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 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 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 investigate the prevalence of genes conferring aminoglycoside resistance in multidrug-resistant strains of Acinetobacter baumannii (MDR-ABA).Methods Multidrug-resistant A.baumannii strains were isolated during the period from August to November 2012 from patients in the affiliated hospital of Jiangsu University and the First Hospital of Zhen-jiang.Kirby-Bauer diffusion method was used to determine the susceptibility of these strains to antimicrobial agents.PCR was performed to detect the aminoglycoside resistance genes.Results The 36 MDR-ABA strains showed high resistance rates to most antimicrobial agents except cefoperazone-sulbactam.The prevalence of the genes conferring aminoglycoside resistance, aac (3)-I,aac (6’)-Ib,aph (3’)-I and armA,was 72.2% (26/36),72.2% (26/36),80.6% (29/36)and 80.6% (29/36), respectively.Conclusions The MDR-ABA strains in this study are highly resistant to antimicrobial agents,which is closely as-sociated with the genes conferring aminoglycoside resistance.

Objective The aim of this study was to study the antibiotic resistance and resistance genes of methicillin-resistant Staphylococcus aureus (MRSA) in children from Shanghai area,and to determine the relationship between phenotypic and genotypic resistance profiles.Methods In this study,a total of 37 MRSA strains isolated from clinical specimens of hospitalized patients in Children's Hospital of Fudan University from March 2009 to November 2011 were collected.The mecA,ermA,ermB,ermC,aac (6') /aph (2),aph (3')-Ⅲ,ant (4',4),and qacA genes were detected by polymerase chain reaction (PCR).Resistance to antibiotics was detected by agar dilution tests.The data analysis was done by chi square test.Results Among the 37 MRSA isolates,all (100.0 ％) were mecA gene positive,9 (24.3％) were ermB gene positive,none was ermA/C gene positive,21 (56.8％) were aac (6')/aph (2) gene positive,10 (27.0％) were aph (3')-Ⅲ gene positive,6 (16.2％) were ant(4',4) gene positive,and 9 were qacA gene positive (24.3％).The positive rate of aac(6')/aph(2) in hospital acquired methicillin-resistant Staphylococcus aureus (HA-MRSA) was significantly higher than that of community acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) (85.7％ vs18.8％,x2=60.340,P=0.000).Among the 37 MRSAisolates,37 (100.0％) were resistant to penicillin,ampicillin-sulbactam,cefazolin,cefoxitin and cefuroxime.The 37 isolates were all susceptible to teicoplanin,vancomycin,and linezolid.The resistant rates to gentamicin,erythromycin,clindamycin,sulfamethoxazole,fosfomycin,rifampicin,and levofloxacin were 51.4％ (19/37),81.1％ (30/37),51.4％ (19/37),16.2％ (6/37),27.0％ (10/37),37.8％ (14/37) and 54.0％ (20/37),respectively.Compared with CA-MRSA,HAMRSA isolates had significantly higher resistance rates to gentamicin (12.5％ vs 81.0％; x2 =17.033,P=0.000),levofloxacin (31.2％ vs 71.4％; x2 =5.903,P=0.017),and rifampin (6.2％ vs 61.9％; x2=11.959,P=0.001).The rate of gentamicin resistance in aac(6')/aph(2) gene carrying strains was significantly higher than strains not carrying the gene (x2 =29.757,P=0.000).Conclusions MRSA in children carry a variety of drug-resistant genes,showed multi-drug resistance.HA-MRSA carries more resistance genes,and has higher rates resistance to antimicrobials than CA-MRSA.

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 prevalence of multidrug resistant genes in Klebsiella pncumoniae.MethodsTwenty strains of multidrug resistant Klebsiella pneumoniae were isolated from burn patients.Susceptibility of these strains to 14 antibiotics was detected by KB method.PCR was used to detect oqxA,smrKpn,qacE,tehA,mdfA and qacEΔl-sul1 genes.ResultsThe antibiotic sensitivity rates of 20 multidrug resistant Klebsiella pneumoniae isolates to antibiotics tested were ＜ 30％ except that to imipenam.The positive rates of efflux pump genes mdfA,qacEΔl-sull and oqxA were 65％,100％ and 100％,respectively; while those ofsmrKpn,qacE and tehA were 0％,0％ and 15％.ConclusionoqxA gene has been detected in multidrug resistant Klebsiella pneumoniae from burn patients with high positive rate.

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 investigate the molecular mechanism of Klebsiella pneumoniae resistant to carbapenem. Methods The minimal inhibitory concentrations ( MICs) of the antimicrobial agents were determined by E-test. The 23 β-lactamase genes and 2 porin genes were amplified by polymerase chain reaction (PCR) , then the products were purified and their sequences were analyzed. Results The MICs of piperacillin, piperacillin/sulbactam, amoxicillin/clavulanic acid, cefoperazone/sulbactam, cefotaxime, cefepime and aztreonam to 5 strains of Klebsiella pneumoniae were all higher than 128 μg/mL, and those of imipenem or meropenem were higher than 32 μg/mL. All isolates carried blaTEM-1 and blaDHA-1 genes. Deletion of ompK35 and ompK36 were observed in Kp01 and Kp03, and the deletion of ompK35 was also observed in Kp02 and Kp05. Base insertion of ompK36 occurred in Kp02, Kp04 and Kp05. Compared with GenBank (GU945384) , ompK35 gene mutations of G→C at base 465 and T → C at base 466 in Kp04 lead to Gln to His substitution at position 155 and Tyr to its substitution at position 156, and it might be a new subtype. Conclusion The production of DHA-1 β-lactamase combined with the loss of OmpK36 or OmpK35 in porin genes may contribute to high-level carbapenem resistance in Klebsiella pneumoniae.

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.