ObjectiveTo investigate the prevalence of 16S rRNA methylase genes, aminoglycoside modifying enzymes (AMEs) genes and small multidrug resistance efflux pump gene smr-2 in Klebsiella pneumoniae. MethodsTotally 138 Klebsiella pneumoniae isolates were collected in the First Affiliated Hospital, college of medicine, Zhejiang University from January 2007 to December 2009. Polymerase chain reaction (PCR) and DNA sequencing were performed to screen the presence of six 16S rRNA methylase genes ( rmtA, rmtB, rmtC, rmtD, armA and npmA), seven AMEs genes[aac ( 3 )- Ⅰ , aac ( 3 )- Ⅱ,aac(6′)- Ⅰ b, aac(6′)-Ⅱ, ant(2″)- Ⅰ , ant(3″)- Ⅰ , aph(3′)-Ⅵa]and small multidrug resistance efflux pumps gene (smr-2).Results Thirteen (9. 4％) isolates were found to carry rmtB gene, whereas 87 (63.0％) isolates were found to carry at least one kind of AMEs genes but no smr-2 was detected. The positive rates of aac(3)-Ⅱ, aac(6′)- Ⅰ b, ant (3″)- Ⅰ and ant(2″)- Ⅰ were 40.6％ (56/138), 31.9％ (44/138), 28.3％ (39/138) and 2.2％ (3/138), respectively. All strains harboring rmtB gene carried one to three AMEs genes. Among 44 aac(6′)- Ⅰ b positive strains, 37 (84. 1％ ) were confirmed to carryaac(6′)- Ⅰ b-cr. ConclusionFor Klebsiella pneumoniae, rmtB is the predominant subtype in 16S rRNA methylase genes, accompanying with several AMEs genes.

Objective To investigate ehloramphenicol,tetracycline,rifampicin and trimethoprimsulfamethoxazole resistance and the related genes in multi-drug resistant Acinetobacter baumannii(MDR-ABA).Methods Sixty-two strains of MDR-ABA were isolated from clinical samples,and their susceptibilities to 22 antimicrobial agents were detected by Kirby-Bauer disk diffusion tests.Genes related to chloramphenicol(catB and cmlA),rifampicin(arr-2/3),tetracycline(tetA and tetB)and trimethoprimsulfamethoxazole(sull,dfrA1,dfrA5,dfrA7/17,dfrA12,dfr85)resistance and drug emux genes(tehA,emrB,emrD,emrE,smr-2,mdfA)were analyzed by PCR and verified by DNA sequencing.Results Resistant rates of these MDR-ABAs to chloramphenicol,rifampicin,tetracycline and trimethoprimsulfamethoxazole were 100.0%(62/62),100.0%(62/62),90.3%(56/62)and 82.3%(51/62)respectively,while62 strains(100.0%),46 strains(74.2%),36 strains(58.1%)and 8 strains (12.9%)were detected to carry mafA,tetB,sull and tehA genes,respectively.The lest 13 genes were all negative.tetB,sull,tehA and mafa genes(2 for each)chosen optionally from positive ones were verified by DNA sequencing and BLASTn.and all were identified as the same sequences in GenBank.Conclusions MDR-ABAs show hish resistance to chloramphenicol,tetracycline, rifampiein and trimethoprimsulfamethoxazole.Multi-drug resistant phenotypes of MDR-ABAs may be closely related to mdfA genes harboring in strains.

Objective To analyze molecular evolution of carbapenemase KPC-12 and its binding free energies with β-lactams.Methods Class A beta-lactamases were divided into 2 clusters:those with carbapenemase activities and those without.Minimum Evolution method in MEGA4.1 software was used to analyze molecular evolution of class A beta-lactamases with carbapenemase activity,including KPC-2 to KPC-13,SFC-1,SME-1,IMI-1,NMC-A,and class A beta-lactamases without carbapenemase activity,including TEM-1,SHV-1.Then,tertiary structure of KPC-12 was predicted by homology modeling as reported in SWISS-MODEL database depending on tertiary structure of KPC-2.Moreover,DOCK module in ArgusLab 4.1 software was used to perform molecular docking of KPC-12 to 10 kinds of beta-lactams substrates,and the binding free energies (△ G) were calculated.Results Molecular evolution between KPC-12 and KPC-2 was the closest.The top three decline in binding free energies of β-lactams were penicillin G sodium salt (△G =-8.45149 kcal/mol),ertapenem (△G =-8.36383 kcal/mol) and ampicillin (△G =-8.19326 kcal/mol),while the last two decline in binding free energies of β-lactams were aztreonam (△G =-6.50614 kca]/mol) and clavulanic acid (△G =-6.88533 kcal/mol).Conclusion Carbapenemase KPC-12 has high catalytic activities to penicillin G sodium salt,ertapenem and ampicillin,while has low catalytic activities to aztreonam and clavulanic acid.

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.

Objective To investigate the synergistic efficacy of different antibiotic combinations against KPC-2 carbapenemase producing Klebsiella pneumoniae strains in vitro and search for effective antibiotic combination.Methods During 2008 - 2009,a total of 24 strains of K.pneumoniae producing KPC-2 carbapenemase were collected from 8 hospitals in the First Affiliated Hospital of Medical School of Zhejiang University,Ningbo LiHuiLi Hospital,Zhejiang People's Hospital,Hangzhou Third Hospital,the Second Hospital of Shaoxing,Hangzhou First Hospital,Fudan University Huashan Hospital,General Hospital of Nanjing Military Region.MLST technique was used for epidemiological analysis.The MIC of antibiotics,such as amikacin,minocycline,imipenem,amoxicillin/clavulanic-acid,ceftazidime,meropenem,gentamicin,cefoxitin,cefepime,rifampicin,polymyxinB,ciprofloxacin were determined by an agar dilution method,the MIC of tigecycline and piperacillin/tazobactain were determined by Etest.The antibacterial activities of cefepime in combination with amoxicillin/clavulanic-acid,amikacin,or ciprofloxacin,amikacin with ciprofloxacin,imipenem with amikacin,ciprofloxacin,polymyxinB,or minocycline,polymyxin B with rifampicin,ceftazidime with amoxicillin/clavulanic-acid were assessed by chequerboard synergy agar dilution tests against all the isolates.Results MLST showed 5 STs among 24 strains of KPC-2 carbapenemase producing K.pneumoniae,and the most prevalent clone was ST11 (15 strains).All isolates were susceptible to polymyxin B and tigecycline,and the resistance rate of minocycline was 4.2％.The synergetic effects were observed in cefepime-amoxicillin/clavulanic acid,imipenem-amikacin,ceftazidime-amoxicillin/clavulanic acid combinations as 19 isolates,13 isolates,and 13 isolates,respectively.Conclusions KPC-2 carbapenemase producing K.pneumoniae is sensitive to polymyxin B,tigecycline and minocycline.The synergetic effect is predominant in cefepime-amoxicillin/clavulanic acid,imipenem-amikacin ceftazidime-amoxicillin/clavulanic acid combinations in vitro,their clinical efficacy are worthy of further observation.

Objective To investigate the distribution of 22 beta-lactamase genes and enzyme activities in multi-drug resistant Acinetobacter baumannii (MDRAB). Methods Sixty-two MDRAB strains were isolated from the First Affiliated Hospital, College of Medicine, Zhejiang University. Twenty-two beta-lactamase genes were analyzed by PCR and verified by DNA sequencing. Enzyme activities of extended spectrum beta-lactamases (ESBLs) , cephalosporinase ( AmpC) and metallo-beta-lactamases ( MBL) were detected by the modified three-dimensional method using enzyme extraction. Results In 62 MDRABs, 51 (82.3% ) , 50 (80.6% ) and 36 (58.1% ) isolates were found to carry blaTEM, blaOXA-23 cluster, and blaADC, respectively. The rest 19 genes were not detected in this study. DNA sequencing and genomic comparison showed that 5 isolates carrying blaTEM had the same genotype as blaTEM-l , 6 isolates carrying blaOXA-23 cluster had the same genotype as blaOXA-23 carbapenemases (accession; CAB69042. 1) , and 2 isolates carrying blaADC had the same genotype as blaADC-like (accession: EU081908). Thirty-two isolates (51.6% ) produced ESBLs and AmpC, 19 isolates (30.6% ) produced ESBLs only, and 1 isolate (1.6%) produced AmpC only; and no isolate produced MBL. Conclusion MDRAB carrying blaOXA-23 carbapenemase and blaADC AmpC in this study are of high drug resistance.

Objective To investigate ADC type cephalosporinase genes in muhidrug-resistant strains of Acinetobater baumannii (MDR-ABA). Methods Sixty-two ABA strains were collected during November 2007 to February 2008 from the First Affiliated Hospital, College of Medicine, Zhejiang University. Kirby Bauer disk diffusion method was used to detect the susceptibilities of the strains to antimicrebial agents. PCR and DNA sequencing was performed to analyze the ADC type cephalosporinase gene. Results Sixty-two MDR-ABA strains showed high drag-resistance rate to most antimicrobial agents expect cefoperazone/sulbactam (69.4%), and 36 strains were ACD positive (58.1%). Conclusion MDR-ABA strains in this study are of high drug resistance, which is closely related to ADC type cephalosporinase.

Objective To investigate the correlation between drug-resistant genes and mobile genetic elements in multi-drug resistant Escherichia coli,and to explore phylogeny among the strains.MethodsTotally 20 strains of multi-drug resistant Escherichia coli were collected from Pan' an Hospital,Zhejiang Province during June 2009 and June 2010.Beta-lactam-resistance genes,aminoglycoside-resistance genes,genetic markers of mobile genetic elements were analyzed by PCR.Index and sample cluster analysis were performed on above results. Results In 20 strains of Escherichia coli,4 kinds of beta-lactamresistance genes,4 kinds of aminoglycoside-resistance genes,and 5 kinds of genetic markers of mobile genetic elements were detected.Index cluster analysis showed that correlation existed between resistance genes TEM,CTX-M-1,aadA5 and mobile genetic elements traA,IS26,ISEcpl; and correlation also existed between resistance genes OXA-1,aac(6′)-Ⅰ b,ant(3)-Ⅰ,rmtB and mobile genetic elements trbC,IS903.Sample cluster analysis showed that this group of Escherichia coli could be divided into 2 groups which were genetically different.ConclusionsDrug-resistant genes in multi-drug resistant Escherichia coli are correlated with mobile genetic elements.Sample cluster analysis can reveal phylogeny among the strains,which is important for hospital infection control.

ObjectiveTo investigate correlation between drug-resistance related genes and mobile genetic elements of Klebsiella pneumoniae resistant to β-lactams. Methods Forty-seven strains of multidrug-resistant Klebsiella pneumoniae were collected from 6 hospitals in Hangzhou and Huzhou of Zhejiang province from August 2008 to May 2010.Modified Hodge test was performed to detect phenotypes of carbapenemases.Forty kinds of β-lactamases (class A-D),ompK35,ompK36,and 12 kinds of mobile genetic elements were detected by PCR,and the results were analyzed by index cluster.ResultsThirty-five strains were positive in modified Hodge test,and 5 kinds of β-lactamases gene ( including KPC-2-like,GenBank:HQ258934) and 9 kinds of mobile genetic elements were detected.Mutations were observed in ompK35 and ompK36 when compared with sensitive strains.Index cluster analysis showed that correlation existed between KPC-2,KPC-2-like and ISKpn6,between TEM-1 and ISEcpl,IS26,int Ⅰ 1,trbC,IS903,and between CMY-2,OXA-30,DHA-1 and tnpU,tnp513,trbC.ConclusionsFive kinds of β-1actamases genes,and mutations in ompK35 and ompK36 may be associated with the resistance to β-1actams in multidrug-resistant Klebsiella pneumoniae.

OBJECTIVE To investigate in vitro activities of 12 antimicrobial agents including cefmetazole against extended-spectrum beta-lactamases-producing Escherichia coli(528 strains),Klebsiella pneumoniae(311 strains) and Proteus mirabilis(15 strains).METHODS They all collected from 15 teaching hospitals in China during 2005 and 2006 and included in the study.The levels of minimal inhibitory concentration(MIC) of 12 antimicrobial agents were determined by agar dilution method.WHONET 5.4 Software was used to analyze the data.RESULTS Against ESBLs-producing E.coli and ESBLs-producing K.pneumoniae,carbapenems were the most active antimicrobial agents(all 100.0% susceptible),followed by cephamycins(80.1-97.3%).Piperacillin/tazobactam(78.5-95.1%)showed a higher activity than cefoperazone/sulbactam(44.1-56.2%).The susceptible rate to ceftazidime against ESBLs-producing E.coli was remarkably higher than the other three cephalosporins,however the differences did not happen to ESBL-producing K.pneumoniae obviously.The susceptible rate to cefuroxime was below 1.6%.ESBLs-producing K.pneumoniae showed high sensitivity to carbapenems,cephamycins and ?-lactam/lactamase inhibitor combinations(all 100% susceptible),however the susceptible rates to cephalosporins were relatively lower.CONCLUSIONS Carbapenems and cephamycins remain the relatively high activity against ESBLs-producing Enterobacteriaceae.