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 mechanisms of aminoglycoside resistance in pan-drug resistant Pseudomonas aeruginosa(PDRPA).METHODS The 11 genes of 16S rRNA methylase and aminoglycoside-modifying enzymes were detected by polymerase chain reaction(PCR) and verified by DNA sequencing.RESULTS In 33 strains of PDRPA,the positive rates of rmtB,aac(3)-Ⅱ,aac(6′)-Ⅰb,aac(6′)-Ⅱ,ant(3″)-Ⅰ and ant(2″)-Ⅰ were 42.4%,51.5%,42.4%,57.6%,48.5% and 63.6%.A total of 32 strains identified aminoglycoside modified genes,one strain aminoglycoside-modifying enzyme gene did not discovered,but rmtB positive.CONCLUSIONS The aminoglycoside resistance mechanisms of the PDRPA are the production of 16S rRNA methylase and aminoglycoside-modifying enzymes related.

Aim In order to detect Mycoplasma fermentans (Mr)and Mycoplasma penetrans (Mpe) at the same time dual nested-PCR(DN-PCR) technique was established Method Mpe the same time PCR Primers were designed according to published 16s rRNA Sequences of Mf and Mpe, the outer primers were univevsal to both Mf and Mpe and the inner was Mf、Mpe speciespecific respectively. Two-step amplification procedures were perfomed. Result Chromo some DNAS of Mf and Mpe gave characteristic DN-PCR prafiles which were differeht from other my coplasmas or microorganism tested.Conclusion The DN-PCR method was established for the detection of Mf and Mpe at the same time which was sensitive specific and rapid.

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 the genes associated ?-lactamases in Acinetobacter baumannii(ABA)isolated and preliminary discovery of Acinetobacter-derived cephalosporinases(ADC)-subtype in Shaoxing,Zhejiang.METHODS Eighteen ?-lactamases genes were detected by PCR,and a new ADC ?-lactamases gene fragments produced by PCR were sequenced and analyzed.RESULTS The positive rates of ADC-gene,TEM-gene and OXA-23 group-gene were 85.9%,27.1% and 58.8%,respectively.The other genes were not found in all 85 isolates tested.CONCLUSIONS There are very high positive percentages of ADC,TEM and OXA-23 group genes in A.baumannii isolated clinically and that is the important mechanism of multi-resistance.The ADC gene may be a novel subtype in lactamases(GenBank accession number:EF569590).

OBJECTIVE To investigate the resistance and the genes of quaternary ammonium compounds-resistance(qacE△1) and classⅠintegrase genes(intⅠ1)in continuously isolated Acinetobacter baumannii(ABA).METHODS K-B tests were performed to detect the susceptibility of 85 strains of ABA to 13 kinds of antimicrobial agents.The qacE△1 and intⅠ1 genes were analyzed by PCR.RESULTS The resistant rates to antimicrobial agents were between 8.2% and 81.2%.qacE△1 And intⅠ1 genes were 77.6%.CONCLUSIONS The 85 ABA strains are multiple-drug-resistant.There are very high positive percentages of qacE△1 and intⅠ1 genes in ABA isolated from Shaoxing,Zhejiang Province.

OBJECTIVE To investigate mechanisms of drug resistance in multidrug-resistant strains of Acinetobacter baumannii(MDR-ABA).METHODS The resistant genes related to 5 kinds of antibiotics were detected by using polymerase chain reaction and verified by DNA sequencing.RESULTS In 30 strains of MDR-ABA,the positive rates of TEM,ADC,armA,aac(3)-Ⅰ,ant(3″)-Ⅰ,dfrA12 and sul1 were 63.3%,70.0%,30.0%,90.0%,90.0%,53.3% and 80.0%,respectively.CONCLUSIONS There are high positive percentages of TEM,ADC,armA,aac(3)-Ⅰ,ant(3″)-Ⅰ,dfrA12 and sul1 genes in MDR-ABA.

OBJECTIVE To investigate the quinolone-resistance mechanisms of multi-drug-resistant Klebsiella pneumoniae(MDRKP).METHODS Seven kinds of chromosome and plasmid mediated quinolone-resistance genes were analyzed by PCR and verified by DNA sequencing in 25 strains of MDRKP.RESULTS In 25 strains of MDRKP,the positive rate of genes of gyrA,aac(6′)-Ⅰb-Cr,qnrA1,qnrB4-like,qnrS1,mdfA,and qepA were 76.0%,36.0%,8.0%,8.0%,12.0%,100.0%,and 0,respectively.CONCLUSIONS The mutation of gyrA gene is the main cause of the resistance of quinolone in the 25 strains of MDRKP.

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.