不同疾病标本中铜绿假单胞菌分离鉴定与多重耐药性分析

目的 了解不同疾病铜绿假单胞菌的感染状况以及该菌对不同抗菌药物的耐药性。方法 采用VITEK-2 Compact全自动细菌鉴定仪联合GN卡和16S RNA基因PCR对不同疾病标本中铜绿假单胞菌进行分离鉴定。分别采用VITEK-2 Compact仪联合AST-GN卡K-B纸片扩散法对铜绿假单胞菌分离株进行药物敏感试验。结果 仅检出铜绿假单胞菌199例病人所患主要疾病为下呼吸道感染(χ²=24.8,P<0.05),病人以≥60男性为主(χ²=26.1,P<0.05)且主要来自重症监护病房(ICU)和普外科(χ²=17.7,P<0.05)。临床常用抗菌药物中,铜绿假单胞菌分离株对氨苄西林、头孢唑林和头孢曲松耐药率较高(耐药率95.48%～98.99%),但对哌拉西林、庆大霉素和左旋氧氟沙星相对敏感(敏感率70.51%～88.44%)。55.3%铜绿假单胞菌分离株(110/199)株多重耐药,优势多重耐药模式为青霉素+头孢菌素+碳青霉烯类(30.9%,34/110)和菌株对青霉素+头孢菌素+碳青霉烯+喹诺酮类(39.1%,43/110)耐药(P<0.05)。结论 铜绿假单胞菌是不同感染性疾病常见病原体,老年男性是该菌感染高危人群。临床标本中分离的铜绿假单胞菌有较强的耐药性,尤其对多种临床常用抗菌药物呈现多重耐药。     

糖尿病足创面感染铜绿假单胞菌的耐药性及其毒力基因分析

目的了解糖尿病足创面感染铜绿假单胞菌耐药特点并分析其与毒力基因的关系。方法连续收集2018-01-01～2018-12-31在天津医科大学代谢病医院糖尿病足病科住院的糖尿病足患者中54株细菌培养报告为铜绿假单胞菌的菌株,在药物敏感试验及菌落复种培养后,交南开大学微生物实验室逐一进行铜绿假单胞菌固有pcrV基因检测,以明确铜绿假单胞菌。另收集非糖尿病创面感染铜绿假单胞菌21株作为对照组。对铜绿假单胞菌的3型分泌系统中毒力基因exoS和exoU进行检测及分析其与耐药的关系。对创面中连续2次(每次间隔1个月)及以上培养出的铜绿假单胞菌进行随访。结果 54株中筛查出8株不是铜绿假单胞菌,46株明确为铜绿假单胞菌。糖尿病足组与对照组中,主要致病基因均为含有exoS的铜绿假单胞菌,糖尿病足组为91.3%(42/46),耐药率为11.9%;对照组为76.2%(16/21),耐药率为6.3%。含有exoU的铜绿假单胞菌在糖尿病足组与对照组中均占比较少,糖尿病足组中为8.7%(4/46),耐药率为25.0%;对照组中为23.8%(5/21),耐药率为20.0%。在糖尿病足组中,有6例患者连续2次及以上培养出铜绿假单胞菌,合计19株。有5例患者感染含有exoS的铜绿假单胞菌,其中有2例培养出耐药铜绿假单胞菌,耐药率为23.5%(4/17),均出现在治疗的初期和中期,在末次培养时均转为敏感铜绿假单胞菌,有1例患者创面中含有exoU的铜绿假单胞菌,耐药率为50.0%(1/2),首次培养为耐药菌,末次培养为敏感菌。结论糖尿病足创面中取得的铜绿假单胞菌中,以含有exoS的铜绿假单胞菌为主,但是在耐药菌方面,含有exoU的铜绿假单胞菌的耐药率更高。同一患者创面的铜绿假单胞菌不会因为耐药与否出现exoS或exoU基因型改变。     

成都地区老年人下呼吸道铜绿假单胞菌感染的耐药分析

目的 探讨成都地区老年人下呼吸道铜绿假单胞菌感染的耐药状况。方法 采集本院2011年1月~2013年12月356例符合老年人下呼吸道感染患者的痰标本,采用全自动微生物分析系统(VITEK-2 COMPACT)对铜绿假单胞菌进行分析。结果 2011年、2012年、2013年共分离出的356株铜绿假单胞菌对多粘菌素B的耐药率分别为1.65%、2.01%、1.11%。2011年分离出的121株铜绿假单胞菌对环丙沙星的耐药率较低,为20.66%。2012年分离出的145株铜绿假单胞菌对头孢吡肟的耐药率较低,为22.07%。2013年分离出的90株铜绿假单胞菌对阿米卡星的耐药率较低,为15.55%。结论铜绿假单胞菌是老年人下呼吸道感染常见病原菌,监测其耐药性,可为早期治疗控制感染提供依据,为减少并发症、延长患者生命提供保证。     

361株呼吸道铜绿假单胞菌体外药敏试验分析

目的分析门诊及住院患者呼吸道铜绿假单胞菌的药敏结果,指导临床合理使用抗生素。方法采用液体稀释法检测2006年1月1日至2008年2月1日解放军第210医院从呼吸道标本中分离的361株铜绿假单胞菌对19种抗生素的最小抑菌浓度(MIC)值,依据NCCLS/CLSI推荐标准分析药敏结果。结果361株铜绿假单胞菌对阿米卡星敏感率最高,达84.2%;其次为亚胺培南,敏感率80.1%;第3位为环丙沙星,敏感率67.9%。美罗培南耐药率仅14.1%,但中介率25.5%。抗假单胞菌广谱青霉素、三代头孢菌素、四代头孢菌素及β-内酰胺/β-内酰胺酶抑制剂耐药率较高。72株亚胺培南中介、耐药铜绿假单胞菌对阿米卡星敏感率达81.9%。结论亚胺培南、环丙沙星等抗菌药可经验性地用于本院铜绿假单胞菌引起的呼吸道感染,最好与阿米卡星联用。亚胺培南不敏感铜绿假单胞菌对阿米卡星敏感率相对较高,但治疗难度大。     

产金属β-内酰胺酶铜绿假单胞菌的基因检测及耐药性研究

目的　研究本地区铜绿假单胞菌临床分离株中产金属 β -内酰胺酶流行株的主要基因型和耐药性。 方法　用纸片扩散法进行抗生素协同敏感试验 ,检测产金属 β -内酰胺酶的铜绿假单胞菌 ,用琼脂稀释法检测其最低抑菌浓度 (MIC) ,分别用金属酶IMP - 1,IMP - 2 ,VIM - 1和VIM - 2引物进行聚合酶链反应 (PCR)并对扩增产物进行序列分析。结果　在 82株耐亚胺培南铜绿假单胞菌中 ,抗生素协同敏感试验阳性 7株 ,PCR检测金属 β -内酰胺酶阳性株也为这 7株 ,且基因型均为VIM - 2型。产酶株对亚胺培南、头孢噻肟、头孢他啶、哌拉西林 /他唑巴坦等常用抗铜绿假单胞菌 β -内酰胺类抗生素不敏感 ,对氨曲南较为敏感。结论　本地区铜绿假单胞菌所产金属 β-内酰胺酶为VIM - 2型 ,且产酶株耐药性强 ,应注意对其进行临床检测和监控。     

胸外科院内感染铜绿假单胞菌耐药性与耐药机制研究北大核心CSCD

目的 分析胸外科院内感染患者的病原菌分布情况、铜绿假单胞菌耐药性及耐药机制。方法 收集1 095例接受胸外科手术患者的送检标本,采用全自动细菌鉴定仪鉴定病原菌。采用K-B纸片扩散法对临床常见的12种抗生素进行药敏试验,测定52株铜绿假单胞菌的耐药性。采用PCR对18株耐氨基糖苷类铜绿假单胞菌的氨基糖苷类修饰酶基因和16SrRNA甲基化酶基因扩增,通过扩增产物分析耐药机制。结果 1 095例患者入院诊断主要为肺癌(30.32%)与食管癌(25.11%)。93例发生术后感染,感染98例次,感染率为8.49%,主要为下呼吸道感染(52.04%)。93例胸外科院内感染患者中,培养分离病原菌共115株。86株革兰阴性菌,主要为铜绿假单胞菌;22株革兰阳性菌,主要为金黄色葡萄球菌;7株真菌。52株铜绿假单胞菌对临床常见抗生素药敏试验显示,氨曲南、头孢他啶、环丙沙星、左氧氟沙星的耐药率>30%,多粘菌素B的敏感性为100.00%。52株铜绿假单胞菌中,18株对氨基糖苷类耐药,15株产氨基糖苷类修饰酶,主要为acc(6′)-Ⅱ阳性株。产氨基糖苷类修饰酶阳性基因模式主要为acc(6′)-Ⅰ+acc(6′)-Ⅱ+ant(2″)-Ⅰ+ant(3″)-Ⅰ。18株耐氨基糖苷类铜绿假单胞菌中,16SrRNA甲基化酶基因型72.22%为armA阳性。结论 本院胸外科院内感染率为8.49%,主要为下呼吸道感染,铜绿假单胞菌为主要病原菌。耐氨基糖苷类铜绿假单胞菌的耐药基因型主要为armA、acc(6′)-Ⅱ。     

肺结核合并肺部感染铜绿假单胞菌的药敏分析

目的调查分析肺结核合并肺部感染铜绿假单胞菌的耐药性。方法在342例肺结核合并肺部感染患者中共分离出115株铜绿假单胞菌,进行细菌鉴定及药物临床分析。结果肺结核合并肺部感染患者铜绿假单胞菌的感染率为33.6%,50岁以上者PA感染率(49.1%)明显高于50岁以下者(25.7%)(P<0.05);115株铜绿假单胞菌中对头孢唑林耐药率最高(80.0%),其次为氯霉素(75.65%);对碳青霉烯类药物亚胺培南的敏感率最高(86.96%),其次为阿米卡星(68.70%)、左氧氟沙星(64.35%)和环丙沙星(61.74%)。结论肺结核病患者由于长期服药,免疫功能低下,合并感染铜绿假单胞菌易产生抗药性,用药应根据药敏结果选择合适抗生素。     

铜绿假单胞菌耐药性及产碳青霉烯酶研究

目的了解医院2010年分离的铜绿假单胞菌的耐药性及耐亚胺培南菌株产碳青酶烯酶情况。方法 K-B纸片扩散法进行药敏试验;碳青霉烯酶检测采用改良Hodge试验。结果 2010年分离铜绿假单胞菌145株,主要来自呼吸道标本、脓汁及分泌物。药敏结果显示铜绿假单胞菌氨苄西林耐药率最高,达88.97%。对阿莫西林/克拉维酸、头孢噻肟、环丙沙星和头孢他啶的耐药率均超过50%。对洛美沙星和头孢哌酮/舒巴坦较敏感;耐亚胺培南铜绿假单胞菌31株,耐药率21.38%,改良Hodge试验阳性10株,阳性率为32.26%。结论分离的铜绿假单胞菌耐药性和产碳青霉烯酶率均较高,铜绿假单胞菌耐药严重     

老年铜绿假单胞菌感染的临床特点及耐药性分析

目的探讨2015年我院老年患者铜绿假单胞菌感染的临床特点及耐药性,为临床治疗提供参考依据。方法收集2015年1~12月我院老年患者临床送检标本中分离出的铜绿假单胞菌233株,全部细菌严格按照《全国临床检验操作规程》进行分离与培养,采用Vitek 2 Compact全自动细菌鉴定和药敏分析系统进行细菌的鉴定及药敏试验,并用Excel软件进行数据统计分析。结果纳入研究的233株铜绿假单胞菌主要来源于痰液(72.10%),其余依次为尿液(7.73%),引流液(4.72%),全血(4.29%),导管(2.58%),腹水(2.58%),胆汁(2.15%),胸水(1.29%),切口分泌物(1.29%),咽刷、体液及脓液分别占0.43%;铜绿假单胞菌感染的主要科室是呼吸科和重症监护室,分别占33.05%及22.32%;铜绿假单胞菌对头孢曲松、头孢噻肟、头孢唑啉及头孢西丁等抗生素的耐药率达100%,对氨苄西林/舒巴坦和复方新诺明耐药率亦达100%,仅对少数β-内酰胺类、喹诺酮类及氨基糖苷类抗生素耐药率30%。结论老年患者铜绿假单胞菌感染以呼吸道感染为主,由于老年感染患者的临床用药存在局限性,加之铜绿假单胞菌对绝大多数抗生素都耐药,因此必须更加重视老年患者铜绿假单胞菌感染的有效预防与合理治疗。     

铜绿假单胞菌近7年的耐药性变迁及其抗生素应用分析

目的分析铜绿假单胞菌近7年的耐药性变迁情况。方法收集2001年1月至2007年6月我院分离的铜绿假单胞菌1076株,分析其耐药性变迁及临床抗生素的应用情况;结果对铜绿假单胞菌保持抗菌活性较强而耐药率30%的抗菌药物依次为美罗培南、亚胺培南及阿米卡星,对常用抗菌药物的耐药率有普遍增高的趋势;抗铜绿假单胞菌所用抗生素单用455例(44.4%);二联521例(50.9%);三联48例(4.7%)。结论铜绿假单胞菌耐药率高,耐药率有普遍增高的趋势;对严重铜绿假单胞菌感染、多药耐药(MDRP)或泛耐药(PDRP)的治疗,宜采用联合用药,β-内酰胺类+阿米卡星为较优化的组合治疗方案之一。     

N-乙酰半胱氨酸对铜绿假单胞菌生物被膜影响的实验研究

目的研究N-乙酰半胱氨酸(NAC)单独作用对铜绿假单胞菌(PA)生物被膜(BF)形成的影响,以及与左氧氟沙星(LFX)合用对BF菌的清除作用。方法2005年6月至2006年6月在中南大学湘雅医院中心实验室中进行试验。扫描电镜(SEM)观察BF的形态,最小抑菌浓度(MIC)采用试管双倍稀释法测定;用甲基四氮唑蓝(MTT)比色法测定BF中活菌数。结果NAC能减少BF中基质样物质以及被膜的厚度,单用LFX需1MIC以上浓度才能使BF内活菌数明显下降。小剂量NAC(0.5g/L)与1/2MIC的LFX联合应用可使BF内活菌数明显下降,NAC对LFX的协同杀菌作用呈浓度依赖性。结论NAC能够抑制BF形成,并可增强LFX对PA生物被膜菌的杀菌活性。     

糖尿病足创面铜绿假单胞菌三型分泌系统和生物膜特点及与抗生素耐药性的关系

目的:探讨糖尿病足(diabetic foot, DF)创面中铜绿假单胞菌(Pseudomonas aeruginosa)三型分泌系统、生物膜特点,分析其与抗生素耐药性的关系。方法:收集2018年2月1日至2018年12月31日天津医科大学朱宪彝纪念医院糖尿病足科住院DF患者创面中铜绿假单胞菌33株,作为DF组,同时收...     

铜绿假单胞茵在急性下呼吸道感染患者中生物被膜形成与群体感应系统基因相关性分析

目的：探讨在急性下呼吸道感染患者体内铜绿假单胞菌群体感应系统中Las系统和Rhl系统的表达及与其生物被膜形成的相关性。方法：以铜绿假单胞菌临床分离株为研究对象,检测群体感应系统中Las系统和Rhl系统相对表达量及生物被膜形成能力．分析Las系统和Rhl系统表达量与生物被膜形成能力的相互关系。结果：测定的120株铜绿假单胞菌临床菌株中．Las信号系统的Lasl和LasR基因相对表达与生物被膜形成均呈正相关fP〈0．001）,其中第1天分离的临床株中Lasl和LasR基因相对表达量与生物被膜生成量呈正相关（P〈0．01）．第14天分离的临床株中Lasl基因相对表达量与生物被膜生成量有密切正相关性（P〈0．001）．LasR基因相对表达量与生物被膜生成量呈正相关（P〈0．05）。Rh1信号系统的Rh11和．R^艉基因相对表达量与生物被膜生成量密切正相关（P〈0．001）．其中第1天分离的菌株中Rh11和Rh1R基因相对表达量与生物被膜生成量密切正相关（P〈0．001）,而第14天分离的菌株中Rh11和Rh1R基因相对表达量与生物被膜生成量无相关性。结论：铜绿假单胞菌的群体感应系统中Las信号系统和Rh1信号系统与生物被膜形成密切相关．在生物被膜的形成过程起到关键作用．     

Cloning and surface expression of Pseudomonas aeruginosa O antigen in Escherichia coli.

As a step toward developing recombinant oral vaccines, we have explored the feasibility of expression of O polysaccharide antigens from Pseudomonas aeruginosa by Escherichia coli. We cloned in E. coli HB101 a 26.2-kilobase DNA fragment from P. aeruginosa strain PA103 that specifies the production of the O polysaccharide of Fisher immunotype 2 (IT-2) strains. The recombinant organism incorporated the P. aeruginosa IT-2 O polysaccharide onto the core of the E. coli lipopolysaccharide (LPS). Transfer of the recombinant plasmid to three LPS-rough strains of P. aeruginosa resulted in synthesis of IT-2 O antigen, and two of these transconjugant strains also synthesized a second O polysaccharide, presumably representing expression of a repressed, or an incomplete, set of genes for an endogenous O polysaccharide. Rabbits injected with the purified recombinant LPS made antibody specific for P. aeruginosa IT-2 O side chains, as did mice fed the recombinant E. coli strain. Expression of P. aeruginosa O antigens by enteric bacteria makes it possible to study these recombinant strains as oral vaccines to prevent P. aeruginosa infections.     

铜绿假单胞菌生物膜形成过程观察

目的观察铜绿假单胞菌实验室保存株与临床分离的菌株体外细菌生物膜的形成过程。方法通过FITC标记的刀豆球蛋白(FITC-ConA)和碘化丙啶(PI),结合激光共聚焦显微镜(confocal laser scanning microscopy,CLSM)摄取生物膜形成阶段不同层面的图片。结果培养3 d后可观测到生物膜中的多糖开始形成,培养第7 d,多糖基质逐渐增多,细菌趋向化聚集,膜结构致密,其内部存在相互交通的间隙及孔道;CLSM结合FITC-ConA和PI标记,实现了对铜绿假单胞菌菌株生物膜动态形成过程的观察。结论铜绿假单胞菌生物膜可在体外支持物上形成并成熟;临床新分离株较实验室保存株更易形成生物膜。     

The roles of the quorum-sensing system in the release of extracellular DNA, lipopolysaccharide, and membrane vesicles from Pseudomonas aeruginosa

Biofilms play an important role in the establishment of chronic infection caused by Pseudomonas aeruginosa. It has been suggested that membrane vesicles (MVs) are released into the surrounding medium during normal growth and might supply the bacterial extracellular DNA that is required for early biofilm formation, as MVs released from the bacterial outer membrane are suspected to be the source of extracellular DNA. MVs possess lipopolysaccharide (LPS), extracellular DNA, and several hydrolytic enzymes. It is well known that the quorum-sensing (QS) system is important in controlling virulence factors in P. aeruginosa and biofilm formation. In the current study, we investigated extracellular LPS and DNA in the supernatants of culture solutions from PAO1, the wild-type P. aeruginosa, and those of QS mutants. As compared to that of las QS mutants, the amount of LPS and DNA released was significantly higher in PAO1 and in las QS mutants complemented with N-(3-oxododecanoyl) homoserine lactone. Our study indicated that the QS is among the regulators involved in the release of extracellular DNA and LPS. It is possible that these extracellular components are supplied from MVs. Investigation of the mechanism of biofilm formation is of particular interest, as it may be useful for designing treatments for severe P. aeruginosa infection.     

The assay of beta-lactamase activity in Pseudomonas aeruginosa biofilm]

OBJECTIVE: To analyze the resistance mechanism of Pseudomonas aeruginosa biofilm bacteria. METHODS: An in vitro model of Pseudomonas aeruginosa bacterial biofilm was established in silicon disk with modified flat-board method and a rapid staining procedure of AgNO3 was used to verify it. Biofilm was observed under scanning electron microscopy. The test was carried out in three groups. Group A was planktonic bacteria of Pseudomonas aeruginosa, group B biofilm bacteria of Pseudomonas aeruginosa and group C biofilm bacteria induced by imipenem. The activity of beta-lactamase was quantitated with a spectrophotometric assay method and beta-lactamase quantitation was determined by using Bio-Rad protein assay. RESULTS: The activity and protein content of beta-lactamase in group B was higher than that in group A by 4.67 and 2.09 times. The activity of group C was 21.86 times as much as that in group A and 4.68 times as much as that in group B. The quantitation in group C was 6.28 times as much as that in group A and 3.00 times as much as that in group B. The activity and quantitation of the three groups were different significantly from each other (P < 0.01). CONCLUSION: The production of beta-lactamase in Pseudomonas aeruginosa biofilm bacteria is one of the main reasons of its resistance.     

A polyphosphate kinase 1 (ppk1) mutant of Pseudomonas aeruginosa exhibits multiple ultrastructural and functional defects

Pseudomonas aeruginosa, of medical, environmental, and industrial importance, depends on inorganic polyphosphate (poly P) for a wide range of functions, especially survival. Mutants of PAO1 lacking poly P kinase 1, PPK1, the enzyme responsible for most poly P synthesis in Escherichia coli and other bacteria, are defective in motility, quorum sensing, biofilm formation, and virulence. We describe here multiple defects in the ppk1 mutant PAOM5, including a striking compaction of the nucleoid, distortion of the cell envelope, lack of planktonic motility and exopolymer production, and susceptibility to the beta-lactam antibiotic carbenicillin as well as desiccation. We propose that P. aeruginosa with reduced poly P levels undergoes ultrastructural changes that contribute to profound deficiencies in cellular functions.     

Alginate is not a significant component of the extracellular polysaccharide matrix of PA14 and PAO1 Pseudomonas aeruginosa biofilms

The bacterium Pseudomonas aeruginosa causes chronic respiratory infections in cystic fibrosis (CF) patients. Such infections are extremely difficult to control because the bacteria exhibit a biofilm-mode of growth, rendering P. aeruginosa resistant to antibiotics and phagocytic cells. During the course of infection, P. aeruginosa usually undergoes a phenotypic switch to a mucoid colony, which is characterized by the overproduction of the exopolysaccharide alginate. Alginate overproduction has been implicated in protecting P. aeruginosa from the harsh environment present in the CF lung, as well as facilitating its persistence as a biofilm by providing an extracellular matrix that promotes adherence. Because of its association with biofilms in CF patients, it has been assumed that alginate is also the primary exopolysaccharide expressed in biofilms of environmental nonmucoid P. aeruginosa. In this study, we examined the chemical nature of the biofilm matrix produced by wild-type and isogenic alginate biosynthetic mutants of P. aeruginosa. The results clearly indicate that alginate biosynthetic genes are not expressed and that alginate is not required during the formation of nonmucoid biofilms in two P. aeruginosa strains, PAO1 and PA14, that have traditionally been used to study biofilms. Because nonmucoid P. aeruginosa strains are the predominant environmental phenotype and are also involved in the initial colonization in CF patients, these studies have implications in understanding the early events of the infectious process in the CF airway.     

Changes in serotypes of clinical isolates of Pseudomonas aeruginosa by anti-pseudomonal drugs]

Forty-two isolates of P. aeruginosa from various infections were each incubated in Mueller-Hinton broth including piperacillin, cefsulodin, ceftazidime, imipenem, gentamicin or norfloxacin (1MIC-4MIC) at 35 degrees C for 18 hours, and serotyped using monoclonal antibodies. Serotypes of 4 (9.5%)-8 (19.0%) of the 42 isolates each changed to different groups after incubation. No relationship was found between serotypes of the formed variants and anti-pseudomonal drugs used. When P. aeruginosa TA-2 was exposed to cefsulodin at different concentrations (1MIC and 2MIC) under the above conditions, the distinct variants different in serotypes were formed according to the drug concentrations. Furthermore, P. aeruginosa TA-2 and TA-13 were incubated in Mueller-Hinton broth including cefsulodin (1/2 or 2MIC) and gentamicin (1/2MIC), respectively, and the growth curves of parent and variant cells were determined. In the experiments, the variants appeared 6 hours after onset of the incubation and grew with the parents. The present results may explain our findings previously reported; coexistence of colonies different in serotype of P. aeruginosa isolated from some individual patients. The results indicate the possibility that alteration in bacterial surfaces in association with changes in serotypes might occur in vivo in these patients infected with P. aeruginosa and treated with anti-pseudomonal drugs.