耐碳青霉烯铜绿假单胞菌耐药性的基因学研究进展

碳青霉烯类抗生素是治疗铜绿假单胞菌感染的有效药物,但随着此类抗生素的广泛应用,铜绿假单胞菌对碳青霉烯类抗生素的耐药率呈上升趋势。本文从药物的主动转运系统、抗菌药物渗透障碍、产生药物灭活酶及形成生物被膜这4个方面对耐碳青霉烯铜绿假单胞菌耐药性的基因学研究进行综述,为临床合理应用抗生素和进行感染防控提供理论依据。     

铜绿假单胞菌对碳青霉烯类抗生素耐药性

目的 探讨铜绿假单胞菌对碳青霉烯类抗生素的耐药性和耐药机制。方法 选取温州医学院附属第一医院162株铜绿假单胞菌临床分离株,采用琼脂稀释法检测抗生素亚胺培南和美罗培南对铜绿假单胞菌的最低抑菌浓度(MIC);PCR扩增分离株外膜蛋白基因OprD₂和碳青霉烯酶基因VIM、IMP、SPM、KPC,对阳性产物测序确定基因亚型;羰基氰氯苯腙(CCCP)协同抑制试验检测膜外排机制。结果 162株分离菌株中,亚胺培南和美罗培南的耐药率分别为37.0%和30.9%;60株耐亚胺培南和/或美罗培南菌株(简称耐药株)中,50株对2种抗生素均耐药,另10株仅对亚胺培南耐药;耐药株中有18株OprD₂基因缺失,102株不耐亚胺培南或美罗培南菌株(简称敏感株)中有20株OprD₂基因缺失;耐药株中检出13株碳青霉烯酶阳性,其中5株产VIM型,8株产IMP型,敏感株中未检出VIM、IMP基因,所有菌株中未检出SPM-1、KPC基因;耐药株有48.3%外排泵表型试验阳性,以亚胺培南和美罗培南作为底物的分别有19株和24株,敏感株有10.8%外排试验阳性。结论 铜绿假单胞菌对碳青霉烯类抗生素耐药严重,与外膜蛋白OprD₂缺失、产碳青霉烯酶和主动外排共同作用有关。     

铜绿假单胞菌1238株对碳青霉烯类抗生素耐药性分析

目的调查武汉大学人民医院近5年铜绿假单胞菌对碳青霉烯类抗生素的耐药率及其变迁情况。方法收集临床分离的铜绿假单胞菌进行培养并行药敏试验。结果分离出铜绿假单胞菌的标本以痰为主(74.80%),主要分布于神经外科(20.27%)和呼吸内科(13.00%)。2006—2010年铜绿假单胞菌对亚胺培南的耐药率依次为17.4%、38.1%、55.5%、27.8%、37.9%(P0.01),对美罗培南的耐药率依次为11.1%、41.0%、41.5%、23.3%、33.1%(P0.01)。结论铜绿假单胞菌对碳青霉烯类抗生素耐药率自2006—2008年呈明显上升趋势,2008—2010年有所下降,临床应根据药敏结果合理用药,以减少和延缓耐药菌株的产生。     

耐碳青霉烯类铜绿假单胞菌耐药基因与药敏分析

目的调查医院耐碳青霉烯类铜绿假单胞菌(Carbapenem-resistant Pseudomonas aeruginosa,CRPA)的耐药特性,为指导临床抗菌药物应用、降低细菌耐药性和防控医院感染提供参考。方法收集2017年3-12月在南昌大学第一附属医院分离的CRPA 95株,检测其耐药性;聚合酶链式反应(PCR)检测碳青霉烯类相关基因及外膜蛋白基因OprD2的携带情况。结果测试菌株对亚胺培南和美罗培南的耐药率分别为97.89%和95.79%,对哌拉西林/他唑巴坦的耐药率最低为50.53%,对阿米卡星、头孢吡肟、头孢他啶、左氧氟沙星的耐药率在51%～60%间,对氨曲南、环丙沙星、哌拉西林、庆大霉素、妥布霉素、头孢哌酮/舒巴坦的耐药率均≥60%。PCR法检测出67株(70.53%)OprD2基因缺失菌株,12株(12.63%)bla_(GES)阳性菌,10株(10.53%)bla_(VIM)阳性菌,bla_(IMP)、bla_(GIM)、bla_(SIM)、bla_(TEM)、bla_(PER)、bla_(VEB)、bla_(OXA-10)阳性菌均未检出。结论 CRPA的耐药形势严峻,其耐药主要与细菌产生bla_(GES)基因、bla_(VIM)基因和缺失OprD2基因相关。临床应加强对CRPA的监测,合理使用抗菌药物,最大限度地降低细菌耐药。     

耐碳青霉烯类铜绿假单胞菌的耐药机制及特征分析

目的 探究本院耐碳青霉烯类铜绿假单胞菌(carbopenem-resistant pseudomonas aeruginosa, CRPA)的耐药机制及特征。方法 用BD phoenix M50全自动鉴定药敏分析仪对菌株进行鉴定及药敏分析,微量肉汤稀释法测定头孢他啶/阿维巴坦的最低抑菌浓度(minimal inhibitory concentration, MIC),采用改良碳青霉烯类失活法(modified carbapenem inactivation method, mCIM)和胶体金免疫层析法对菌株进行碳青霉烯酶表型检测,采用全基因组测序对筛查阳性菌株进行碳青霉烯酶耐药基因检测及ST分型。结果 (1)收集22株临床分离CRPA,其中耐药率最低的抗菌药物为头孢他啶/阿维巴坦(22.7%),然后依次为庆大霉素和阿米卡星(27.3%),哌拉西林/他唑巴坦(59.09%),头孢吡肟(63.6%),头孢他啶和氨曲南(77.27%),环丙沙星(86.36%),左氧氟沙星(95.45%)。(2)22株CRPA表型检测结果显示产碳青霉烯酶共5株(22.7%)。(3)全基因组测序结果显示,有4株...     

儿科铜绿假单胞菌的OprD基因突变及表达对碳青霉烯类抗生素耐药的作用

目的:探讨我院儿科病区分离的碳青霉烯类耐药铜绿假单胞菌(CRPA)流行情况及OprD基因突变及表达在碳青霉烯类耐药中的作用。方法:儿科分离的30株碳青霉烯类耐药的铜绿假单胞菌,随机扩增DNA多态性(RAPD)进行同源性分析;PCR法检测KPC、IMP、VIM、SPM等产酶基因和OprD基因并双向测序及比对;FQ-RT-PCR测定OprD基因的mRNA相对表达量;SDS-PAGE电泳分析OprD外膜蛋白缺失情况。结果:儿科病区30株CRPA中,检出IMP基因阳性菌株1株,接合试验阴性。oprD基因缺失4株(4/30),突变率100%(9/9),碱基突变主要集中在1010 bp~1136 bp和1212 bp~1355 bp区域。CRPA菌株的oprD基因相对表达量与非耐药组差异有显著性(t=6.145,P<0.05),oprD基因突变位点与其表达量之间无相关性。结论:oprD基因发生广泛突变及表达量降低或缺失是本院儿科CRPA菌株对碳青霉烯类抗生素耐药的主要原因之一。     

烧伤患者耐碳青霉烯类铜绿假单胞菌感染影响因素分析

目的 探讨烧伤患者耐碳青霉烯类铜绿假单胞菌(carbapenem-resistant aeruginosa,CRPA)感染发生和死亡的影响因素。方法 回顾性分析2017年1月-2018年12月烧伤科108例铜绿假单胞菌感染患者的临床资料,对患者性别、年龄、基础疾病、使用抗菌药物情况、细菌药敏结果、预后情况等资料进行分析,归纳烧伤患者CRPA感染和死亡的影响因素。结果 108例铜绿假单胞菌感染患者中,CRPA感染者33例占30.56%。入住ICU和白蛋白<30 g/L是CRPA感染的影响因素(P<0.05)。CRPA感染是铜绿假单胞菌感染患者死亡的影响因素(P<0.05)。结论 烧伤科患者CRPA感染发生率较高,临床应结合细菌特点及抗菌药物的敏感性予抗菌药物治疗,针对感染和死亡影响因素进行预防和控制,提高患者的生命质量。     

农村井水中耐碳青霉烯类铜绿假单胞菌及耐药基因分子流行病学特征

目的:分析农村井水中耐碳青霉烯类铜绿假单胞菌（PA）及耐药基因的分子流行病学特征。方法:依据《饮用天然矿泉水检验方法》（GB 8538-2016）对山东省巨野县农村井水采集112份水样进行检测,并对检出的PA分别进行PFGE分型和药物敏感性试验。PCR鉴定碳青霉烯类耐药基因后,采用S1-PFGE和Southern杂交确...     

耐碳青霉烯类铜绿假单胞菌的耐药性分析

目的了解医院耐碳青霉烯类铜绿假单胞菌耐药性,指导临床合理使用抗菌药物。方法统计分析2010年1月-2011年1月30例耐碳青霉烯类铜绿假单胞感染患者感染部位、科室分布及耐药性;细菌鉴定采用VITEK-32细菌鉴定系统,药敏试验采用纸片扩散(K-B)法,采用WHO细菌耐药监测网提供的WHONET5.4软件进行统计分析。结果全年共分离出铜绿假单胞菌1 144株,其中耐碳青霉烯类铜绿假单胞菌30株,检出率为2.6%;30株耐碳青霉烯类铜绿假单胞菌以痰液检出率最高,占66.6%;科室分布以重症监护病房为主,其次是神经内科,分别占50.0%、26.7%;耐碳青霉烯类铜绿假单胞菌仅对多黏菌素B的耐药率为0,对阿米卡星耐药率为17.7%,对其他抗菌药物耐药率均>80.0%。结论耐碳青霉烯类铜绿假单胞菌临床分布比较集中,对常用抗菌药物呈高度耐药,给临床治疗带来严重挑战,提示临床加强抗菌药物的管理和对耐碳青霉烯类铜绿假单胞菌感染病例的实时监控,防止和减少此类耐药菌在医院内传播。     

铜绿假单胞菌主动外排系统MexAB-OprM与碳青霉烯类药物耐药的关系研究

目的:探讨临床分离的铜绿假单胞菌的主动外排系统MexAB-OprM与其对碳青霉烯类药物耐药之间的关系,研究其耐药分子机理。方法:测定临床分离的铜绿假单胞菌对亚胺培南和美罗培南的体外抗菌活性,根据耐药情况,分为碳青霉烯类药物敏感组和碳青霉烯类药物耐药组;应用RT-PCR方法,研究两组菌株的主动外排系统MexAB-OprM中结构基因oprM的mRNA表达水平;用PCR法扩增两组菌株的泵调节基因mexR,对扩增产物进行DNA双向测序。结果:耐药组oprM的mRNA表达水平明显高于敏感组(P0.05);一株菌发现MexR第126位氨基酸突变(缬氨酸→谷氨酸),两株菌发现MexR第70位氨基酸突变(精氨酸→谷氨酰胺)。结论:山西医科大学第一医院临床分离铜绿假单胞菌的主动外排系统MexAB-OprM参与碳青霉烯类药物的耐药;MexAB-OprM高表达的分子机理可能与泵调节基因mexR的突变以及其他突变型有关。     

Pseudomonas aeruginosa orbital cellulitis in four neutropenic patients

Four neutropenic patients on a haematology ward developed orbital cellulitis due to different strains of Pseudomonas aeruginosa over a 7-month period. Investigation of patients and the ward environment revealed two P. aeruginosa isolates indistinguishable from the infecting strains in a plastic washing bowl and a sink in a single cubicle respectively. These items were unlikely to have been the sources of the infecting strains but were a potential cross infection hazard. Treatment of orbital cellulitis is discussed briefly.     

Development of resistance to chlorhexidine diacetate in Pseudomonas aeruginosa and the effect of a 'residual' concentration

Stable resistance in Pseudomonas aeruginosa NCIMB 10421 was obtained by step-wise exposure to gradually increasing concentrations of chlorhexidine diacetate (CHX). Repeated exposure to a proposed 'residual' (sub-MIC) concentration of CHX also created stable resistance. Resistance was also developed by a single exposure to the 'residual' concentration of CHX, but this was unstable. Similar experiments with Escherichia coli and CHX or cetylpyridinium chloride resulted in no significant increase in resistance. Antibiotic susceptibility profiles of the CHX-resistant P. aeruginosa cultures showed no cross-resistance, although some of the cultures were resistant to benzalkonium chloride.     

Neonatal infections with Pseudomonas aeruginosa associated with a water-bath used to thaw fresh frozen plasma

In our 15-bed neonatal intensive care unit (NICU), four new-borns were found to be colonized or infected with Pseudomonas aeruginosa within a period of one week. To identify the outbreak source, three independent studies were performed: epidemiological investigation, environmental surveillance and genotypic typing of isolates. Although epidemiological investigation by a case—control study revealed no conclusive results, the transfusion of fresh frozen plasma (FFP) and human albumin (HA) appeared to be the factor with highest risk. Environmental surveillance and random amplification of polymorphic DNA (RAPD) of isolates identified a water-bath used to warm FFP and HA as the likely reservoir for the outbreak. Further spread of the organism did not occur after elimination of this water-bath from the NICU. RAPD identified in addition an isolate from an infant hospitalized in the NICU five months before the outbreak with a pattern matching the one of the outbreak cluster.     

Outbreak of nosocomial urinary tract infections due to Pseudomonas aeruginosa in a paediatric surgical unit associated with tap-water contamination

An outbreak of 14 cases of urinary tract infections by Pseudomonas aeruginosa, including six symptomatic infections, occurred from September to November 1994 in a paediatric surgical unit. During the outbreak, urine samples from patients and multiple samples from the environment of patients were tested for the presence of P. aeruginosa. Bacterial isolates were studied by pulsed field gel electrophoresis. Genotypic analysis showed that most of the isolates from children were different. Multiple P. aeruginosa isolates were also found in the tap water, as the only putative source of contamination. Two of these isolates were identified in two infected patients, indicating possible direct contamination of patients via tap water and this was related to the distal colonization of faucets. Bacteria were eradicated from tap water by replacement of taps. The cluster of cases of P. aeruginosa urinary infection was, therefore, related to multiple contaminations through tap water. These results illustrate an unexpected risk of nosocomial infection and emphasizes the importance of checking tap water to prevent bacterial contamination through handwashing in contaminated water.     

2014—2017年上海市某医院铜绿假单胞菌不同耐药性菌株的临床分布

目的 了解上海市某三甲医院铜绿假单胞菌（PA）临床分离株的分布，以及多重耐药PA（MDR-PA）、泛耐药PA（XDR-PA）和耐碳青霉烯类PA（CRPA）流行情况，为抗菌药物合理应用提供依据。方法 收集2014-2017年临床科室培养标本中分离PA的标本信息及其药敏数据，分析PA的标本、病区来源，以及与患者年龄、性别间的关系，并计算MDR-PA、XDR-PA、CRPA的分离率。结果 4年间共分离PA 1 218株，分离株数呈逐年递增趋势；菌株主要分离自痰（75.0%），其次为尿（10.6%）。PA最常见于老年医学科（44.9%），其次为重症监护病房和呼吸科（各占8.9%），多分离于60岁以上人群，以男性为主。PA菌株中MDR-PA菌株的比率达30.5%，XDR-PA仅为2.5%，CRPA比率高达38.8%，均呈逐年递增趋势。MDR-PR、XDR-PA、CRPA标本来源及科室分布与PA相仿，即以痰及老年医学科为主。结论 该院MDR-PA、CRPA分离率高，主要分离自痰、老年医学科，应针对不同科室及人群加强医院感染防控措施，强化抗菌药物临床使用管理，预防和控制MDR-PA、CRPA的产生和流行。     

Vegetables as a source of infection with Pseudomonas aeruginosa in a University and Oncology Hospital of Rio de Janeiro

Samples of fresh vegetables fed to patients in an Oncology and a University Hospital were examined for frequency of recovery and counts of Pseudomonas aeruginosa. Thirty-eight isolates from vegetables as well as 98 clinical isolates recovered during the same period of vegetable collection were serotyped and assayed for pyocin production in order to evaluate the role of vegetables as a source of microorganisms. Pseudomonas aeruginosa was recovered from 19·0% of the vegetable samples. Although 1% hypochlorite solution was used as a sanitizer, 50% of the positive samples were found to harbour more than 100 colony-forming units (ctu) g⁻¹. Lettuce, chicory and watercress yielded the highest frequencies of isolation (P < 0·05). The pyocin typing and serotyping of clinical strains revealed some types identical to those recovered from vegetables. Among those found in the University Hospital, serotype O4 and pyocin type PT10/b were detected in vegetables and in clinical specimens whereas types O1-PT22/e, O2a-PT10/a, O2a-PT10/b, O4-PT10/a, O11-PT10/a and O11-PT10/b were common in both groups of strains isolated in the Oncology Hospital. Our results strongly suggest that vegetables represent a source of endemic infection with P. aeruginosa for hospitalized patients.     

Effect of three plastic catheters on survival and growth of Pseudomonas aeruginosa

The effect of Polyvinylchloride (PVC), polyurethane (PU) and siliconized latex (SL) catheters on the survival and growth of six non-mucoid and three mucoid strains of Pseudomonas aeruginosa was evaluated. Pseudomonas aeruginosa (1 × 10⁸) was incubated in PBS alone (control) or with 30 1-cm length segments of each catheter and the number of viable microorganisms was determined after 8 h, 1, 2, 5, 7 and 10 days. The presence of PVC catheters significantly favoured the survival and growth of non-mucoid strains in comparison to the control (P < 0·05 at 5 days, P < 0·01 at 7 days and thereafter); a similar result was observed with SL catheters (P < 0·05 at 2 days, P < 0·01 at 5 days and thereafter). No differences were observed with PU catheters. The number of mucoid microorganisms decreased with time in all controls and suspensions containing segments of catheter, but non-mucoid revertants appeared and quickly increased in the presence of PVC and SL (but not PU) catheters. Eluates of PBS previously containing PVC or SL segments induced a 100- to 500-fold increase in the growth of a non-mucoid strain in comparison with PBS alone. It is concluded that some plastic catheters can release substance(s) that favour the viability of P. aeruginosa.     

磷酸二酯酶PA4781对铜绿假单胞菌运动能力及毒力的影响

目的 探讨磷酸二酯酶PA4781对细菌运动能力及毒力的影响。方法 通过测量菌苔的直径检测细菌的运动能力,利用刚果红染色和苯酚-硫酸显色法检测细菌生物膜分泌胞外多糖的能力。通过实时荧光定量PCR检测介导蹭行运动的基因PilZ、与胞外多糖合成有关的基因PelA以及控制绿脓菌素的基因PhzM的mRNA表达量。利用琼脂糖凝胶电泳检测细菌不同时间段内产生的胞外DNA量,通过测量菌斑在牛奶平板中透明圈的直径检测胞外蛋白酶的总活性。结果 相比于PA03野生型菌株,PA4781敲除菌株的运动能力及PilZ的mRNA表达量降低,PA4781过表达菌株的运动能力及PilZ的mRNA表达量增高,差异均有统计学意义(均P<0.05);相比于PA03野生型菌株,PA4781敲除菌株分泌的胞外多糖及PelA的mRNA表达量增多,PA4781过表达菌株分泌的胞外多糖及PelA的mRNA表达量减少,差异均有统计学意义(均P<0.05); PA4781敲除菌株和PA4781过表达菌株的胞外DNA分泌量、绿脓菌素基因PhzM的表达量及胞外蛋白酶的水解能力均低于PA03野生型菌株,差异均有统计学意义(均P<...     

Epidemiology of Pseudomonas aeruginosa in a tertiary referral teaching hospital

A genotypically indistinguishable strain of Pseudomonas aeruginosa (Australian epidemic strain III: AES III) has previously been found in a proportion of adults with cystic fibrosis (CF) in Tasmania, Australia. The aim of this study was to identify a source of these infections within the major tertiary referral hospital for the State of Tasmania, and to determine if this strain could be isolated from settings other than the CF lung. A total of 120 isolates of P. aeruginosa were collected from clinical and environmental sources within the hospital and from environmental locations in the hospital vicinity. These isolates were genotyped by random amplification of polymorphic DNA (RAPD)–polymerase chain reaction (PCR) and antimicrobial susceptibility testing was performed using the Clinical and Laboratory Standards Institute method. Confirmation of similar genotypes identified by RAPD–PCR was performed using pulsed-field gel electrophoresis with restriction enzyme SpeI. AES III was not recovered from any source other than the respiratory secretions of CF patients. P. aeruginosa in the non-CF settings was found to be panmictic, and no cross-infection or acquisition of hospital environment strains by patients was observed.