Objective To investigate the distribution of virulence genes and resistance genes in a group of Staphylococcus aureus clinical isolates.Methods Forty strains of Staphylococcus aureus isolated from Ningbo First Hospital during July and September 2013 were collected.Forty-two kinds of virulence genes and 11 kinds of resistance genes were analyzed by polymerase chain reaction (PCR),and binary typing were performed based on 10 classes of virulence genes and resistance gene mecA.Results Among 40 Staphylococcus aureus strains,5 (12.5％) were sensitive to penicillin,and 17 (42.5％) were sensitive to erythromycin; The sensitive rates to the remaining 15 antibiotics were all higher than 65.0％.The positive rates of adhesins,cytotoxins,capsular antigens,superantigens,serine proteases were 2.5％-100.0％; While map gene was not detected.Resistance genes to β-lactam,aminoglycoside,erythromycin,tetracycline,polymer disinfectant and antibacterial peptide were also positive with positive rates of 2.5％-37.5％.By binary typing,40 strains of Staphylococcus aureus were divided into 16 kinds of positive modes.At least 3 classes of virulence genes were positive in all strains,and 7 classes of virulence genes and resistance gene mecA were positive in strain No.36.Conclusion The phenotypes of antibiotic resistance are well correlated with genotypes in this group of Staphylococcus aureus isolates,which carry several virulence genes and resistance genes.

Objective To investigate resistant mechanisms of a pandrug-resistant Acinetobacter baumannii (js01) to β-1actams.Methods Strain js01 isolated from sputum sample of an inpatient from Ningbo First Municipal Hospital in December 2011 was confirmed by PCR amplifying and sequencing of gyrA and parC,and aligning with BLASTn.Thirty-three kinds of β-lactamase genes ( 13 kinds of class A,10kinds of class B,2 kinds of class C,8 kinds of class D),linkage detection of insertion sequences and β-lactamase genes,as well as outer membrane porin gene carO were analyzed by PCR.Genes encoding PBPI A were divided into three fragments,PCR amplified and bidirectional sequenced,and ligated to the full-length gene.Results Four kinds of β-lactamase genes were positive in js01:TEM-I,ADC-30, OXA-23 and OXA-66.Linkage detection of insertion sequences and β-lactamase genes showed that ISabal-ADC-30 and ISabal-OXA-23 were positive. When compared with sensitive strain (SDF) of Acinetobacter baumannii,sense mutations were found in carO gene of js01,and identity of amino acid sequence of carO gene between js01 and SDF was 76.0％ (189/249),and differences owed to loss of 3 amino acids.Sense mutations were also found in genes encoding PBP1A of js01,and identity of amino acid sequence of genes encoding PBP1A between js01 and SDF was 99.6％ ( 848/851 ),and differences owed to variations of 3amino acids.However,compared with three-dimensional structure of PBP1 A of SDF,PBP1 A of js01 lost 2helixes.Conclusion In strain js01,mutations of housekeeping genes ( genes encoding PBP1A and CarO),and genes producing β-lactamase mediated by mobile genetic elements,may play a key role in resistance to β-lactams.

Objective To investigate the distribution and variety of quinolone-resistance genes in multi-drug resistant strains of Escherichia coli (E. coli) isolated from urine. Methods From October 2008 to March 2009, 28 strains of multi-drug resistant E. coli isolated from urine were collected from Ningbo No. 1 Hospital, China. One kind of chromosome-mediated quinolone-resistance gene(gyrA) and 5 kinds of plas-mid-mediated quinolone-resistance genes[qnrA, qnrB, qnrS, aac(6')- Ⅰb-Cr, qepA]were analyzed by PCR and verificated by DNA sequencing. Results In 28 strains ofE. coli, only 1 strain was detected to harbor aac(6')-Ⅰb-Or (confirmed by DNA sequencing and genomic comparison with sequence registered in NC-BI), but qnrA, qnrB, qnrS, qepA could not be detected. Furthermore, all 28 strains(100.0%) contained mutations at 83rd coden in gyrA: TCG→TTG(mutations at 83rd amino acid: S83L). However, 22 strains (78.6%) contained mutation at 87th ceden in gyrA. Among them, 21 strains(75.0% ) contained muta-tions: GAC→AAC(mutations at 87th amino acid: D87N) ; while gyrA gene of NB005 (3.6%) contained mutations: GAC→TAC(mutations at 87th amino acid: D87Y), which was a new subtype(GenBank Acces-sion No. GQ286174). And other 6 strains contained no mutation at 87th coden. Conclusion All isolates of E. coli contained mutations in gyrA(100.0%), which play a key role in resistance to quinolones antimi-crobial agents, but positive rate of other resistance genes is low.

Objective To investigate the binding capacities of two variants of quinolone resistance-determining region in the DNA gyrase subunit A to substrates in Klebsiella pneumonia (K.pneumonia).Methods Tertiary structures of two variants (type Ⅰ and type FH) of quinolone resistance-determining region in the DNA gyrase subunit A in K.pneumonia were predicted by homology modeling referring to that of wild type.Then,DOCK module in ArgusLab 4.1 software was used to perform molecular docking of two variants and wild type to seven kinds of quinolones substrates,and calculate binding free energies (△G).Moreover,numbers and distances of interaction between amino acid residues of DNA gyrase subunit A and ciprofloxacin were calculated.Results Molecular docking showed that binding free energies of type Ⅰ and type FH to pipemidic acid,ciprofloxacin,gatifloxacin were-26.607 50,-29.530 39,-29.493 09 kJ/mol and-26.696 44,-28.972 83,-29.590 50 kJ/mol,respectively,which declined greater than those of wild type (-27.188 82,-30.872 00 and-30.244 04 kJ/mol,respectively) and showed drug resistance.While binding free energies of type Ⅰ and type FH to levofloxacin were-29.013 81 and-29.497 57kJ/mol,respectively,and that of wild type was-28.016 20 kJ/mol.The binding free energies of type Ⅰ and type FH to nalidixic acid,norfloxacin,ofloxacin increased or declined.Moreover,if distance was less than 5 angstroms,atom pairs formed between wild type of DNA gyrase subunit A and ciprofloxacin had 16 pairs,while type Ⅰ and type FH had 2 pairs and 4 pairs,respectively.If distance was less than 4 angstroms,atom pairs formed between wild type and ciprofloxacin had 8 pairs,while type Ⅰ and type FH had no atom pairs.Conclusion Decline of binding capacities of two variants of DNA gyrase subunit A in K.pneumonia to ciprofloxacin played a role in drug resistance.

Objective To perform molecular evolution analysis of mazEF gene in genome sequenced strains of Escherichia coli and Shigella. Methods Pathway Tools (version 13.5) provided by BioCyc was used to search encoding gene mazF of toxin MazF ( chpA) and encoding gene mazE of antitoxin MazE (chpR) in genome sequenced 10 strains of Escherichia coli, 6 strains of Shigella and 1 strain of unkown Enterobacteria. Then Minimum Evolution method in MEGA4. 1 software was used to analyze molecular evolution of MazE and MazF. Results Encoding gene mazF of toxin MazF was found in 12 strains, and encoding gene mazE of antitoxin MazE was found in 11 strains, while neither mazE nor mazF was found in rest 5 strains. Both mazE and mazF had good conservation in molecular evolution analysis. Conclusions MazEF is the first toxin-antitoxin system found in prokaryotic chromosomes, but not in all strains of Escherichia coli and Shigella. MazEF deletion is associated with antibiotic resistance and it also mediates programmed cell death in bacteria, so MazEF might be a new target for antimicrobial agents.

Objective To analyze molecular evolution and binding free energies in substrates of KPC-2,KPC-5 and KPC-10 carbapenemases.Methods Minimum Evolution method in MEGA 4.1 was used to analyze molecular evolution of KPC-2,KPC-5 and KPC-10 carbapenemases,Dock module in ArgusLab 4.1 was used to perform molecular docking of these 3 carbapenemases to 10 kinds of β-lactams substrates,and calculate binding free energies(△G).Results Ambler Class A β-lactamases with carbapenemase activities were grouped in the same cluster and had good conservation,while ordinary Ambler Class A β-lactamases without carbapenemase activities were groupod in the other cluster.Binding free energies of KPC-2,KPC-5 and KPC-10 carbapenemases were lower to carbapenem antibiotics than the thirdgeneration cephalosporins,while binding free energies to aztreonam and clavulanic acid were of comparatively higher levels.Conclusion Catalytic activities of KPC to carbapenem antibiotics are higher than those to the third-generation cephalosporins,but the activities to aztreonam and clavulanic acid are low.

We investigated molecular identification of a group of 14 strains of Aeromonas sp .,and genetic background of re‐sistance to beta‐lactams ,aminoglycosides .From January to December 2012 ,14 strains of Aeromonas sp .were collected from stool from diarrheal patients in enteric clinics in Ningbo First Hospital in Zhejiang Province ,China .Then ,molecular identifica‐tion by 16SrDNA ,23 kinds of beta‐lactamase genes ,6 kinds of aminoglycoside modifying enzyme genes ,6 kinds of 16srRNA methylase genes ,and 6 kinds of mobile genetic elements were analyzed by PCR .In addition ,genotyping and sample cluster a‐nalysis were performed .Results showed that 10 strains of A .hydrophila ,1 strain of A .aquariorum ,A .sobria ,A .entero‐pelogenes ,A .punctata were confirmed by 16SrDNA sequencing and arithmetic .Five kinds of beta‐lactamase genes ,4 kinds of aminoglycoside modifying enzyme genes ,and 3 kinds of mobile genetic elements were positive .BlaAQU of strain No .4(AQU‐2) and strain No .11(AQU‐3) were new subtypes .It’s suggested that identification of Aeromonas sp .should be performed by molecular identification method .This group of 14 strains of Aeromonas sp .conferred multidrug resistance .

Objective To investigate the distribution of acquired resistance-related genes and markers of mobile genetic elements, and their relationships in multidrug-resistant Escherichia coli. Methods From October 2008 to March 2009, 28 strains of multidrug-resistant Escherichia coli isolated from urine were collected from the Ningbo First Hospital. Then, 47 kinds of acquired resistance genes to beta-lactams, aminoglycosides, quinolones, 2 kinds of acquired drug efflux gene and 13 kinds of genetic markers of mobile genetic elements: conjugal plasmids, transposons, insertion sequences, and integrons were analyzed by PCR. The index cluster analysis was used to investigate their relationships. Results In 28 strains of Escherichia coli, 7 kinds of acquired beta-lactam-resistance genes, 8 kinds of acquired aminoglycosideresistance genes, 1 kind of acquired drug efflux gene, 2 kinds of genetic markers of conjugal plasmids, 3 kinds of genetic markers of transposon and insertion sequences, 1 kind of genetic marker of integron were detected; but other 46 kinds of genes were not detected. Two clusters, A and B, were divided by index cluster analysis depending on positive genes. Conclusions In this group of Escherichia coli, acquired resistance related genes may be associated with resistant phenotypes of antimicrobial agents. Horizontal transfer of mobile genetic elements may bring rapid spread of resistance of bacterial pathogens, not only among the same kind of pathogens, but also among the different kinds. In addition, index cluster analysis suggests that correlation might exist between acquired resistance-related genes and mobile genetic elements.

Objective To investigate the clinical distribution and the drug resistance of carbapenemresistant Proteus mirabilis strains isolated from 2009 to 2012 ; and to study carbapenemase genotypes of the isolates.Methods A total of 15 non-repetitive carbapenem-resistant Proteus mirabilis strains were selected from 422 Proteus mirabilis strains isolated from Ningbo First Hospital during January 2009 to December 2012.The minimal inhibitory concentrations (MIC) of 6 antibacterial agents,including imipenem (IPM),meropenem (MEM),ertapenem (ETP),ciprofloxacin (CIP),amikacin (AK) and minocycline (MIN),against 15 isolates were determined by E-test.The modified Hodge test (MHT) was used to detect the carbapenemase production in isolates.The PCR assay was performed to detect drug resistance genes of blaKPC,blaNDM-1,blaGES,blaSME,blaIMI-1/NmcA and blaSHV-38.Plasmids were extracted from the blaKPC-positive strains and then transformed into Escherichia coli J53 strains by electroporation.The transformed and untransformed Escherichia coli J53 strains were tested for MIC values and blaKPC gene by E-test and PCR respectively.Results The resistance rates of the 15 carbapenem-resistant Proteus mirabilis strains to IPM,MEM,ETP,CIP,AK and MIN were 100％,100％,100％,86.7％ (13/15),33.3％ (5/15) and 80％ (12/15),respectively.7 out of 15 strains were Hodge test positive,and 11 strains were blaKPC-2 positive.Results of PCR amplification showed that the transformed Escherichia coli J53 strains,whose MIC values to IPM,MEM,and ETP were increased by 2 to 64 times,were blaKPC-2 gene positive.Conclusion The carbapenem-resistant Proteus mirabilis strains in this study were resistant to many commonly used antibiotics,however,the resistance rates to AK were relatively low.The dominant carbapenemase genotype was blaKPC-2 carried by the plasmid.Attention should be paid to its easily transmissible feature among the strains in clinic.

Objective To investigate the distribution of β-lactamase genes in a pan-drug resistant Klebsiella pneumoniae isolate JM45.Methods Klebsiella pneumoniae JM45 was isolated from the blood sample of a patient admitted in the intensive care unit,the Second Affiliated Hospital,Zhejiang University School of Medicine on April 7,2010.The susceptibilities to 26 antibiotics were tested using E-test method.Cica-β-Test was performed to detect β-lactams,and modified Hodge test was performed to detect carbapenemase.Resistant genotypes were detected using PCR,DNA sequencing and BLAST algorithm.Whole genome sequencing (complete graph) was performed by high throughput Roche 454 sequencing approach to analyze the distribution of β-lactamase genes.Results Except polymyxin B and tigecycline,JM45 was resistant to other 24 kinds of antibiotics including cephalosporins and carbapenems.Several β-lactamases were positive in Cica-β-Test,and modified Hodge test was positive.Based on PCR typing,TEM-1,SHV-11,CTX-M-24 and VEB-3 were positive,but carbapenemase genes and metallo-β-lactamase genes were negative.A complete genome (chromosome) sequence (GenBank accession number:CP006656) and 2 plasmids sequences (GenBank accession number:CP006657,CP006658) were obtained by wholegenome sequencing.CTX-M-24 (Locus tag:N559_5233),TEM-1 (Locus tag:N559_5242) and VEB-3 (Locus tag:N559_5248) were positive in plasmid 1.CTX-M-24 located in insertion sequence (IS903-CTXM-24-ISEcp1),while TEM-1 and VEB-3 located in transposons (tnpA-TEM-1-rmtB and VEB-3-tnpA).SHV-11 (Locus tag:N559_2715) was positive in genome (chromosome),and 4 putative β-lactamase genes or β-lactamase domains were obtained:(1) metallo-β-lactamase domain protein (Locus tag:N559_0119,780 bp) ; (2) putative β-lactamase (Locus tag:N559_1633,1308 bp) ; (3) β-lactamase domain protein (Locus tag:N559_2279,813 bp); (4) β-lactamase domain protein (Locus tag:N559_3769,1101 bp).No insertion sequence or transposase gene was observed near SHV-11.Conclusion The resistance to antibiotics including cephalosporins and carbapenems is correlated with TEM-1,SHV-11,CTX-M-24,VEB-3 and 4 kinds of putative β-lactamase genes or β-lactamase domains.