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 investigate the resistance of Enterobacter cloacae and its gene of chlorhexidine-sulfadiazine-resistance(qacE△1-sul1) isolated from two hospitals. METHODS Microdilute tests were performed to detect the susceptibility of 20 kinds of antimicrobial agents in 74 strains of E.cloacae.The qacE△1-sul1gene was detected by PCR. RESULTS There was no strain resistant to imipenem and meropenem.The resistant rates to other antimicrobial agents were between 44.6% and 94.6%.The positive rate of qacE△1-sul1gene was 74.3%. CONCLUSIONS The 74 strains were multiple-drug-resistant.There was a high positive rate of qacE△1-sul1gene in E.cloacae isolated from two hospitals.

OBJECTIVE To investigate the distribution of multiresistant genes produced by Pseudomonas aeruginosa(PAE) isolated from different areas.METHODS Antimicrobial sensitivities of 104 strains of PAE to 13 kinds of antibiotics were determined by K-B method,of which 16 kinds of drug resistant genes were tested by polymerase chain reaction(PCR) and analyzed by clustering method of multi-molecular markers.RESULTS The resistant detective rates to imipenam in three areas were respectively 51.3%,80%,and 100% and that to the third generation cephalosporin were moving from 48.2% to 100%.The detective rate of oprD2 absent was 90-100%.There were similar phenomena of prevalence between some strains in these areas.oprD2 Absent was the main mechanism of prevailing strains in Shaoxing,however,clone strains in Huzhou were caused by TEM,oprD2 absent,aac(3)-Ⅱ,ant(3″)-Ⅰ and qacE△1.CONCLUSIONS There is a multiresistance to antibiotics in PAE isolated from different areas and the phenomena of prevalence are occurred in these areas,but the mechanisms of drug resistant are different.

OBJECTIVE To investigate the antibiotics-resistant genes in enterococci isolated from the 98th Hospital of PLA,Huzhou,Zhejiang Province,China.METHODS The antibiotics-resistant genes of TEM,aac(6′)/aph(2″),aph(3′)-Ⅲ,ant(2″)-Ⅰ,ant(4′,4″),ant(6)-Ⅰ,ermB,mefA,tetM,vanA,and vanB were analyzed by polymerase chain reaction(PCR) and verified by DNA sequencing in the 15 isolates of Enterococcus faecalis and 9 isolates of E.faecium.RESULTS The positive rate of the resistance genes of TEM,aac(6′)/aph(2″),aph(3′)-Ⅲ,ant(2″)-Ⅰ,ant(4′,4″),ant(6)-Ⅰ,(ermB,) mefA,tetM,vanA,and vanB in the 24 strains of enterococci tested were 37.5%,70.8%,25.0%,0.0%,0.0%,41.7%,75.0%,0.0%,41.7%,4.2%,and 4.2%,(respectively.) CONCLUSIONS The multidrug resistance of enterococci was a serious issue,and harbored antibiotics-resistance genes were the very important reasons of resistance to antibiotics in enterococci.

OBJECTIVE To investigate the ?-lactamase genes in 4 kinds of nonfermenting Gram-negative bacilli isolated from the 98th Hospital of PLA. METHODS Sixty strains of Acinetobacter baumannii,30 strains of Pseudomonas aeruginosa,19 strains of Stenotrophomonas maltophilia,and 15 strains of Flavobacterium were isolated from hospitalized patients.Nine kinds of ?-lactamases genes of TEM,SHV,OXA,CTX-M,PER,VEB,IMP,VIM and GES were analyzed by PCR and verified by DNA sequencing. RESULTS In A.baumannii and(P.aeruginosa),the positive rates of gene of TEM were 100.0% and 66.7%,respectively.SHV gene was positive in 18 of 60 strains of A.baumannii tested,17 of which were SHV-12 subtype ESBLs.The other was a new SHV type ?-lactamase nominated SHV-48.OXA gene was positive in 1 of 30 strains of P.aeruginosa tested,it was an OXA-10 subtype ESBLs.But the rest of genes were all negative. CONCLUSIONS There exist 4 kinds of(?-lactamase) genes at least in nonfermenting Gram-negative bacilli including TEM-1,SHV-12,SHV-48,and(OXA-10.)

OBJECTIVE To investigate the antimicrobial-resistant genes and consanguinity in multi-resistant Pseudomonas aeruginosa(MRPA) isolated from the 98th Hospital of PLA,Huzhou,Zhejiang Province,China.METHODS Thirty strains of MRPA were isolated from hospitalized patients between Sep 2003 and Oct 2004.Twenty four kinds of genes of blaTEM,blaSHV,blaOXA-10 group,blaPER,blaVEB,blaIMP,blaVIM,blaGES,blaCARB,blaDHA,blaMIR,aac(3)-Ⅰ,aac(3)-Ⅱ,aac(3)-Ⅲ,aac(3)-Ⅳ,aac(6′)-Ⅰ,aac(6′)-Ⅱ,ant(3″)-Ⅰ,ant(2″)-Ⅰ,aph(3′)-Ⅵ,oprD,qacE△1-sul1,catB,and cml1 were analyzed by PCR and verified by DNA sequencing.Resistant-genes cluster analysis was performed by Average.RESULTS In 30 strains of MRPA the positive rate of genes of blaTEM,blaOXA-10 group,blaCARB,aac(3)-Ⅱ,aac(6′)-Ⅰ,aac(6′)-Ⅱ,ant(3″)-Ⅰ,ant(2″)-Ⅰ,qacE△1-sul1,and cml1 were 66.7%,3.3%,3.3%,76.7%,3.3%,33.3%,53.3%,26.7%,83.3%,and 3.3%,respectively,and the deficiency rate of oprD gene was 90.0%.The gene of blaOXA-10 group was sequenced and determined to be blaOXA-10 subtype ESBL gene.But the rest of genes were all negative.According to the cluster analysis of resistant-gene,30 strains of MRPA isolated could be classified into four subgroups,which were caused by the infection in hospital.CONCLUSIONS At least 10 kinds of antimicrobial-resistant genes exist in MRPA isolates,and the deficiency rate of oprD gene is very high.MRPA can induce clone transmitted hospital infection and it has fulminant prevalence.

OBJECTIVE To investigate the resistance genes in HZB9055 strain of Enterobacter cloacae isolated from the PLA 98th Hospital,Huzhou District,Zhejiang Province,China.METHODS HZB9055 Strain of E.cloacae was isolated from the inpatient in May,2004,41 kinds(or groups) of genes were analyzed by PCR and verified by DNA sequencing.RESULTS In HZB9055 strain of E.cloacae,3 kinds of genes were positive including blaLAP,blaMIR and qnrS.The 38 kinds of rest genes were all tested negative.The blaLAP gene sequence including 858 nucleotides,which had the amino acid mutation in position 193 compared with LAP-1 type narrow-spectrum ?-lactamase(GenBank accession number:EF026092),and was nominated LAP-2(GenBank accession number:EU159120).CONCLUSIONS At least 3 kinds of resistance genes exist in HZB9055 strain of E.cloacae,and blaLAP-2 is a novel subtype of ?-lactamases gene.

OBJECTIVE To investigate the produce of extended-spectrum ?-lactamases(ESBLs) and the presence of genotype of the ?-lactamases-encoding genes in Klebsiella pneumoniae isolated from the 98th Hospital of PLA,Huzhou,Zhejiang Province,China.METHODS Twenty-five strains of K.pneumoniae were isolated from the inpatients between Sep 2005 and Apr 2006.ESBLs were tested by phenotypic confirmatory tests recommended by CLSI.Twenty-one kinds of ?-lactamases genes of blaTEM,blaSHV,blaLEN,blaOKP,blaCTX-M-1 group,blaCTX-M-2 group,blaCTX-M-9 group,blaOXA-1 group,blaOXA-2 group,blaOXA-10 group,blCARB,blaPER,blaVEB,blaGES,blaLAP,blaDHA,blaACT/MIR,blaCMY/MOX,blaFOX,blaCMY/LAT,and blaACC were analyzed by PCR and verified by DNA sequencing.RESULTS In 25 strains of K.pneumoniae,the positive,negative,and "uncertainty" rates of ESBLs were 56.0%,20.0%,and 24.0%,respectively.The positive rate of genes of blaTEM,blaSHV,blaCTX-M-1 group,blaOXA-10 group,blaLAP,and blaDHA were 80.0%,4.0%,4.0%,80.0%,4.0% and 32.0%,respectively.The 15 kinds of rest genes were all tested negative.The total positive rate of 21 kinds of ?-lactamases gene was 92.0%.Among them,the blaLAP-2 gene sequence of the HZ12593 strain has been registered in GenBank(GenBank Accession Number: EU529981).CONCLUSIONS There are higher rate of ESBLs-producing strains in K.pneumoniae isolated from the inpatients,and at least 6 kinds of ?-lactamases gene existed.Both genes of blaTEM and blaOXA-10 group are the most common genotypes.Carring blaDHA Gene may influence the result of phenotypic confirmatory test for ESBLs in K.pneumoniae.

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.