Objective To study the molecular characters of porA and porB genes which encode outer membrane proteins (OMP), and predominated clonal complex of Neisseria meningitidis isolates from Guangdong province. Methods Eighteen Neisseria meningitidis isolates from Guangdong province during year 1967 to 2007 were recovered and reconfirmed by API NH biochemical system, and serogrouped by antiserum. The characters of porA and porB gene were analyzed by DNA sequencing. The allele profiles and the sequence types (ST) were determined by multilocus sequence typing (MLST). Based on their allelic profiles, the evolution relationship was analyzed by PHYLIP software. The predominant clonal complex was determined through comparing with the information of reference strains from the PubMLST database. Results For porA gene, type 20 was more frequently in the variable region (VR) 1 and type 9 in VR2 before year 2004. However, for porB gene, type 4 was more frequently in VR Ⅰ, type7 in VR Ⅳ, type 11 in VR Ⅴ,and type 10 in VR Ⅵ, respectively. The multi-types character was presented in VR Ⅴ and VR Ⅵ after2004. VR Ⅶ and VR Ⅷ can not be found among all the isolates except for one W135 isolate in 2007. Among the seven housekeeping genes, the polymorphism of abcZ was the lowest one with 4 allele numbers, while pgm was the highest one with 13 allele numbers. The predominant clonal complex was ST-5 before 2004. The ST-4821 clone complex appeared in 2004 and caused cases every year since then. More important, highly invasive ST-11 clonal complex firstly appeared in Guangdong in 2007. Conclusion The molecular characteristic of OMP genes presents polymorphism for the Neisseria meningitidis isolates from patients in Guangdong province during 1967 to 2007. ST-5 is the predominant clonal complex before 2004 and the highly invasive clonal complex is circulating in recent 3 years. It suggests that the surveillance based on laboratory should be further enhanced.

Objective To investigate the polymorphism of Brucella melitensis biovar 3 ( B.melitensis biovar 3) strains isolated from Guangdong province .Methods PCR assays followed by agar-ose gel electrophoresis and capillary electrophoresis based on the multiple locus variable number of tandem repeats (VNTR) analysis (MLVA) were performed to analyze 43 clinic isolates of B.melitensis biovar 3 strains isolated from clinical patients in Guangdong province .Results MLVA typing showed that the simi-larities of the analyzed locus among 43 strains of Brucella ranged from 68.2%to 100%.32 genotypes identi-fied among the isolates were identical (100%similarity).27 out of 43 strains (62.8%) were single geno-types, while the other 16 strains (37.2%) belonged to 5 other genotypes with 2 to 5 strains in each of them . Conclusion B.melitensis biovar 3 isolates showed polymorphism distribution in Guangdong province as in-dicated by MLVA typing analysis .Single-genotype isolates accounted for 62.8% of all studied strains.No predominant genotype was found among all isolates .

ObjectiveTo study the toxin genes and pandemic group distribution as well as the genetic correlation between the major serotypes( O3:K6,O1:Kut,O4:K8 ) of Vibrio parahaemolyticus (VP) isolated from the outbreaks of Guangdong province.MethodsThe tdh and trh genes,GS-PCR and orf8 gene were detected on the 62 isolates sourced from patient and seafood occurred in the 23 outbreaks during 2008-2010.44 isolates of which were analyzed on PFGE digested by Not Ⅰ enzyme.ResultsToxin genes distributions suggested that 96.8% (60/62)isolates were tdh+,trh-.Three tdh+ isolates sourced from seafood were found.Pandemic group distribution suggested that 97.2% (35/36) O3:K6,5.88% (1/17) O4:K8,66.7% (8/9)O1:Kut serotype was GS-PCR+ and/or orf8+,respectively.PFGE analysis suggested that 44 isolates were separated into 3 clusters,of which the similarity of PFGE profile was 80.5% in the pandemic group cluster constructed by 28 isolates,the similarity between pandemic group and non-pandemic group was 59.5%.Pandemic group of O3:K6,O1:Kut as well as 04:K8 isolated on some outbreaks were processing the same PFGE profiles.ConclusionThe characteristic of toxin genes of major serotypes VP isolated in the outbreaks of Guangdong province form 2008-2010 was tdh- present and trh- absent.Within the pandemic group,O3:K6 and O1:Kut were the major serotypes.In single outbreak,isolates belongs to pandemic group but with different serotype seem to be close correlations.

Objective To investigate the etiological characteristics of Streptococcus pyogenes that caused scarlet fever in different periods in Guangdong province.Methods 22 isolates from different periods were analyzed through emm typing,PCR detection for super antigen genes,antibiotic susceptibility test and pulsed-field gel electrophoresis (PFGE).Results All isolates were susceptible to cefotaxim,levofloxacin and penicillin.Streptococcus pyogenes isolated after the year 2000 were 100％ resistant to erythrocin and clindamycin,but the resistant rate for strains isolated before the year 2000 was 9.1％ (1/11).There were 3 emm types indentified from 22 isolates including emm12.0 (59.09％,13/22),emm6.0 (36.36％,8/22) and emm1.0 (4.55％,1/22),which were detected in the isolates from the year 1997 and 2011,from 1978 and 1986,and from 2008,respectively.The positive rates for speA,speB,speC,speF,speG,speH,smeZ,and ssa genes detected by PCR were 54.55％,100％,100％,100％,100％,54.55％,0％,and 86.36％ respectively.Among all strains,95.45％ of the isolates carried 6 superantigen genes simultaneously.Three clusters of 10 PFGE subtypes were identified in 22 isolates.Cluster Ⅰ consisted of all strains from 1997 and one strain from 2011.Cluster Ⅱ consisted of strains isolated from 1978 and 1986.Cluster Ⅲ consisted of nine strains from 2011 and one from 2008.Conclusion S.pyrogenes isolates in Guangdong province were susceptible to penicillin but resistant to erythrocin.emm 12.0 accounted for the majority of the three types and there was a high frequency of super antigen genes.

To explore the possibility to type the Brucella strains isolated in Guangdong province with analytical method to detect the fatty acid components and to collect the basic data of fatty acid components of Brucella strains, 29 strains of Brucella were selected for analysis on the bacterial fatty acid components and the cluster analysis on the collected data was performed with Sherlock analysis soft-ware (MIDI). It was demonstrated that the main fatty acid components of Brucella strains isolated in Guangdong province were 19∶0 cycloω8c acid, 16∶0 acid and 18∶0 acid. The content of 19∶0cycloω8c acid was highest in B.abortus, followed by B.melitensis and lowest in B.suis.-In addition, the content differences of 19∶0cycloω8c and 18∶0 acid between B. melitensis and Brucella suis were statistically significant; and that of 19∶0cycloω8c and 18∶0 acid between strains isolated in 1965 and those isolated in recent 3 years was statistically significant. It was also shown that the fatty acid components of Brucella strains were stable, but the contents of fatty acid components were different in different species.-It is evident that at certain euclidean distance, 3 species of Brucella can be differentiated in species level.

Objective To develop a rapid and sensitive DNA microarray for Listeria monocytogenes detection.Methods A DNA microarray was developed using gyrB,ISR,16S rRNA,23S rRNA,hlyA,iap and prfA as the target genes and tested against 18 different species of known reference for repeatability,sensitivity,and specificity to verify the effectiveness of the chip.Results After testing of samples by the LM array,results show that the 70 mer Oligos synthesized by IDT are superior to the Oligos synthesized by Sagon with respect to both probe spotting or samples detection.The comparison of 3 spotting probe concentrations of 10 μmol/L,40 μmol/L and 80 μmol/L demonstrated that the 10 pmol/L probes result in good detection signals equivalent to the 40 μmol/L and 80 μmol/L probes.The repeatability and sensitivity evaluated by sample testing on the LM array revealed that the chips developed in this study have good repeatability and the lower limit of sample detection is 0.9 ng DNA.The LM array can distinguish clearly and definitively between Listeria and non-Listeria bacteria in the sample.Conclusion The microarray is able to rapidly detect and identify Listeria monocytogenes.

To compare and evaluate the discriminatory ability and potential value of pulsed field gel electrophoresis (PF-GE) and multiple locus VNTRs analysis (MLVA) on the genotyping of Brucella ,a total of 60 strains of Brucella and three standards (16M ,544A ,1330S) were genotyped simultaneously by PFGE and MLVA .The result indicated that the similarity coefficient among the 63 isolates was from 72 .1-100 .0% by PFGE ,and could distinguish three species of B .melitensis ,B .su-is and B .abortus at the similarity level of 94 .4% .There were 14 clusters and 29 PFGE types identified by PFGE with discrim-inatory index (DI) of 0 .957 5 at the similarity level of 100% ;the similarity coefficient among the 63 isolates was from 16 .9-100 .0% by MLVA ,and could distinguish three species of Brucella at the similarity level of 52 .3% .There were 8 clusters and 47 MLVA types identified by MLVA with discriminatory index (DI) of 0 .985 2 at the similarity level of 100% .It's suggested that PFGE and MLVA could be used to distinguish three species of Brucella in the similarity coefficient of certain ,but could not effectively distinguish the type in the same species .Both of these two methods could be used for Brucella molecular typing , but MLVA is better than PFGE for its relatively higher discriminating ability .

Objective To investigate the antimicrobial resistance pattern of non-typhoidal Salmonella isolated from diarrhea cases in Guangdong province,China.The multidrug-resistant strains were analyzed by pulsed field gel electrophoresis(PFGE) typing.Methods All the non-typhoidal Salmonella strains isolated between 2009 and 2011 were serotyped,then the antimicrobial resistance was detected by the disk diffusion method and molecular typed by PFGE.Results 91.76％ (256/279) S.typhimurium isolates were multiple resistant to 3 and more antimicrobials.Forty S.typhimurium isolates were multiple resistant to 9 and more antimicrobials and 3 out of which were multiple resistant to all the 12 antimicrobials in vitro.96.91％ (94/97) Salmonella I4,5,12:i:-isolates were multiple resistant to 3 and more antimicrobials.Nine Salmonella I4,5,12:i:- isolates were multiple resistant to 9 and more antimicrobials and I out of which was multiple resistant to all the 12 antimicrobials1 in vitro.47％ (47/100) S.enteritidis isolates were multiple resistant to 3 and more antimicrobials.Only 1 S.enteritidis isolates was multiple resistant to 9 and more antimicrobials.4.27％ (27/632) non-typhoidal Salmonella isolates was resistant to ciprofloxacin,including 17 S.typhimurium and 6 Salmonella 14,5,12:i:- isolates.Also,there were 3 1.96％ ( 202/632 ) non-typhoidal Salmonella isolates was intermediary to ciprofloxacin.The PFGE patterns of the predominant strains which were highly resistant and multidrug-resistant had different genotypes and demonstrated significant genetic diversity.Conclusion The situation about the multiple antimicrobial resistances of non-typhoidal Salmonella in Guangdong province has showed the prevalent problem.The PFGE types of the multiple drug-resistant strains prompted these strains were come from different clones.This requires that we continue to strengthen the resistance monitoring and control of the rational use of antibiotics.

Objective To prepare simulated stool specimens for proficiency testing ( PT) by mix-ing lentils with Salmonella, Shigella and Escherichia coli strains and to establish an assessment scheme for the detection of Salmonella and Shigella in clinical samples. Methods Salmonella, Shigella and Escherich-ia coli strains were respectively spiked to lentils in Cary-Blair transport medium to create simulated stool specimens. Various ratios of Escherichia coli to Salmonella strains were spiked to lentils to prepare mixed simulated stool specimens. The accuracy and stability of prepared stool samples for PT were tested in-house. Results of sample detection were collected from participating laboratories for further external quality assess-ment. Results The Escherichia coli and Salmonella strains mixed at ratios of 100 ∶ 1 to 106 ∶ 1 could be ef-ficiently isolated from the media. Enrichment was needed in order to effectively isolate Salmonella strains from the media when the ratios of Escherichia coli to Salmonella strains were 104 ∶ 1 to 106 ∶ 1. Of the16 participating laboratories, 14 laboratories (87. 5%) received a grade of“satisfactory” and the other 2 labo-ratories (12. 5%) received a grade of “mainly satisfactory”. Conclusion The simulated stool specimens and the PT procedures designed in this study were suitable for proficiency testing program on the detection of Salmonella, Shigella and other similar microbes.

Objective To analyze the serotype distribution and antibiotic resistance characteristics of Salmonella strains isolated in Guangdong province for better understanding the condition of Salmonella infection in patients with diarrhea.Methods Fecal samples collected from patients with diarrhea in Guangdong province were used to isolate Salmonella strains.Biochemical analysis was performed to identify these isolated strains.Serotyping and antimicrobial susceptibility testing were carried out for further analysis of the isolated Salmonella strains.Results The rate of Salmonella infection was 7.64％in 2015, and the male to female patient ratio was 1.52∶1.A total of 2 377 patients of all age groups were positive for Salmonella infection and the patients aged 0-6 years accounted for 81.74％.The isolation rate of Salmonella strains in the summer and autumn was higher than that in the winter and spring (10.73％ vs 4.24％;X2=463.77, P<0.01).The Salmonella isolation rates in different areas were as follows: 16.82％ in Zhuhai, 15.85％ in Heyuan, 11.81％ in Yangjiang, 10.68％ in Jiangmen, 8.49％ in Zhongshan, 8.07％ in Maoming, 8.05％ in Jieyang, 7.35％ in Shaoguan, 6.97％ in Foshan, 6.03％ in Dongguan, 5.48％ in Guangzhou and 0.00％ in Zhanjiang.And the differences between different regions were statistically significant (X2=367.67, P<0.01).The 2 377 isolated Salmonella strains were classified into 108 serotypes except for oneSalmonella strain that could not be classified.The top four predominant serotypes were 4,5,12:i:-, Salmonella enteritidis,Salmonella stanley and Salmonella typhimurium.Most Salmonella strains were sensitive to imipenem, azithromycin, ceftazidime, cefotaxime and trimethoprim/sulfamethoxazole, but multidrug resistance was common among those strains.Conclusion Salmonella serotypes of 4,5,12:i:-and Salmonella enteritidis are the predominant pathogens causing human Salmonella infections in Guangdong province.Ceftazidime and cefotaximeare are preferred in the treatment of Salmonella infections.Surveillance for drug resistance in Salmonella should be strengthened as multidrug resistant strains have become a serious problem in Guangdong province.