Objective To isolate and identify the pathogenic bacteria from peripheral blood of a patient with septicemia of unknown etiology and to analyze their drug resistance genes.Methods Two peripheral blood samples were collected from the patient after having fever.Several assays including culturing bacteria on blood agar plates by using streaking technique,Gram-staining of bacterial colonies and microscopic observation,VITEK 2-compact automatic bacterium detection and analysis system as well as a sequencing analysis of the 16s rRNA gene were performed to identify the bacterial pathogens in blood samples.Microdilution test was performed to detect the drug susceptibilities of isolated bacteria to antibiotics.Confirmatory tests were performed to detect the production of β-lactamase and extended spectrum β-lactamase by the isolated strains and the phenotypes of AmpC enzyme and carbapenemase.PCR was used to identify the β-lactamase-encoding genes in the isolated strains by using the primers of 19 common β-lactamase-,AmpC enzyme-and carbapenemase-encoding genes in Enterobacteriaceae strains and the primers of 21 annotated gene sequences encoding the β-lactamase of a Ralstonia mannitolilytica strain.The PCR products were sequenced and analyzed after T-A cloning.Results Ralstonia mannitolilytica strains were isolated from the two peripheral blood samples.The isolated strains were sensitive to ceftriaxone,cefepime,ciprofloxacin,ofloxacin,tigecycline and compound sulfamethoxazole (SMZ-TMP),but resistant to the other 11 tested antibiotics.Results of PCR amplification by using the primers of common genes encoding β-lactamase of Enterobacteriaceae strains were all negative.Fragments of genes encoding the β-lactamase of the isolated Ralstonia mannitolilytica strain were successfully amplified,which were TK49_09850,TK49_12955,TK49_14470,TK49_14495and TK49_18990.The sequences of the amplified gene fragments were not similar to those of the common β-lactamase-encoding genes in Enterobacteriaceae strains.Conclusion The patient was infected with Ralstonia mannitolilytica.The isolated Ralstonia mannitolilytica strain showed a high-level drug resistance with a noticeable diversity against different β-lactam antibiotics.The genes encoding β-lactamase of the isolated Ralstonia mannitolilytica strain were completely different to those of Enterobacteriaceae strains.

We isolated and identified the bacterial pathogen in a pyogenic balanoposthitis patient and investigated the drug resistance and its mechanism of the isolate.Urethral secretions and balanus pustule liquids were collected for microscopic examination after Gram-staining and detection of mycoplasma using Mycoplasma IST 2 kit.The two samples were inoculated on Columbia blood plate,N.gonorrhoeae selective plate and chromID Candida plate for isolation.The obtained colonies were identified by VITEK 2-compact automatic bacterial detection and analysis system.Moreover,PCR was performed to detect 16S rRNA gene of N.gonorrhoeae in the samples and colonies.KB method was applied for detecting susceptibility of five common antibiotics against the isolate.The β-lactamase and extended spectrum β-lactamase confirmatory tests were used to investigate the enzyme production of the isolate as well as drug resistance-associated tetM,TEM,mefA and ermF genes in the isolate were detected by PCR.Results showed that all the clinic samples showed negative for mycoplasma.All the isolating cultivation results of urethral secretions were negative while the balanus pustule liquids provided positive isolating cultivation in the blood and selective plates.The VITEK 2-compact system and 16S rRNA-PCR revealed that the isolated strain belongs to N.gonorrhoeae.The isolate can produce β-lactamases and resist to penicillin G,ciprofloxacin and tetracycline.The tetM,TEM,mefA and ermF genes could be found in the isolate's genome.The patient's balanoposthitis is caused by infection of N.gonorrhoeae.The multidrug resistance of Neisseria gonorrhoeae isolate is closely associated with its carried resistant genes.

Objective To develop a simple high-resolution melting ( HRM) analysis method for differentiation of Pc and P2 variants in class 1 integron.Methods DNA fragments containing Pc and P2 variants were amplified from plasmids pACW ( PcW ) and pACWP2 ( PcW-P2 ) respectively , then these purified PCR products and P 2 promoters were analyed full-length amplicon by HRM .Eight DNA fragments containing different Pc promoters were amplified and site-specific mutated from plasmids pACS ( PcS ) , pACH2 ( PcH2 ) , pACH1 ( PcH1 ) , pACW ( PcW ) , genomic DNA of Klebsiellar pneumonia HS07-68 (PcWTGN-10)and HS05-1792(PcH2TGN-10)respectively.The purified PCR products and eight Pc variants were characterized by HRM analyses of an unlabeled probe and full-length amplicon.This assay was applied to the differentiate Pc and P2 variants in 109 class 1 integrons from 95 urine clinical Escherichia coli isolates in Huashan Hospital during 2004 -2007.The differentiation results were compared with that determined by direct sequencing .Results P2 promoter with a significant higher melting temperature ( Tm ) can be identified by HRM analysis clearly .P2 promoters were identified in 2 class 1 integrons and consistent with direct sequencing results .Eight Pc variants were classified into three groups: PcS, PcSTGN-10 , PcW, PcWTGN-10, PcH1, PcH1TGN-10.Using direct HRM analysis.PcH2, PcH2TGN-10 were classified into four groups:PcS, PcH1, PcH2, PcW, PcSTGN-10 , PcH1TGN-10 , PcH2TGN-10 , PcWTGN-10 according to the melting curves of the unlabeled probe .Combined the HRM analyses of the whole amplicon and unlabeled probe , the eight Pc variants can be differentiated from each other .Five different Pc variants, PcS, PcW, PcH1, PcH2TGN-10 and PcWTGN-10 , were identified and consistent with direct sequencing results .Conclusions This developed a simple Pc and P 2 variants differentiation method via simultaneous HRM analyses of an unlabeled probe and full-length amplicon .This method is cost-effective and accurate , could be used in differentiation of Pc and P2 variants of class 1 integrons in clinical isolates .

Objective To establish a high resolution melting based method for the rapid identifica-tion of functional class 2 integron.Methods Nighty-nine non-repetitive Proteus spp.strains positive for genes encoding class 2 integrase were isolated from August, 2011 to August, 2012.The genomic DNAs were extracted and used as templates to amplify 60 base pair fragments containing the mutated point in class 2 in-tegrase gene by PCR.The high resolution melting analysis was conducted to identify the functional class 2 in-tegrons that were further compared by sequence analysis.Results There were remarkable differences with the high resolution melting curves between the functional class 2 integrons and the ordinary class 2 integrons. The results of high resolution melting analysis were consistent with those by using sequence analysis.Con-clusion High resolution melting analysis could be used for the rapid and accurate identification of functional class 2 integron.

The aim of this study is to determine the diversity of virulence genes carried by different vancomycin-resistant enterococci (VRE),which will provides a basis for studying pathogenic mechanism of VRE.Microdilution-based drug sensitivity test was applied to detect the vancomycin resistance of 490 Enterococcus faecium isolates and 862 Enterococcus faecalis isolates in Zhejiang area.The seven virulence genes (ace,asa1,cylA,efaA,esp,gelE and hyl) in the isolates of VRE were detected by PCR.According to the results of drug sensitivity test,10％ of the E.faecium isolates (49/490) and 0.8％ of the E.faecalis (7/862) were identified as VRE.In the vancomycin-resistant E.faecium isolates,five isolates were negative for any of the target genes and the other 44 isolates were positive for asa1,esp,gelE and hyl genes alone,in which the esp (73.5％,36/49) and hyl (53.1％,26/49) were the predominant genes and single or double virulence genes acted as the major carrying models.Except for the hyl gene,the vancomycin-resistant E.faecalis isolates were positive for the other six pathogenic genes,and the isolates could carry 3-6 pathogenic genes.All the data indicate that E.faeciurn is the major species of VRE in the local area,and the carrying rate,types and models of virulence genes in the vancomycin-resistant E.faecium and E.faecalis isolates are obviously different.

We investigated correlation between the level of miR-16 expression and the severity of Staphylococcus aureus sepsis,and further explored its potentially clinical significance.Blood samples were collected from 32 patients,including each 8 cases of septic shock,severe sepsis and general sepsis,as well as 8 cases of healthy volunteers.Blood samples from 24 cases of healthy subjects with different ages were measured,and additionally 8 cases of blood samples from gram negative bacteria sepsis were also determined in current study as a control.Trizol solution for the whole blood lysis was added into blood samples,and followed by the extraction of microRNA.The expression levels of miR-16 in different groups were measured by fluorescence quantitative PCR,in which 2-Delta Ct method was used.SPSS software (version 13.0) was used to analyze the statistical differences between the groups,and further analyze the correlation between miR-16 value and the corresponding CRP and PCT values.Results showed that the expression level of miR-16 was negatively correlated with the severity of Staphylococcus aureus sepsis.There were statistically significant differences in experimental groups when compared with the control (P＜0.001),and there was also a statistically significant difference between each experimental group (P＜0.01).We found that the expression level of miR-16 was negatively correlated with CRP and PCT,the correlation coefficients were-0.561 and-0.769 respectively,and trend analysis showed that there was a significantly negative correlation.A significantly negative correlation was found between the miR-16 expression level and severity of sepsis,suggesting that miR-16 may serve as a biomarker for the severity of Staphylococcus aureus sepsis.

Objective To investigate the molecular types of carbapenem-non-susceptible Esche-richia coli ( E.coli) isolates and their mechanism of carbapenem resistance .Methods Twenty-two carbap-enem-non-susceptible E.coli strains were isolated from 3 hospitals in Hangzhou from 2007 to 2011.The mini-mum inhibitory concentrations ( MICs) of antimicrobials to those isolates were determined by agar dilution method and E-test.The molecular mechanisms of carbapenem resistance of E.coli isolates were analyzed by conjugation experiment,PCR and DNA sequencing.Pulsed-field gel electrophoresis (PFGE),multilocus se-quence typing ( MLST ) , and phylogenetic typing were performed to analyze the molecular epidemiology of those isolates.Results The MICs of imipenem and meropenem to 22 E.coli isolates were ranged from 1 μg/ml to 16 μg/ml,and the MICs of ertapenem were 2 μg/ml to 64 μg/ml.All E.coli isolates produced the KPC-2 carbapenemase and various β-lactamases , and some of them also produced plasmid-mediated AmpC enzymes.Carbapenem resistance was transferred by conjugation and transformation from 22 E.coli iso-lates to E.coli EC600 strains.The E.coli transconjugants or transformants acquired the blaKPC-2 gene and showed similar antibiotic susceptibility patterns in comparison with donor strains .Only a few isolates were in-distinguishable or closely related as indicated by PFGE .Four sequence types including ST131 (9 isolates), ST648 (5 isolates),ST38 (2 isolates) and ST405 (2 isolates) were identified by MLST.Phylogenetic analy-sis indicated that 9 ST131 isolates belonged to phylogenetic group B 2 and the other isolates belonged to group D (11 isolates),group B1 (1 isolate) and group A (1 isolate),respectively.Conclusion The sequence type of prevalent E.coli isolates producing KPC-2 from Hangzhou was ST131,which is an international epi-demic,multidrug-resistant clone,followed by ST648.

OBJECTIVETo investigate the drug resistance, β-lactamase-encoding genes and protoporphyrin ferrochelatase-encoding genes of, and to explore its structure and pathogenic function.

METHODSThe strain was isolated by plate streaking method and identified by automatic bacteria detection system and 16S RNA gene PCR. Microdilution method was applied for drug susceptibility test. β-lactamases, extended spectrum β-lactamases (ESBL) and carbapenemases were detected using nitrocefin-disk, Kirby-Bauer disk, and Hodge test, respectively. Five β-lactamase-encoding genes and protoporphyrin ferrochelatase-encoding gene of the isolate were amplified by PCR for sequencing. Bioinformatic softwares were used to analyze the structure and function of the product of protoporphyrin ferrochelatase-encoding gene.

RESULTSA strain belonging towas isolated. This isolate was sensitive to cefepime, ciprofloxacin, ofloxacin and tigecycline, but resistant to five penicillins, four cephalosporins and two carbapenems antibiotics. The isolate produced β-lactamases but did not produce ESBL and carbapenemases. The isolate had five distinct β-lactamase-encoding genes and protoporphyrin ferrochelatase-encoding gene. The product of protoporphyrin ferrochelatase-encoding gene contained two functional domains of protoporphyrin ferrochelatase belonging to type Ⅱ chelatase superfamily that presented the most closely genetic relationship with the protoporphyrin ferrochelatase of.

CONCLUSIONSThe isolate ofhas a higher resistance to β-lactam antibiotics and its β-lactamase-encoding genes are different with the common bacterial β-lactamase-encoding genes. Protoporphyrin ferrochelatase may act as an important virulence factor of.