婴幼儿眼部感染肺炎链球菌的群体结构研究

目的 研究婴幼儿眼部感染肺炎链球菌的群体结构,包括对抗菌药物的耐药性、耐药基因、血清型和分子分型.方法 用k-b法和etest检测39株肺炎链球菌对10种抗菌药物的耐药性,乳胶凝集试验检测血清型,pcr法检测大环内酯类药物耐药基因mefe和ermb,并选择20株菌株进行多位点序列(mlst)分型.结果 39株肺炎链球菌中,30株(76.9％)对3种或3种以上药物耐药,未检测到对万古霉素、青霉素和头孢噻肟耐药的菌株.33株检出ermb耐药基因,4株检出mefe基因.共检出12种血清型,主要为19型( 8/39)和14型(4/39),17株包含在pcv7疫苗内,疫苗覆盖率为43.6％.mlst分型发现国际耐药克隆taiwan19f-14和spain23f-1.结论婴幼儿眼部感染的肺炎链球菌包含国际流行耐药克隆,血清型和分子分型较为分散,克隆分布以散发为主,对眼部常用抗菌药物有较高的耐药性. abstract： objective to determine the population structure of streptococcus pncumoniae isolates collected from infants with eye infections,including drug resistance,resistance genes, serotypes and molecular types.methods the susceptibility of 39 isolates to 10 antibacterial agents was tested by k-b disk diffusion and etest.latex agglutination test was performed to determine the serotype of the strains,and pcr was carried out to detect macrolides resistance genes mefe and ermb.molecular types of the 20 strains were determined by multilocus sequence typing (mlst).results a total of 39 streptococcus pneumoniae isolates were obtained,in which 30 (76.9％) were resistant to 3 or more antibacterial agents,and no vancomycin,penicillin or cefotaxime resistant strain was found.ermb gene was found in 33 strains and mefe gene was found in 4 strains.twelve serotypes were found,and the most frequent serotypes were 19 (8/39) and 14 (4/39). seventeen strains (43.6％) were covered in pcv7 vaccine. the international clone taiwan19f-14 and spain23f-1 were found by mlst. conclusions streptococcus pneumoniae isolates from infants with eye infections include international resistance clones.the distribution of serotype and molecular type are dispersed, and the clones are sporadic. the isolates are highly resistant to commonly used antibacterial agents.     

阴沟肠杆菌外膜孔蛋白基因缺失合并β-内酰胺酶和Ⅰ类整合子所致多重耐药性分析

目的 了解临床分离阴沟肠杆菌膜孔蛋白基因缺失合并β-内酰胺酶和Ⅰ类整合子所致耐药的特点及其分布.方法 收集2008年1月至2011年5月自临床感染患者分离的112株阴沟肠杆菌.应用vitek-2 compact鉴定细菌,k-b法进行药敏试验,改良hodge试验、美罗培南改良三维实验和edta-美罗培南纸片协同试验筛选产碳青霉烯酶菌株.多重pcr分别检测4种esbls基因、6种ampc酶基因、4种碳青霉烯酶基因及4组oxa-like基因,单一pcr检测isecpl、ompk35/36、ndm-1、oxa-48,并对Ⅰ类整合子可变区进行pcr扩增及序列分析.结果 6株(5.4％)改良hodge试验阳性,14株(12.5％)美罗培南改良三维试验阳性,未检出碳青霉烯酶基因.29株(25.9％)esbls基因阳性,其中,ctx-m基因上游均检测到isecpl,并检测到2株产oxa-1型esbls阴沟肠杆菌.ampc酶基因阳性45株(40.2％),以mir-3型为主(37/45,82.2％).共检出22株Ⅰ类整合子基因阳性菌株,其中16株可变区扩增阳性.扩增出4种耐药基因盒,aadb-aada2(1 000 bp)9株,dfra 15 (700 bp)5株,aadal(1 000 bp)2株,有l株菌同时携带4种耐药基因盒.所有菌株均合并有膜孔蛋白ompk35和/或ompk36基因缺失,并呈现多重耐药.结论 阴沟肠杆菌对碳青霉烯类抗生素的敏感性降低主要与其高产ampc酶、esbls并存在膜孔蛋白ompk35和/或ompk36基因缺失有关,且Ⅰ类整合子参与阴沟肠杆菌的多重耐药. abstract： objective to investigate the drug resistance and its distribution induced by β-lactamases and class Ⅰ integrons with the deficiency of omp genes in enterobacter cloacaes.methods totally 112 strains of enterobacter cloacaes were isolated during january 2008 and may 2011.the identification of strains was performed by using vitek-2 compact automatic system; and antibiotic susceptibility was determined by k-b method.isolates of e.cloacae were screened for carbapenemases by modified hodge test,improved three dimensional test and edta-meropenem synergy test.genes encoding ampc β-lactamase,metallo-β-1actamases (mbls) and oxa-like β-1actamases were screened by multiple pcr.single pcr was used to detect isecpl,ompk35/36,ndm-1 and oxa-48.the variable regions of class Ⅰ integrons were amplified and sequenced.results among 112 isolates,6 (5.4％) demonstrated positive in the modified hodge test and 14 ( 12.5％ ) were positive in the improved three-dimensional test.no carbapenemases gene was found.there were 29(25.9％ ) strains positive for esbls genes,isecpl was found in the upstream of all the ctx-m-type esbls; oxa-1 esbls were detected in 2 isolates.ampc β-lactamase genes were positive in 45 (40.2％) strains,and 82.2％ (37/45) were mir-3 type.twenty two isolates carried class Ⅰ integrons,and four different cassettes arrangements were identified within 16 strains:9 isolates harbored aadb-aada2 ( 1 000 bp),5 isolates with dfral5 (700 bp),2 isolates with aadal ( 1 000 bp).one isolate harbored all the above gene cassettes.the deficiency of ompk35/36 was found in all strains.conclusion esbl,ampc β-lactamase and the deficiency of ompk35/36 are correlated with the resistance to carbapenems in enteobacter clocace,and class Ⅰ integrons may also partly account for the multidrug-resistance.     

多重耐药大肠埃希菌毒力基因研究

目的 探讨多重耐药大肠埃希菌中7种相关毒力基因存在情况.方法 用pcr方法检测多重耐药大肠埃希菌中7种毒力基因(papa、cnf1、cnf2、cfab、ipab、hofq和ompt),并研究pcr阳性基因在biocyc基因组数据库31株已完成全基因组测序的大肠埃希菌中的分布.结果 20株多重耐药大肠埃希菌中仅检测到hofq、ompt两种毒力基因,检出率分别为95.0％ (19/20)和55.0％( 11/20).biocyc基因组数据库的31株大肠埃希菌中,21株存在hofq基因,阳性率为67.7％;15株存在ompt基因,阳性率为48.4％.结论 多重耐药大肠埃希菌中毒力基因hofq和ompt的携带率较高. abstract： objective to investigate the distribution of virulence-related genes in multidrug resistant escherichia coli.methods seven virulence genes papa,cnf1,cnf2,cfab,ipab,hofq and ompt were detected by pcr in 20 strains of multidrug resistant escherichia coli clinically isolated,and the positive genes were further searched in 31 strains of escherichia coli in biocyc database whose genomies had been fully sequenced.results virulence genes hofq and ompt were detected in 20 strains of escherichia coli with a positive rate of 95.0％ (19/20) and 55.0％ ( 11/20),respectively.among 31 strains of escherichia coli in biocyc,21 (67.7％) were positive for hofq gene and 15 (48.4％) were positive for ompt gene.conclusion hofq and ompt genes are prevalent in multidrug resistant escherichia coli.     

肺炎克雷伯菌与植生克雷伯菌喹诺酮类耐药基因研究

目的 分析宁波大学医学院附属医院克雷伯菌属的耐药性及其对喹喏酮类药物的耐药机制.方法 收集2009年10月-2011年3月分离出的20株克雷伯菌属菌株,经鉴定确认18株为肺炎克雷伯菌,2株为植生克雷伯菌.采用k-b纸片扩散法检测其对药物的敏感性.pcr法检测染色体介导的gyra、parc基因和质粒介导的aac(6’)-Ⅰ b-cr、qnra、qnrb、qnrs、qepa基因,pcr阳性产物采用pcr直接全自动荧光法测序.结果 20株克雷伯菌属菌对β-内酰胺类、氨基糖苷类、喹诺酮类抗菌药物均表现为多重耐药性(耐药率均在80％以上),其中植生克雷伯菌对亚胺培南和美罗培南敏感.18株(90％)菌株存在gyra、parc基因突变;aac(6’)-Ⅰ b-cr阳性12株(60％),qnrb和qnrs阳性均为4株(20％).结论 本组20株克雷伯菌属菌对喹诺酮类药物耐药主要与gyra和parc基因突变相关. abstract： objective to investigate the multi-drug resistance of klebsiella strains and its mechanism.methods twenty strains of klebsiella were isolated from the affiliated hospital of medical college,ningbo university from october 2009 to march 2011,in which 18 isolates were klebsiella pneumonia and 2 were klebsiella planticola. drug sensitivity was determined by k-b tests. drug resistant genes gyra,parc (chromosome mediated) and aac( 6′)-i b-cr,qnra,qnrb,qnrs,qepa (plasmid mediated) were amplified by pcr and verified by direct automated fluorogenic sequencing. results resistance to β-1actams,aminoglycosides and quinolones was observed in 20 strains,and resistant rates were all above 80％.klebsiella planticola strains were sensitive to imipenem and meropenem.mutations of gyra and parc genes existed in 18 strains (90％),and the positive rates of aac (6') -i b-c r,qnrb and qnrs were 60％ (12/20),20％ (4/20) and 20％ (4/20),respectively.conclusion the mutations ofgyra and parc genes may be the main cause of the resistance to quinolones in these strains.     

肺炎克雷伯菌氨基糖苷类耐药基因研究

目的 了解肺炎克雷伯菌16s rrna甲基化酶基因、氨基糖苷类修饰酶(ames)基因和小多重耐药外排泵基因smr-2的存在状况.方法 收集2007年1月- 2009年12月浙江大学医学院附属第一医院连续非重复分离肺炎克雷伯菌138株,采用聚合酶链反应(pcr)及序列分析法检测6种16s rrna甲基化酶基因(rmta、rmtb、rmtc、rmtd、arma和npma),7种ames基因[aac(3)-Ⅰ、aac(3)-Ⅱ、aac(6′)-Ⅰ b、aac(6′)-Ⅱ、ant(2″)-Ⅰ、ant(3″)-Ⅰ和aph(3′)-Ⅵa]和小多重耐药外排泵基因smr-2.结果 138株肺炎克雷伯菌中13株(9.4％)检出rmtb基因,未检出rmta、rmtc、rmtd、arma 和npma基因,所有rmtb阳性的菌株均同时携带1～3种ames基因;87株(63.0％)至少检出1种ames基因,检出率从高到低依次为aac(3)-Ⅱ56株(40.6％)、aac(6′)-Ⅰb44株(31.9％)、ant(3″)-Ⅰ39株(28.3％)、ant(2″)-Ⅰ3株(2.2％).对44株aac(6′)-Ⅰb基因pcr阳性产物进行测序,证实37株(84.1％)携带aac(6′)-Ⅰ b-cr双功能酶基因.未检到小多重耐药外排泵基因smr-2.结论 肺炎克雷伯菌存在多种氨基糖苷类耐药基因,rmtb是主要的16s rrna甲基化酶基因,并与多种氨基糖苷类修饰酶基因协同存在. abstract： objective to 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. methods totally 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. conclusion for klebsiella pneumoniae, rmtb is the predominant subtype in 16s rrna methylase genes, accompanying with several ames genes.     

耐药铜绿假单胞菌临床分离株发现aac(6')-Ⅰb-cr基因

目的 调查耐药铜绿假单胞菌中氨基糖苷类修饰酶基因(ame)和16s rrna甲基化酶基因的存在情况.方法 对分离自江苏省淮安市第一人民医院住院患者送检痰液和伤口分泌物样本的耐药铜绿假单胞菌共20株,采用聚合酶链反应(pcr)及序列分析研究8种氨基糖苷类修饰酶基因[aac(3)-Ⅰ、aac(3)-Ⅱ、aac(6')-Ⅰ b、aac(6')-Ⅱ、ant(2")-Ⅰ、ant(3")-Ⅰ ant(4')-Ⅰ、aph(3')-Ⅱb]和6种16s rrna甲基化酶基因(arma、rmta、rmtb、rmtc、rmtd、npma).结果 20株铜绿假单胞菌共检出4种ame基因:aac(6')-Ⅱ8株(40.0%)、ant2"-Ⅰ 8株(40.0%)、aac(3)-Ⅱ5株(25.0%)、aac(6')-Ⅰ b 2株(10.0%),以及1种16s rrna甲基化酶基因:rmtb 1株(5.0%),其余9种基因未检出.对2株aac(6')-Ⅰ b基因pcr阳性产物进行测序,证实有1株单独携带aac(6')-Ⅰ b经典型,1株单独携带aac(6')-Ⅰ b-cr双功能酶基因.结论 在耐药铜绿假单胞菌中存在aac(6')-Ⅰ b-cr型基因,且对氨基糖苷类和喹诺酮类药物均有修饰作用. abstract： objective to investigate the distribution of aminoglycoside modifying enzyme genes (ames) and 16s rrna methylase genes in drug-resistant strains of pseudomonas aeruginosa. methods twenty strains of drug-resistant pseudomonas aeruginosa were isolated from sputum and wound secretion samples collected from the first people's hospital of huai' an in jiangsu province. eight ames [aac(3)-Ⅰ , aac(3)-Ⅱ, aac(6')-Ⅰb,aac(6')-Ⅱ, ant{2")-Ⅰ ,ant(3")-Ⅰ , ant(4')-Ⅰ , aph(3')-Ⅱb] and 6 16s rrna methylase genes (arma, rmta, rmtb, rmtc, rmtd, npma) were analyzed by pcr and verified by dna sequencing. results out of 20 strains of pseudomonas aeruginosa, aac(6')-Ⅱ was positive in 8 strains (40.0% ) , ant2"- Ⅰ in 8 strains (40.0% ) , aac(3)-Ⅱ in 5 strains (25.0% ) , aac(6')- Ⅰ b in 2 strains (10.0% ) and rmtb in 1 strains (5.0% ) , respectively. the rest 9 genes were not detected. among 2 strains harboring aac(6')- Ⅰ b, dna sequencing confirmed that 1 was aac(6')- Ⅰ b (the clssical type) and another was aac(6')- Ⅰ b-cr. conclusion gene aac(6')- Ⅰ b-cr exists in drug-resistant pseudomonas aeruginosa, and it has modifying effect on both aminoglycosides and quinolones.     

国内首次从多重耐药肺炎克雷伯菌中检出oqxa基因

目的 研究烧伤科多重耐药肺炎克雷伯菌抗菌制剂外排泵基因的流行情况.方法 收集从烧伤患者分离的多重耐药肺炎克雷伯菌20株,采用kb法检测其对14种常用抗菌药物的敏感性.用聚合酶链反应(pcr)检测抗菌制剂外排泵基因oqxa、smrkpn、qace、teha、mdfa和qaceΔlsul1.结果 20株多重耐药肺炎克雷伯菌除了对亚胺培南具有较高敏感性外,对其他抗菌药物的敏感率均在30％以下.抗菌制剂外排泵基因oqxa、qaceΔl-sul1和mdfa的检出率很高,分别为100％、100％和65％,smrkpn、qace和teha检出率很低,分别为0％、0％和15％.结论 从烧伤科分离的多重耐药肺炎克雷伯菌中检出oqxa基因,且携带率非常高. abstract： objective to investigate the prevalence of multidrug resistant genes in klebsiella pncumoniae.methods twenty 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.results the 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％.conclusion oqxa gene has been detected in multidrug resistant klebsiella pneumoniae from burn patients with high positive rate.     

泛耐药鲍曼不动杆菌获得性耐药基因和可移动遗传元件检测与指标聚类分析

目的 调查泛耐药鲍曼不动杆菌获得性耐药基因和可移动遗传元件携带情况,并探讨二者之间的相关性.方法 收集临床样本中分离的20株泛耐药鲍曼不动杆菌,采用pcr法检测53种水平转移获得性耐药基因(与β-内酰胺类、氨基糖苷类、喹诺酮类耐药相关)以及12种可移动遗传元件(接合性质粒、转座子、插入序列、整合子等),并对检测结果进行指标聚类分析.结果 本组菌株检出3种β-内酰胺酶类药物耐药基因tem-1、adc-30和oxa-23,4种氨基糖苷类修饰酶基因aac(3)-Ⅰ、aac(6′)-Ⅰb、ant(3″)-Ⅰ和aph(3′)-Ⅰ,以及5种可移动遗传元件intⅠ1、tnpu、tnp513、is26和isaba1.指标聚类分析显示、tem-1、adc-30等2种β-内酰胺酶基因,aac (6′)-Ⅰb、ant(3″)-Ⅰ等2种氨基糖苷类修饰酶基因,abeb和qace Δ1外排泵基因与intⅠ1、tnpu、tnp513、is26、isaba1等可移动遗传元件高度相关.结论 临床分离的pdr-aba可携带多种获得性耐药基因和可移动遗传元件,且二者之间高度相关. abstract： objective to investigate the correlation between acquired drug resistance-related genes and mobile genetic elements from pandrug-resistant acinetobacter baumannii. methods fifty-three horizontal transfer drug resistance-related genes ( β-lactamases,aminoglycoside and quinolones resistance related) and 12 mobile genetic elements (including zygosity plasmid,transposon,insertion sequence and integron) were detected by polymerase chain reaction (pcr) in 20 clinical isolates of pandrug-resistant acinetobacter baumannii.index cluster analysis was performed to explore the correlation.results in 20 strains of pandrug-resistant acinetobacter baumannii,there were 3 types of β-lactamases related genes (tem-1,adc-30,oxa-23 ),4 types of aminoglycoside modifying enzyme genes [ aac (3)-Ⅰ,aac(6′)-Ⅰ b,ant( 3″)-Ⅰ and aph( 3′)-Ⅰ ],and 5 kinds of mobile genetic elements ( int Ⅰ 1,tnpu,tnp513,is26 and isaba1 ). index cluster analysis showed high correlations between resistance genes [tem-1,adc-30,aac( 6′)-Ⅰ b,ant( 3″)-Ⅰ,abeb,qace Δ1] and mobile genetic elements ( int Ⅰ 1,tnpu,tnp513,is26,isaba1 ).conclusion clinical isolated pandrug-resistant acinetobacter baumannii carries several acquired drug resistance-related genes and mobile genetic elements,and there may be a close association between them.     

广州地区儿童偏肺病毒感染的分子流行病学研究

目的 了解广州地区婴幼儿患者呼吸道中人偏肺病毒(hmpv)感染的分子流行病学特点.方法 采集2010年1-12月到广州市妇女儿童医疗中心就诊的呼吸道感染患儿的咽拭子等样本1840份,对临床样本进行hmpv检测,对f基因进行扩增,并对pcr产物进行测序.结果 1 840份临床样本中66份hmpv检测阳性,阳性率为3.59％.全年除9、10月份外每月均有hmpv检出,其中4月份检出阳性率最高,为6.09％.随机选取3株hmpv,将其f基因核苷酸序列与a1、a2、b1、b2、c型和2008年分离的hmpvgz01株的f基因序列进行clustal w比较,发现与a2b组的bj1887同源性最高,为97％,4株广州分离的病毒株之间f基因的同源性为99％.序列与进化树分析表明广州地区流行的是a2b型.结论 2010年广州地区hmpv流行季节是春夏季,广州地区流行株的型别为a2b亚型. abstract： objective to conduct a molecular epidemiological study on human metapneumovirus (hmpv) among pediatric patients in guangzhou. methods a total of 1 840 clinical specimens were obtained from pediatric patients with respiratory infections in guangzhou women and children' s medical center in 2010. hmpv was detected by real-time taqman rt-pcr in clinical specimens. f gene was amplified and the pcr-products were directly sequenced. results in 1 840 clinical specimens, 66 werehmpv-positive with a positive rate of 3.59％. hmpv was detected in all specimens except those collected in september and october, and the highest positive hmpv rate occurred in april (6.09％). the f genes of 3 randomly selected strains and hmpvgz01 ( isolated in 2008) were compared with subgroups a1, a2, b1,b2 and c, and the highest homology was with bj1887 strain of genotype a2b (97％). the f genes of the randomly selected strains and hmpvgz01 were 99％ identical to each other. sequences and phylogenetics analysis revealed that the epidemical strain in guangzhou belonged to genotype a2b. conclusion hmpv is prevalent in spring and summer among children in guangzhou, and a2b is the predominant genotype.     

膜孔蛋白基因缺失与肺炎克雷伯菌耐碳青霉烯类抗生素的关系

目的 研究肺炎克雷伯菌对碳青霉烯类抗生素耐药的分子机制.方法 采用浓度梯度法(e-test)测定细菌对各种抗菌药物的最低抑菌浓度(mic),聚合酶链反应(pcr)检测23种β-内酰胺酶基冈和2种膜孔蛋白基因,并对扩增产物进行基因测序.结果 5株肺炎克雷伯菌对哌拉西林、哌拉西林/他唑巴坦、阿莫西林/克拉维酸、头孢哌酮、头孢噻肟、头孢吡肟和氨曲南的mic值均≥128 μg/ml,而对亚胺培南和美罗培南的mic值均≥32 μg/ml.5株肺炎克雷伯菌均携带blatem-1和bladha-1基因.kp01和kp03同时出现ompk35和ompk36基因缺失.kp02、kp04、kp05的ompk36出现碱基插入,kp02、kp05的ompk35碱基缺失.与genbank(g0945384)比较,kp04的ompk35基因发生第465位g→c和466位t→c突变,致gln 155 his、tyr 156 his氨基酸替换,可能为新的亚型.结论 β-内酰胺酶的产生合并ompk36或ompk35膜孔蛋白基因的缺失可引起肺炎克雷伯菌对碳青霉烯类抗生素耐药. abstract： 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.