multi-antibiotic Resistant Cronobacter spp. Infections of Infants in China.

Infections by Cronobacter spp. are hazardous to infants since they can lead to neonatal meningitis, bacteremia, and necrotizing enterocolitis. Cronobacter spp. are frequently resistant to ß-lactam derivatives, macrolides, and aminoglycosides. In addition, multi-resistant strains have also been detected. In China, the isolation rate of Cronobacter spp. from commercial powdered infant formula (PIF) or follow-up formula (FUF) is relatively high. Nevertheless, clinical cases of Cronobacter infection have been ignored to date. Here we describe two cases of Cronobacter infection detected at the Wuhan Women and Children Medical Care Center Hospital (Wuhan City, China). We provide the genomic analysis of the isolates and the antibiotic-resistance profiles of the two strains. The Cronobacter strains identified in this study were not susceptible to third-generation cephalosporins, aminoglycoside, and/or trimethoprim-sulfamethoxazole. Whole genome sequencing revealed various genes known to encode antibiotic resistance. Future studies are needed to determine whether the genes predicted in this study are functional. As with Enterobacter spp., the antibiotic resistance of Cronobacter is a serious issue that requires more attention.

Effects of common polymorphisms in miR-146a and miR-196a2 on lung cancer susceptibility: a meta-analysis.

BACKGROUND: MicroRNAs (miRNAs) may play an important role in organ development, cell differentiation, apoptosis, proliferation, cell growth regulation and act as tumor suppressor genes or proto-oncogenes. Single nucleotide polymorphisms (SNPs) in miRNAs are considered to be genetic factors to influence the susceptibility to lung cancer (LC). Rs2910164 in miR-146a and rs11614913 in miR-196a2 are shown to be associated with increased/decreased LC risk. The aim of this meta-analysis was to systematically summarize the possible association. METHODS: The relevant articles were retrieved from several important databases. Studies were selected using specific inclusion and exclusion criteria. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated to assess the strength of association between miRNA polymorphism and susceptibility to LC. All analyses were performed using the Stata software. RESULTS: Seven studies were included in this meta-analysis. There were 3,225 cases and 3,268 controls for SNP rs2910164 and 2,794 cases and 2,840 controls for SNP rs11614913. The significant associations between SNP rs2910164 and LC risk were observed (CC vs. GG: OR =1.30, 95% CI: 1.13-1.50; CC + GC vs. GG: OR =1.15, 95% CI: 1.02-1.29; CC vs. GC + GG: OR =1.27, 95% CI: 1.13-1.42; C vs. G: OR =1.15, 95% CI: 1.08-1.24). SNP rs11614913 was found to be associated with LC risk in most genetic models (TC vs. TT: OR =1.16, 95% CI: 1.02-1.32; CC vs. TT: OR =1.24, 95% CI: 1.06-1.44; CC + TC vs. TT: OR =1.19, 95% CI: 1.06-1.34; C vs. T: OR =1.11, 95% CI: 1.03-1.20). In the subgroup analysis by ethnicity, genotyping method and control characteristics, significantly affected LC risks were also suggested. CONCLUSIONS: The rs2910164 in miR-146a and the rs11614913 in miR-196a2 are likely to be associated with LC risks.

Multilocus sequence typing analysis of Cronobacter spp. isolated from China.

Multilocus sequence typing (MLST) has proven to be an effective approach for the subtyping isolates of the Cronobacter genus and to exhibit a high level of discrimination between isolates. In this study, 151 Cronobacter strains were isolated from different sources and provinces across China from 2010 to 2012 and analyzed by MLST. Their sequence type profiles were compared with strains from other countries which were widely geographically and temporally distributed. Out of 151 strains in this study, the majority of strains were Cronobacter sakazakii (70.9 %), C. malonaticus (15.9 %), C. dublinensis (10.6 %), C. turicensis (2.0 %), and C. muytjensii (0.7 %). The strains were divided into 85 sequence types (STs), among which only 17 had previously been reported in other countries. The 85 identified STs for the Cronobacter genus were grouped into 14 clonal complexes and 47 singletons according to eBURST algorithm. The Cronobacter isolated from China showed a high diversity when they were subtyped using the MLST method. When compared to the Cronobacter PubMLST database, some sequence types of strains cultured from food and/or water in this study were also the same with strains isolated from patients in other countries as reported previously. This result showed the potential hazard of strains contaminating water and weaning food from China.

TP63 gene polymorphisms, cooking oil fume exposure and risk of lung adenocarcinoma in Chinese non-smoking females.

BACKGROUND: Genetic polymorphisms of TP63 have been suggested to influence susceptibility to lung adenocarcinoma development in East Asian populations. This study aimed to investigate the relationship between common polymorphisms in the TP63 gene and the risk of lung adenocarcinoma, as well as interactions of the polymorphisms with environmental risk factors in Chinese non-smoking females. METHODS: A case-control study of 260 cases and 318 controls was conducted. Data concerning demographic and risk factors were obtained for each subject. The genetic polymorphisms were determined by Taqman real-time PCR and statistical analyses were performed using SPSS software. RESULTS: For 10937405, carriers of the CT genotype or at least one T allele (CT/TT) had lower risks of lung adenocarcinoma compared with the homozygous wild CC genotype in Chinese nonsmoking females (adjusted ORs were 0.68 and 0.69, 95%CIs were 0.48-0.97 and 0.50-0.97, P values were 0.033 and 0.030, respectively). Allele comparison showed that the T allele of rs10937405 was associated with a decreased risk of lung adenocarcinoma with an OR of 0.78 (95%CI=0.60-1.01, P=0.059). Our results showed that exposure to cooking oil fumes was associated with increased risk of lung adenocarcinoma in Chinese nonsmoking females (adjusted OR=1.58, 95%CI=1.11-2.25, P=0.011). However, we did not observe a significant interaction of cooking oil fumes and TP63 polymorphisms. CONCLUSION: TP63 polymorphism might be a genetic susceptibility factor for lung adenocarcinoma in Chinese non-smoking females, but no significant interaction was found with cooking oil fume exposure.

Lack of influence of an XRCC3 gene polymorphism on oral cancer susceptibility: meta-analysis.

BACKGROUND: To systematically summarize the association between the X-ray repair cross complementing 3 (XRCC3) gene polymorphism and oral cancer susceptibility by meta-analysis. MATERIALS AND METHODS: Databases including PubMed, EMbase, CNKI, VIP and WanFang Data were searched to identify case-control studies concerning the association between an XRCC3 gene polymorphism and the risk of oral cancer from the inception to June 2014. Two reviewers independently screened the literature according to the criteria, extracted the data and assessed the quality. Then meta-analysis was performed using Stata 11.0 software. RESULTS: Seven published case-control studies including 775 patients with oral cancer and 1922 controls were selected. Associations between the rs861539 polymorphism and overall oral cancer risk were not statistically significant in all kinds of comparison models (CT vs CC: OR=0.94, 95%CI=0.74-1.18; TT vs CC: OR=0.94, 95%CI=0.64- 1.38; dominant model: OR=0.95, 95%CI=0.76-1.18; recessive model: OR=0.94, 95%CI=0.69-1.29; allele T vs C: OR=0.97, 95%CI=0.84-1.11). In the stratified analysis by ethnicity, no significant associations were found among Asians and Caucasians. On stratification by tumor type, no significant associations were found for cancer and oral premalignant lesions. CONCLUSIONS: The XRCC3 gene polymorphism was not found to be associated with the risk of oral cancer. Considering the limited quality of the included case-control studies, more high quality studies with large sample size are needed to verify the above conclusion.

Cronobacter carriage in neonate and adult intestinal tracts.

A total of 7 Cronobacter strains were isolated from 703 fecal samples collected in Jinan from June 13 to December 30, 2011, with the positive rate of Cronobacter spp. being 1.0% (95% confidence interval 0.6%-1.4%). Three Cronobacter sakazakii stains were isolated from 157 fecal samples of healthy neonates (95% confidence interval 0.4%-5.5%). This number was slightly higher than that isolated from 273 fecal samples of healthy adults, in which 1 strain of C. sakazakii and 1 strain of Cronobacter malonaticus were isolated, and that from 173 fecal samples of adults with acute diarrhea, in which 1 strain of C. sakazakii and 1 strain of C. malonaticus were isolated, but the differences were not statistically significant (P>0.05). The Cronobacter isolates were all from different genetic sources. It should be noted that Cronobacter carriage may cause infection under certain conditions, especially in neonates.

[Molecular characterization of Vibrio parahaemolyticus collected from human infections in Shenzhen, between 2002 and 2008].

OBJECTIVE: To determine the occurrence and distribution of specific clones of pathogenic Vibrio parahaemolyticus(VP)isolated in Shenzhen and to assess the relationship between serotype O3:K6 and the globally distributed pandemic clone. METHODS: A total of 1005 VPs isolated from diarrhea patients in 2002-2008 were sero-typed. Real-time PCR was used to detect the virulence genes tlh, toxR, tdh, trh and orf8 in 281 isolates from 68 different serotypes. The main serotypes were typed by pulsed field gel electrophoresis(PFGE). Strains with dominant serotypes and PFGE patterns were assayed by GS-PCR and toxRS sequencing for the identification of pandemic clone. Multilocus sequence typing(MLST)analysis was reserved for exemplary 41 O3 : K6 and O1 : K25 isolates. RESULTS: Seventy-nine serotypes were observed among the 1005 isolates, including O3 : K6(57.9%), O4 : K8(8.16%), O1 : KUT(5.87%), O1 : K25(5.27%), O4 : K68(1.39%), O1 : K56(1.39%) and O9 : K44(0.99%). Most of the strains(99.36%)showed PCR positive to tlh, toxR, and tdh but eleven strains were tdh negative. MLST showed that all the 36 O3 : K6 isolates belonged to ST3 and all the 5 O4 : K8 strains were ST189. These results matched the description of the pandemic VP clone. CONCLUSION: A recognizable burden of diarrheal illness caused by VP had been seen in Shenzhen. Results from serotyping indicated that although there existing a large variety of diversities, the dominant serotype appeared to be O3 : K6. VP isolates identified in Shenzhen mainly showed as tdh positive but trh negative, in consistent with the current pandemic O3 : K6 clone. The pandemic O3 : K6 clone did appear to co-exist with other clones of O3 : K6, as well as O4 : K8,O1 : K25. Potential outbreak of VP could be monitored through the laboratory-based surveillance programs, suggesting that the strategies related to prevention and control of VP should be prioritized in Shenzhen.

[The fundamental role of stage control technology on the detectability for Salmonella networking laboratory].

OBJECTIVE: To evaluated the fundamental role of stage control technology (SCT) on the detectability for Salmonella networking laboratories. METHODS: Appropriate Salmonella detection methods after key point control being evaluated, were establishment and optimized. Our training and evaluation networking laboratories participated in the World Health Organization-Global Salmonella Surveillance Project (WHO-GSS) and China-U.S. Collaborative Program on Emerging and Re-emerging infectious diseases Project (GFN) in Shanghai. Staff members from the Yunnan Yuxi city Center for Disease Control and Prevention were trained on Salmonella isolation from diarrhea specimens. Data on annual Salmonella positive rates was collected from the provincial-level monitoring sites to be part of the GSS and GFN projects from 2006 to 2012. RESULTS: The methodology was designed based on the conventional detection procedure of Salmonella which involved the processes as enrichment, isolation, species identification and sero-typing. These methods were simultaneously used to satisfy the sensitivity requirements on non-typhoid Salmonella detection for networking laboratories. Public Health Laboratories in Shanghai had developed from 5 in 2006 to 9 in 2011, and Clinical laboratories from 8 to 22. Number of clinical isolates, including typhoid and non-typhoid Salmonella increased from 196 in 2006 to 1442 in 2011. The positive rate of Salmonella isolated from the clinical diarrhea cases was 2.4% in Yuxi county, in 2012. At present, three other provincial monitoring sites were using the SBG technique as selectivity enrichment broth for Salmonella isolation, with Shanghai having the most stable positive baseline. CONCLUSION: The method of SCT was proved the premise of the network laboratory construction. Based on this, the improvement of precise phenotypic identification and molecular typing capabilities could reach the level equivalent to the national networking laboratory.

[Application of PulseNet China Database in tracking and warning 4 cholerae outbreaks in Anhui province, in 2012].

OBJECTIVE: To track the source of infection regarding 4 Cholerae outbreaks in Anhui province in 2012 through the application of PulseNet China Database (PNCD). METHODS: Cholerae virulence genes were amplified by PCR and typed by pulse field gel electrophoresis(PFGE). Results from electrophoresis were cluster-analyzed by BioNumericsV4.0 software and compared with PNCD. RESULTS: Virulence gene CT and TCP of the tested vibrio cholera showed both positive. Homology of the strains from four cholera outbreaks was more than 98%, based on the homologous and cluster analysis through enzyme digested PFGE electrophoresis. Those strains were highly homologous with the cholera epidemic strains identified in Hunan, Sichuan,Zhejiang, Shanghai and Hubei by PNCD, with the homology as 100% . CONCLUSION: Four cholera outbreaks in Anhui province, 2012 were highly correlated with the outbreaks occurring in Hunan and Sichuan during the same time period, indicating that PNCD could effectively and quickly tracking down the source of infection on the cholera outbreaks and providing early warning of the situation.

[The therapeutic effect analysis of three kinds of methods for the management of post traumatic knee stiffness].

OBJECTIVES: To investigate the therapeutic effect of rehabilitation, arthroscopy and "hybrid technique" for posttraumatic knee stiffness (PTKS), and to make the best choice for the treatment. METHODS: From February 2004 to November 2009, 66 patients suffered from PTKS were treated, and the clinical data were studied retrospectively, 36 male and 30 female patients with an average age of 41 years were analyzed, knee stiffness time averaged 15 months (0.5 - 108.0 months), 21 cases of patients were treated with rehabilitation (rehabilitation group), 22 cases of patients with arthroscopy + rehabilitation (arthroscopy group) and 23 cases of patients with mini-invasive "hybrid technique" + rehabilitation (hybrid technique group). For each case, the difference of range of motion (ROM) and hospital for special surgery (HSS) score of the knee before and after the treatment were analyzed statistically. The characters of PTKS including the course of the disease, the degree of extensor mechanism involving, physical examination and other ancillary data were also analyzed. The management methods for PTKS were summarized. RESULTS: Total 66 cases were followed up ranging from 24.0-72.5 months and the mean time was 34.2 months. The average ROM was improved obviously: rehabilitation group increased from 45° ± 22° to 95° ± 24° (t = -11.2, P < 0.05), arthroscopy group from 47° ± 26° to 118° ± 11° (t = -11.0, P < 0.05) and hybrid technique group from 36° ± 22° to 110° ± 14° (t = -13.4, P < 0.05). Both ROM and HSS score of the knee before and after the treatment for each group showed significant difference statistically (t = -9.1, -6.0, -5.2, P < 0.05). Wound necrosis, tearing, re-fracture and extension lag were not found. According to Judet standard at final follow-up, 15 cases were excellent, 3 cases good and 3 cases normal in rehabilitation group; 15 cases were excellent, 5 cases good and 2 cases normal in arthroscopy group; 14 cases were excellent, 8 cases good and 1 case bad. CONCLUSIONS: Pathology of PTKS is complex, satisfactory result could be obtained through individualized treatment program, which were established depend on the course of the disease, the degree of extensor mechanism involving, physical examination and ancillary data. The timely and effective surgical interference followed by a comprehensive rehabilitation program is the key point for satisfied outcome.

[Multilocus variable-number tandem-repeat analysis for molecular subtypes of Shigella isolates in Beijing].

OBJECTIVE: Selecting variable-number tandem-repeat (VNTR) loci for different serogroups of Shigella spp to explore and establish multilocus variable-number tandem-repeat analysis (MLVA) method, in order to study the molecular characteristic of the isolated strains. METHODS: Of the Shigella strains found by dysentery surveillance in Beijing from 2001 to 2009, 180 strains were selected for this study, according to the number and serotypes of the surveillant strains, at the ratio of 15%; including 50 strains of Shigella sonnei and 130 strains of Shigella flexneri. After screening the polymorphism of the 18 VNTR loci, 10 VNTR loci (sh1-sh10) were retained and constructed three groups of multi-PCR methods to detect all he 180 strains and analyze MLVA molecular subtypes using capillary segments. RESULTS: A range of 2 to 11 alleles were found on the 10 VNTR loci among the 180 Shigella strains, with a diversity index value between 0.158 and 0.766. The 10 loci showed diversity in different serogroups, such as only one allele found in sh6 of Shigella flexneri, sh2 and sh3 of Shigella sonnei individually. The isolated 180 strains were divided into 84 MLVA subtypes, with a resolution ratio D value at 0.967 (95%CI: 0.956 - 0.978). The 130 strains of Shigella flexneri were divided into 63 subtypes, named as TF001-TF063; among which TF001, TF002 and TF 005 were the dominant subtypes, accounting to 17, 16 and 15 strains respectively. The 50 strains of Shigella sonnei were divided into 21 subtypes, named as TS001-TS021; among which TS002 (14 strains) and TS001 (7 strains) were the dominant subtypes. CONCLUSION: MLVA subtyping method including 10 VNTR loci was preliminarily developed. The MLVA cluster analysis revealed that the subtypes of Shigella strains isolated in Beijing were diverse, and suggested the possibility of multiple-clone source.

p-Synephrine stimulates glucose consumption via AMPK in L6 skeletal muscle cells.

Interest in p-synephrine, the primary protoalkaloid in the extract of bitter orange and other citrus species, has increased due to its various pharmacological effects and related adverse effects. The lipolytic activity of p-synephrine has been repeatedly revealed by in vitro and in vivo studies and p-synephrine is currently marketed as a dietary supplement for weight loss. The present study investigated the effect of p-synephrine on glucose consumption and its action mechanism in L6 skeletal muscle cells. Treatment of L6 skeletal muscle cells with p-synephrine (0-100µM) did not affect cell viability and increased basal glucose consumption up to 50% over the control in a dose-dependent manner. The basal- or insulin-stimulated lactic acid production as well as glucose consumption was significantly increased by the addition of p-synephrine. p-Synephrine stimulated the phosphorylation of AMPK but not of Akt. p-Synephrine-induced glucose consumption was sensitive to the inhibition of AMPK but not to the inhibition of PI3 kinase. p-Synephrine also stimulated the translocation of Glut4 from the cytoplasm to the plasma membrane; this stimulation was suppressed by the inhibition of AMPK, but not of PI3 kinase. Taken together, p-synephrine can stimulate glucose consumption (Glut4-dependent glucose uptake) by stimulating AMPK activity, regardless of insulin-stimulated PI3 kinase-Akt activity in L6 skeletal muscle cells.

[Assessment of the application of variable-number tandem repeat loci of Salmonella Enteritidis in subtyping multiple-locus variable-number tandem repeat analysis].

OBJECTIVE: To evaluate the feasibility of the application of variable-number tandem repeat (VNTR) loci of Salmonella Enteritidis (S. enteritidis) in subtyping mutiple-locus variable-number tandem repeat analysis (MLVA). METHODS: A total of 16 isolates of S.enteritidis from different place and time in China were preliminarily assessed by choosing 11 reported VNTR loci, the loci with single amplified bands were picked to subtype all 104 S. enteritidis isolates. The isolates were also analyzed by pulse field gel electrophoresis (PFGE) to compare the superiority or inferiority of MLVA method and PFGE method. RESULTS: Seven VNTR loci were selected from the preliminary screening to expand the analysis, and the 7 VNTR loci had grouped 104 of S.enteritidis isolates into either 16 MLVA subtypes or 22 PFGE subtypes, with the D value at 0.7222 and 0.7974, respectively. Comparing with the isolates in MLVA subtypes, the isolates in PFGE showed a stronger resolving power. Meanwhile the results in PFGE showed a more disperse frequency distribution than those in MLVA. CONCLUSION: These results indicate that some VNTR locus which have shown a good polymorphism internationally, may fail to show polymorphism in China, thereby, more VNTR loci should be included in MLVA and the wide screening may benefit the unity of global laboratorial methods.

[Molecular typing of Legionella pneumophila isolates from China with pulsed field gel electrophoresis].

OBJECTIVE: To analyze the molecular types of Legionella (L.) pneumophila strains isolated in China, and to develop the PulseNet-China Database of L. pneumophila. METHODS: Pulsed field gel electrophoresis (PFGE) was used to analyze 262 L. pneumophila strains collected from 11 provinces between 2004 and 2009 in China. Different kinds of genomic DNA in different L. pneumophila strains were isolated and separated after digesting with Asc I. BioNumerics software was used to analysis the PFGE fingerprints. RESULTS: L. pneumophila strains isolated in China were quite different regarding their PFGE patterns. There were 108 PFGE types among the 262 strains tested in this study. The similarity value of these strains was in the range of 16% - 100% and the same types were discovered in different provinces and years. CONCLUSION: L. pneumophila strains isolated in China were with high genetic variations. There might be different clones existed in China. The development of PulseNet China Database was thus of great significance in monitoring the L. pneumophila strains in the future.

[Epidemiological characteristics and molecular typing of Salmonella in Beijing from 2008 to 2009].

OBJECTIVE: To study the epidemiological characteristics and molecular phenotypes of Salmonella by pulsed-field gel electrophoresis (PFGE) in Beijing from 2008 to 2009. METHODS: A total of one hundred thirty-seven isolates recovered from the WHO Global Salmonella Surveillance system and entero clinic surveillance system were identified by biochemical tests and serotyping. The related epidemiological informations were also analyzed. The isolates were further typed by PFGE. RESULTS: The prevalence of Salmonella from 2008 to 2009 showed obvious seasonal character. High incidence occurred from June to September, and 64.1% (84/131) isolates were recovered in this period. Patients of 18 - 40 year-old were 46.1% (58/128) and 80 patients were male and 40 patients were female with the ratio of 1.57:1. These 137 Salmonella isolates belonged to 20 serotypes, including Enteritidis (46.7%, 64/137) and Typhimurium (17.5%, 24/137) as the dominant serotype. In total, 71 PFGE profiles were identified. Four PFGE patterns of S. Enteritidis isolates (JEGX01.CN0001, JEGX01.CN0003, JEGX01.CN0002, JEGX01.CN0019) and S. Typhimurium pattern of JPXX01.CN0001 were dominant patterns. CONCLUSION: The prevalence of Salmonella from 2008 to 2009 showed distribution characteristics of sex, age and seasons. The numerous PFGE patterns of Salmonella showed diversity of these isolates and different clones existed in Beijing.

cAMP antagonizes ERK-dependent antiapoptotic action of insulin.

Insulin has antiapoptotic activity in various cell types. However, the signaling pathways underlying the antiapoptotic activity of insulin is not yet known. This study was conducted to determine if cAMP affects the antiapoptotic activity of insulin and the activity of PI3K and ERK in CHO cells expressing human insulin receptors (CHO-IR). Insulin-stimulated ERK activity was completely suppressed by cAMP-elevating agents like as pertussis toxin (Ptx) and cholera toxin (Ctx) after 4 h treatment. Insulin-stimulated PKB/Akt activity was not affected at all. Ptx treatment together with insulin increased the number of apoptotic cells and the degree of DNA fragmentation. Ctx or 8-brcAMP treatment also increased the number of apoptotic cells and stimulated the cleavage of caspase-3 and the hydrolysis of PARP. Taken together, cAMP antagonizes the antiapoptotic activity of insulin and the main target molecule of cAMP in this process is likely ERK, not PI3K-dependent PKB/Akt.