β-内酰胺酶合并OmpK36膜孔蛋白缺失引起肺炎克雷伯菌对碳青霉烯类抗生素敏感性的降低

目的 研究肺炎克雷伯菌(Klebsiella pneumoniae,Kp)对碳青霉烯类抗生素敏感性降低的分子机制.方法 临床分离到2株碳青霉烯敏感性降低的Kp Z2554和Z2110,经脉冲场凝胶电泳(PFGE)证实非同一克隆株.对2株细菌进行药物最低抑菌浓度测定(MIC)、接合试验、质粒图谱分析、等电聚焦电泳(IEF)、特异性PCR扩增和DNA序列分析、外膜蛋白分析以及羰酰氰氯苯腙(CCCP)抑制试验等.结果 Kp Z2554和Z2110对亚胺培南和美罗培南的MIC为1～8 μg/ml,前者对青霉素类、头孢菌素类和氨曲南等抗生素高度耐药,后者对上述抗生素的耐药程度稍弱于前者,但对头孢西丁的MIC大于512 μg/ml.IEF、PCR扩增和DNA序列分析证实Kp Z2554产TEM-1(等电点5.4)和CTX-M-14(等电点7.9)2种β-内酰胺酶,Kp Z2110产TEM-1、CTX-M-14和DHA-1(等电点7.8)3种酶.SDS-PAGE分析显示2株细菌均有相对分子质量(Mr)为39×103蛋白条带(OmpK36)的缺失.外膜蛋白基因(ompK35和ompK36)序列分析发现,Kp Z2554和Z2110的ompK35基因的个别位点存在点突变,但氨基酸序列不变.CCCP抑制试验显示CCCP不能提高Kp Z2554和Z2110对碳青霉烯类抗生素的敏感性.结论 β-内酰胺酶的产生合并OmpK36膜孔蛋白的缺失可引起Kp对碳青霉烯类抗生素敏感性的降低.     

北京康复医院耐碳青霉烯类肺炎克雷伯菌临床分布及耐药性分析

目的 分析该院耐碳青霉烯类肺炎克雷伯菌(CR-KP)的临床分布特点及药敏检测结果,为临床合理有效治疗CR-KP感染提供理论依据。方法 分析2021年全年该院检出的非重复分离CR-KP菌株。利用VITEK 2 Compact全自动微生物分析系统进行菌株鉴定和药敏检测试验。药敏结果参照美国临床实验室标准化协会(CLSI M100)标准判读。所有数据使用WHONET 5.6软件进行统计分析。结果 2021年全年该院共分离非重复的肠杆菌科细菌942株,其中CR-KP菌株295株,检出率高达31.3%。CR-KP菌株的标本来源主要是尿液、痰液、血液标本。主要分布科室为神经康复中心、老年康复中心、呼吸康复中心。CR-KP菌株对大部分临床常用的抗菌药物呈现高度耐药性,除对替加环素较为敏感(耐药率为13.5%),对阿米卡星和复方新诺明耐药率在60%以下,对其余检测的抗菌药物耐药率均大于90.0%。结论 该院CR-KP检出占比较高,对大多数抗菌药物耐药情况非常严峻,医护人员应高度重视,合理使用抗菌药物,同时应加强院感防控和手卫生,防止CR-KP暴发流行。     

耐碳青霉烯高毒力肺炎克雷伯菌的临床及分子特点

目的:探讨耐碳青霉烯类高毒力肺炎克雷伯菌(carbapenem-resistant hypervirulent K.pneumoniae,CR-hvKP)感染的临床危险因素和分子流行病学特点,为CR-hvKP菌株的临床预防和治疗提供理论指导.方法:收集徐州医科大学附属医院2017年1月1日至2019年5月31日临床首次...     

对碳青霉烯类耐药的铜绿假单胞菌的金属β-内酰胺酶研究

目的：研究耐碳青霉烯类的铜绿假单胞菌产金属β-内酰胺酶的情况及铜绿假单胞菌的耐药机制。方法：微量二倍稀释法测定亚安培南及美洛培南对铜绿假单胞菌的MIC值；DETA纸片法检测产金属β-内酰胺酶的铜绿假单胞菌。结果：从62株临床分离的铜绿假单胞菌中共检测到28株对亚胺培南耐药，耐药率为45％，18株对美洛培南耐药，耐药率为29．03％。从18株同时对亚胺培南及美洛培南耐药株中共检测到16株产金属β-内酰胺酶，占同期分离铜绿假单胞菌的25．8％。结论：产生金属β-内酰胺酶是铜绿假单胞菌对碳青霉烯类药物耐药的机制之一。     

头孢他啶/阿维巴坦对耐碳青霉烯类肺炎克雷伯菌的体外抗菌活性研究

目的 研究头孢他啶/阿维巴坦(CZA)对耐碳青霉烯类肺炎克雷伯菌(CRKP)的体外抗菌活性并检测其相关耐药基因,为临床治疗CRKP提供参考依据.方法 选取2017年1月至2019年12月北京中医药大学东方医院临床分离的257株CRKP菌株,应用VITEK MS微生物质谱仪和VITEK-2 Compact微生物药敏分析仪进行菌种鉴定和药敏分析,用K-B法检测CZA的药物敏感性.采用碳青霉烯酶抑制剂增强试验对CRKP菌株进行产酶表型检测.通过NG-Test CARBA 5对耐CZA的CRKP菌株进行相关耐药基因检测.结果 257株CRKP菌株对CZA的耐药率为7.8％,对哌拉西林/他唑巴坦、阿莫西林/克拉维酸、头孢哌酮/舒巴坦、头孢呋辛、头孢他啶、头孢曲松、头孢西丁的耐药率均为100％,头孢吡肟97.2％、左氧氟沙星97.1％、阿米卡星48.5％、庆大霉素76.4％、复方新诺明46.5％、替加环素35.5％.碳青霉烯酶抑制剂增强试验结果显示,CRKP菌株耐药主要以产丝氨酸酶为主.20株耐CZA的CRKP菌株中,产KPC 11株,产OXA-481株,产IMP 1株,产NDM 4株,既产KPC又产NDM 2株,不产碳青霉烯酶1株.结论 CZA对CRKP菌株有较强的抗菌效果,抗菌活性明显优于临床常用抗菌药物.CZA体外药敏试验可在临床常规开展,为临床治疗CRKP提供更多的参考.     

2014~2018年我院耐碳青霉烯类肺炎克雷伯菌分布特点及感染患者的临床特点

目的 调查某三甲医院近5年耐碳青霉烯类肺炎克雷伯菌的检出情况、感染患者的临床特点,为临床诊疗提供依据。方法收集2014年1月~2018年12月该院临床分离的肺炎克雷伯菌菌株,分析耐碳青霉烯类肺炎克雷伯菌检出率、标本来源分布、科室分布、耐药情况及预后因素。结果 共检出耐碳青霉烯类肺炎克雷伯菌690株,检出率为14.29%;2018年耐碳青霉烯类肺炎克雷伯菌检出率高于2014~2016年,差异有统计学意义（P＜0.05）;标本来源前5位分别为痰、中段尿、血、切口分泌物、导管,标本科室前5位分别是ICU、呼吸科、泌尿外科、烧伤科、神经外科。16种抗生素药敏检测中,CRKP对15种抗生素耐药率超过50%,耐碳青霉烯类肺炎克雷伯菌对复方新明、米诺环素、替加环素、多粘菌素耐药率相对较低,分别为46.91%、31.86%、10.73%、10.10%。治疗无效患者及治疗有效患者在合并基础病≥3种、患有糖尿病、患有恶性肿瘤、入住ICU、机械通气、保留导尿、其他侵袭性操作、混合感染间比较,差异有统计学意义（P＜0.05）;治疗无效患者及治疗有效患者在性别、吸烟、饮酒、心血管疾病、神经系统疾病、血液病、慢性肺病、慢性肝肾功能不全、近期大手术、既往多次住院或本次就诊前已住院间比较,差异无统计学意义（P＞0.05）。结论 耐碳青霉烯类肺炎克雷伯菌感染呈逐年上升趋势,以ICU中耐青霉烯类肺炎克雷伯菌感染患者最多。临床应针对其可能的预后因素,采取有效、可行的诊治和防控措施预防与减少耐碳青霉烯类肺炎克雷伯菌感染发生几率。     

产超广谱β-内酰胺酶肺炎克雷伯菌药物敏感性和基因型研究

【摘要】 目的 研究产超广谱β-内酰胺酶（ESBLs）肺炎克雷伯菌基因型和药敏特征。方法 对2003年12月～2004年12月哈尔滨地区临床分离的74株产ESBLs肺炎克雷伯菌，测定菌株对常用β-内酰胺类和非β-内酰胺类抗菌药物的敏感性，并采用SHV型和TEM型特异性引物检测ESBLs的基因型别。结果 产ESBLs细菌对头孢吡肟、氨曲南、阿米卡星和环丙沙星的敏感率依次71.6%、20.3%、54.1%、31.1%，对头孢哌酮/舒巴坦的敏感率为54.1%，69株（93.2%）对亚氨培南敏感。74株产ESBLs细菌中携带blaSHV和blaTEM基因的阳性例数分别是45（60.8%）和23（31.1%）。结论 本地区产ESBLs肺炎克雷伯菌对常用β-内酰胺类和非β-内酰胺类抗菌药物的敏感性降低，临床上需慎用酶抑制剂复合物及第四代头孢菌素，亚氨培南仍是治疗产ESBLs菌感染的首选药物。产ESBLs肺炎克雷伯菌blaSHV的流行频率高于blaTEM。     

产生超广谱β-内酰胺酶的肺炎克雷伯菌的药物敏感性及其基因型研究

目的研究产生超广谱β-内酰胺酶（ESBLs）的肺炎克雷伯菌基因型及其药敏特征。方法对2003年12月～2004年12月哈尔滨地区临床分离的74株产ESBLs肺炎克雷伯菌,测定菌株对常用β-内酰胺类和非β-内酰胺类抗菌药物的敏感性,并采用SHV型和TEM型特异性引物检测ESBLs的基因型。结果产ESBLs细菌对头孢吡肟、氨曲南、阿米卡星和环丙沙星的敏感率依次为71．6％、20．3％、54．1％、31．1％,对头孢哌酮／舒巴坦的敏感率为54．1％,69株（93．2％）对亚胺培南敏感。74株产ESBLs细菌中携带blaSHV和6kTEM基因的阳性例数分别是45例（60．8％）和23例（31．1％）。结论哈尔滨地区产ESBLs肺炎克雷伯菌对常用β-内酰胺类和非β-内酰胺类抗菌药物的敏感性降低,临床上需慎用酶抑制剂复合物及第4代头孢菌素,亚胺培南仍是治疗产ESBLs菌感染的首选药物。产ESBLs肺炎克雷伯菌blaSHV的流行频率高于blaTEM.     

blaKPC-2在耐碳青霉烯类肺炎克雷伯菌中的传播机制

耐碳青霉烯类肺炎克雷伯菌(CRKP)在临床高度流行, 由于缺乏有效治疗药物而带来了显著挑战。blaKPC-2编码的肺炎克雷伯菌碳青霉烯酶-2(KPC-2)是CRKP产生碳青霉烯抗性的主要原因之一。在中国和其他亚洲地区, Tn1721和IncFⅡ质粒是blaKPC-2在缺乏规律间隔成簇短回文重复序列(CRISPR)和限制-修饰(R-M)系统的Kpn ST11之间转移的主要载体。转座子的结构对耐药基因的转座频率有明显影响, 这可能和不同转座子的转座模式有关系。blaKPC-2在ST11中有流行优势与ST11免疫缺陷有重要联系, 用重新设计的CRISPR/Cas3系统通过接合实验传递入ST11型肺炎克雷伯菌, 高危IncFⅡ质粒可以被成功剪切, 从而使ST11型CRKP恢复对抗生素的敏感性, 这为CRKP的临床治疗和预防提供了新思路。     

呼和浩特市某三甲医院肺炎克雷伯菌的科室分布及耐药特点

目的:调查2016至2019年呼和浩特某三甲医院肺炎克雷伯菌(Klebsiella pneumoniae,KP)临床分离株的科室、标本分布以及对抗生素的敏感性,为临床制定合理的抗感染方案提供参考.方法:回顾性分析内蒙古医科大学附属医院2016年1月至2019年12月临床分离的2975株KP菌株的临床资料.采用VITEK...     

氟喹诺酮体外诱导耐药肺炎克雷伯菌膜孔蛋白表达的变化

目的 探讨氟喹诺酮体外诱导耐药肺炎克雷伯菌(KPn)膜孔蛋白表达变化.方法 取2008年9月至2009年6月本院临床分离对环丙沙星敏感的KPn 20株,分为对照组和实验组,每组10株.对照组直接涂布于含环丙沙星128 mg/L的平板,观察其对环丙沙星的敏感性.实验组应用不同浓度梯度环丙沙星逐级诱导成为高度耐药株,观察应用环丙沙星前后KPn敏感株和耐药株膜孔蛋白表达的差异.结果 对照组10株KPn直接涂布于含环丙沙星128 mg/L的平板培养后无一存活.3对KPn菌株R9、S9、R4、S4、R3、S3环丙沙星诱导前后膜孔蛋白相对表达量分别是3.86±0.11、6.44±0.26、5.46±0.18、9.58±0.34、1.75±0.06和9.78±0.36,诱导耐药的KPn较相应敏感株膜孔蛋白表达明显减少(均P＜0.05).结论 氟喹诺酮体外诱导耐药KPn膜孔蛋白表达减少,可能通过细菌外膜通透性改变在KPn诱导耐药中起重要作用.     

Study of Klebsiella pneumoniae producing extendedspectrum β-lactamases against aminoglycosides

Klebsiella pneumoniae （ K. pneumoniae） is one of the main gmn-negative bacilli in clinical practice. Nosocomial infections caused by K. pneumoniae producing extended-spectrum β-lactamases （ESBLs） are very difficult to treat. This paper investigated the resistant characteristics of K. pneumoniae producing ESBLs and their aminoglycoside-modifying enzyme gene expressions including Nacetyltransferases and O-adenyltransferases. Bacteria identification and ESBLs confirmatory tests were performed by Phoenix^TM-100 system. And minimum inhibitory concentrations （MICs） of gentamicin, amikacin, kanamycin, tobranycin, netilmicin and neomycin in 53 K. pneumoniae isolates were detected by agar dilution. In addition, six aminoglycoside-modifying enzyme genes were amplified by polymerase chain reaction （PCR） and verified by DNA sequencer. It was found that imipenem and meropenem against 120 K. pneumoniae isolates produced powerful antimicrobial activities. The resistant rates of gentamicin and amikacin were 55.0% and 46.7%, respectively. Except neomycin, MIC50 and MIC90 of gentamicin, amikacin, kanamycin, tobramycin and netilmicin in 53 K. pneumoniae were all 〉 128 μg/ml, and the resistant rates were 83.0%, 52.3%, 75.5%, 81.1% and 69.8%, respectively. However, neomycin was only 39.6%. In addition, five modifying enzyme genes, including aac（3）-Ⅰ, aac（3）-Ⅱ, aac（6＇）-Ⅰb, ant（3＂）-Ⅰ, ant（2＂）-Ⅰ genes, were found in 53 isoaltes except aac （6＇）-Ⅱ, and their positive rates were 11.3%, 67.9%, 47.2%, 1.9% and 39.6%, respectively. It was also confirmed by nucleotide sequence analysis that the above resistant genes shared nearly 100% identities with GenBank published genes. The results obtained in the present study indicated that K. pneumoniae producing ESBLs strains are rapidly spreading in our hospital, and their resistance to aminoglycosides may be associated with aminoglycoside-modifying enzyme gene expressions.     

二家医院肺炎克雷伯菌连续分离株质粒AmpC基因研究

目的了解不同医院间肺炎克雷伯菌连续分离株质粒AmpC基因存在情况和耐药性。方法应用聚合酶链反应（PCR）法对耐药的肺炎克雷伯菌连续分离株进行AmpC酶DHA和ACT-1基因检测和分析。结果PCR结果显示A院44株肺炎克雷伯菌中DHA基因阳性4株（9．1％）；B院25株肺炎克雷伯菌中DHA基因阳性8株（32．0％），ACT-1基因二家均为阴性，二家医院DHA基因检出率有明显差别（P〈0．05）。结论质粒型AmpC酶基因可通过转化、接合等方式转移给其他同种或不同种菌，易于传播。二家医院DHA基因检出率有明显差别，并均己有流行的迹象。     

产AmpC酶大肠埃希菌和肺炎克雷伯菌中整合子与多重耐药的研究

目的探讨整合子介导的耐药机制在产AmpC酶大肠埃希菌和肺炎克雷伯菌多重耐药中的作用.方法5株产AmpC酶大肠埃希菌和肺炎克雷伯菌分离自2002年1月-2004年5月间我院呼吸科住院的患者,采用E-test试验条进行药敏试验、电转化试验,筛选、分离耐药质粒.PCR扩增Ⅰ型整合子基因盒插入序列,分子克隆和序列分析.结果所有产酶菌株通过电转化试验可将头孢西丁耐药性传递给受体菌,5个产AmpC酶耐药质粒中,有4个检出整合酶序列,其中3个携带2种抗药性基因盒,包括氨基糖苷乙酰转移酶基因aacA4;氨基糖苷腺苷转移酶基因aadA5;二氢叶酸还原酶基因dfrA17;氯霉素外排蛋白编码基因cmL44.结论整合子介导的抗药性基因盒参与了产质粒AmpC酶大肠埃希菌和肺炎克雷伯菌多重耐药的形成,应引起高度重视.     

Does the presence of Klebsiella pneumoniae carbapenemase and New Delhi metallo-β-lactamase-1 genes in pathogens lead to fatal outcome?

Introduction: Infections due to multidrug-resistant (MDR) pathogens are a medical challenge. There is considerable apprehension among clinicians regarding pathogens reported as carrying New Delhi metallo-β-lactamase-1 (NDM) and Klebsiella pneumoniae carbapenemase (KPC) genes from their patients. In the face of extremely high rates of antimicrobial resistance, it is essential to gauge the clinical significance of isolation of pathogens carrying these genes from clinical samples. This study compares the outcome of patients infected with pathogens carrying NDM/KPC genes versus those without these genes. Methods: The study was conducted over a 1-year period at a Level-1 trauma centre. Hospital-acquired infections were diagnosed on the basis of CDC’s criteria. The correlation of isolation of a multi-resistant pathogen carrying KPC or NDM genes with the clinical outcome was ascertained. Results: A total of 276 consecutive patients admitted to the Intensive Care Units/wards of the JPNA Trauma Centre were included in this study. Of the 371 isolates recovered from these patients, 116 were from patients who had a fatal outcome. The difference in prevalence of bla_NDM and bla_KPC was not significant in any genera of Gram-negative pathogens isolated from patients who survived versus those who had a fatal outcome. Conclusion: Isolation of MDR pathogens carrying NDM/KPC genes from clinical samples is not always a harbinger of a fatal outcome. Efforts should be made to prevent cross-transmission of these pathogens.     

儿童感染肺炎克雷伯菌产AmpC酶和ESBLs的检测及耐药性分析

目的了解广州地区儿童感染肺炎克雷伯菌产质粒介导的AmpC酶和超广谱β-内酰胺酶（ESBLs）的情况及其耐药特征,为临床合理用药提供参考依据。方法采用标准纸片扩散法检测ESBLs,头孢西丁三维试验法检测AmpC酶,K—B纸片法测定肺炎克雷伯菌对抗菌药物的敏感性。结果共检出248株肺炎克雷伯菌,其中46株产AmpC酶,阳性率为18．5％;157株产ESBLs,阳性率为63．3％;同时产AmpC酶和ESBLs菌株阳性率为18．1％。产AmpC酶肺炎克雷伯菌对第三代头孢菌素高度耐药。耐药率达80％～100％;对头孢吡肟、含酶抑制剂复合药的耐药率也在56．5％～93．5％之间：但对环丙沙星、阿米卡星的耐药率则在30％以下,对亚胺培南全部敏感。产ESBLs菌株对头孢菌素、含酶抑制剂复合药的耐药率也较高,在50％-91．7％之间,但对阿米卡星、环丙沙星、亚胺培南仍高度敏感。ESBLs阴性肺炎克雷伯菌对所测抗生素的敏感率均在81．2％以上。产酶菌株耐药率明显比非产酶菌株高。结论广州地区儿童感染肺炎克雷伯菌产ESBLs和AmpC酶的状况已十分突出：产酶菌株对常用抗生素的耐药率较高;碳青霉烯类抗生素可作为治疗产AmpC酶和／或ESBLs肺炎克雷伯菌感染的经验用药。     

Identification of plasmid-mediated quinolone resistance genes qnrA1, qnrB1 and aac(6')-1b-cr in a multiple drug-resistant isolate of Klebsiella pneumoniae from Chennai

Purpose: Resistance to fluoroquinolones, a commonly prescribed antimicrobial for Gram-negative and Gram-positive microorganisms, is of importance in therapy. The purpose of this study was to screen for the presence of Plasmid-Mediated Quinolone Resistance (PMQR) determinants in clinical isolates of Klebsiella pneumoniae. Materials and Methods: Extended-Spectrum Beta-Lactamase (ESBL) isolates of K. pneumoniae collected during October 2009 were screened by the antimicrobial susceptibility test. The plasmids from these isolates were analysed by specific Polymerase chain Reaction (PCR) for qnrA, qnrB and aac(6’)-1b. The amplified products were sequenced to confirm the allele. Results: Our analysis showed that 61% out of the 23 ESBL K. pneumoniae isolates were resistant to ciprofloxacin and 56% to levofloxacin. The PMQR was demonstrated by transforming the plasmids from two isolates P12 and P13 into E. coli JM109. The PMQR gene qnrA was found in 16 isolates and qnrB in 11 isolates. The plasmid pKNMGR13 which conferred an minimum inhibitory concentration (MIC) of more than 240 µg/ml in sensitive E. coli was found to harbour the qnrA1 and qnrB1 allele. Furthermore, the gene aac(6’)-1b-cr encoding a variant aminoglycoside 6’-N Acetyl transferase which confers resistance to fluoroquinolones was found in the same plasmid. Conclusions: Our report shows the prevalence of PMQR mediated by qnrA and qnrB in multidrug-resistant K. pneumoniae isolates from Chennai. A multidrug-resistant plasmid conferring high resistance to ciprofloxacin was found to harbour another PMQR gene, aac(6’)–1b-cr mutant gene. This is the first report screening for PMQR in K. pneumoniae isolates from India.     

质粒介导KPC-2型碳青霉烯酶肺炎克雷伯菌儿童分离株耐药基因研究

目的 研究碳青霉烯类耐药肺炎克雷伯菌临床儿童分离株的耐药特点及分子流行病学特征.方法 收集温州医学院附属第二医院2010年7月-2011年6月从儿童标本中分离的耐碳青霉烯类肺炎克雷伯菌12株,所有菌株为非重复菌株,菌种鉴定采用全自动微生物分析仪.改良的Hodge试验筛选产碳青霉烯酶阳性菌株,采用PCR法检测KPC、IMP、bla(s)、VIM、SPM和整合酶基因,测序确定基因型.对菌株进行质粒结合试验、质粒消除试验检测质粒的转移性.脉冲场凝胶电泳(PFGE)分析耐药菌株的同源性.结果 12株耐碳青霉烯类肺炎克雷伯菌对庆大霉素、妥布霉素、阿米卡星、环丙沙星、左氧氟沙星、复方磺胺甲噁唑的敏感率分别为8.3％、41.7％、58.3％、8.3％、8.3％、33.3％;12株菌均携带有KPC-2基因,且同时携带有TEM-1和SHV型β-内酰胺酶基因,其中SHV-11-like和SHV-1 2-like各6株;11株携带CTX-M型基因,其中4株为CTX-M-14-like基因,6株CTX-M-15-like基因;2株携带有OXA-10型基因,1株携带有PER-1基因.未检出NDM-1、GIM、SPM、SIM、VIM型碳青霉烯酶基因.12株均为Ⅰ类整合酶基因(int1)阳性.2株通过接合试验把质粒传递给受体菌EC600.所有接合子blaTEM-1基因阳性、超广谱β-内酰胺酶(ESBL)基因阳性及对亚胺培南、庆大霉素、阿米卡星、妥布霉素和头孢噻肟耐药,接合子ESBL基因型与供菌一致.2株菌经质粒消除后对亚胺培南的MIC值均有较大程度降低,消除后KPC-2基因扩增为阴性.12株KPC-2基因阳性菌株经PFGE分成5个基因型,主要为B型和C型.结论 KPC-2型碳青霉烯酶基因已经在儿童肺炎克雷伯菌中播散,常伴随携带多种类型的ESBL基因和Ⅰ类整合酶基因,部分耐药基因可通过质粒播散.     

RAPD typing of Klebsiella pneumoniae, Klebsiella oxytoca, Serratia marcescens and Pseudomonas aeruginosa isolates using standardized reagents

Objective: To perform quality assessment of standardized random amplified polymorphic DNA (RAPD) analysis for epidemiologic typing of Klebsiella pneumoniae, K. oxytoca, Serratia marcescens and Pseudomonas aeruginosa .
Methods: Thirty K. pneumoniae , 15 K. oxytoca , 30 S. marcescens and 33 P. aeruginosa epidemiologically unrelated isolates and four collections of clinically related isolates of each species were included in the study. RAPD analysis was performed using Ready-To-Go RAPD Analysis beads with primer ERIC-1R and Ready-To-Go primer 2 for K. pneumoniae and K. oxytoca , primer set ERIC-2/1026 and Ready-To-Go primer 2 for S. marcescens , and primers D-10514 and D-14306 for P. aeruginosa .
Results: All epidemiologically unrelated K. pneumoniae and K. oxytoca isolates were distinguished. Twenty-nine types were distinguished among the 30 unrelated S. marcescens isolates and 32 types among the 33 unrelated P. aeruginosa isolates. Indistinguishable banding patterns were obtained in repeated analyses of two isolates and from 11 serial subcultures of three isolates of each species included in the study. The RAPD data from the clinically related isolates correlated with the epidemiologic origin of the isolates.
Conclusions: The use of Ready-To-Go RAPD Analysis beads resulted in reproducible and stable banding patterns with a high discriminatory capacity, and the RAPD typing results corresponded with the epidemiologic origin of the isolates.     

Co-existence of NDM-1 and OXA-48 genes in Carbapenem Resistant Klebsiella pneumoniae clinical isolates in Kafrelsheikh,Egypt

BackgroundThe noteworthy spread of carbapenem-resistant K. pneumoniae (CR-KP) isolates represents a significant safety threat.ObjectiveDetermination of the carbapenemase genes incidence among CR-KP clinical isolates in Kafrelsheikh, Egypt.MethodsA total of 230 K. pneumoniae isolates were recovered from four hospitals in Kafrelsheikh, Egypt. Susceptibility testing was conducted using Kirby-Bauer method and automated-Vitek2 system. CR-KP isolates were tested using modified Hodge test (MHT) and combined disk synergy test. PCR and DNA sequencing were conducted for CR-KP isolates to recognize the included carbapenemase-genes.ResultsOut of 230 K. pneumoniae isolates, 50 isolates presented resistance to carbapenem (meropenem). All 50 CR-KP isolates were multidrug-resistant (MDR). Genes like blaNDM-1 and blaOXA-48 were the only detected genes among CR-KP with an incidence of 70.0% and 52.0%, respectively. Up to 74.0% of the tested isolates carried at least one of the two recorded genes, among them 48.0% co-harbored both blaNDM-1 and blaOXA-48 genes. The accession-numbers of sequenced blaNDM-1 and blaOXA-48 genes were {"type":"entrez-nucleotide","attrs":{"text":"MG594615","term_id":"1344457373"}}MG594615 and {"type":"entrez-nucleotide","attrs":{"text":"MG594616","term_id":"1344457375"}}MG594616, respectively.ConclusionThis study reported a high incidence of MDR profile with the emergence of blaNDM-1 and blaOXA-48 genes co-existence in CR-KP isolates in Kafrelsheikh, Egypt. Hence, more restrictions should be applied against the spread of such serious pathogens.