CIP耐药的铜绿假单胞菌两种分子耐药机制关系的研究

目的探讨环丙沙星（CIP）耐药的铜绿假单胞菌临床分离株主动外排药物与gyrA、parC基因突变的关系。方法联合碳酰氰基-对-氯苯腙（CCCP）和CIP对CIP耐药的铜绿假单胞菌株进行主动外排阳性株和阴性株的筛选,并对这些菌株的gyrA,parC基因进行聚合酶链式反应-限制性片段长度多态性分析（PCR—RFLP）。结果57％（55／97）的CIP耐药菌株最小抑菌浓度（MIC）可被逆转,gyrA单基因突变率为65％,gyrA和pa-C双基因突变率为35％,未发现parC单基因突变的菌株。主动外排阳性组与阴性组gyrA、parC基因突变情况差异无显著性。结论在本地区铜绿假单胞菌对CIP的耐药机制中,主动外排系统表达上调与抗菌药物作用靶位的改变均占有重要的地位,两者可能是并存的两种相对独立的机制。     

Analysis of antibiotic resistance gene expression in Pseudomonas aeruginosa by quantitative real-time-PCR

In Pseudomonas aeruginosa many of the clinically relevant resistance mechanisms result from changes in gene expression as exemplified by the Mex drug efflux pumps, the AmpC beta-lactamase and the carbapenem-specific porin OprD. We used quantitative real-time-PCR to analyze the expression of these genes in susceptible and antibiotic-resistant laboratory and clinical strains. In nalB mutants, which overexpress OprM, we observed a four- to eightfold increase in the expression of mexA, mexB, and oprM genes. MexX and mexY genes were induced eight to 12 times in the presence of 2 mg L(-1) tetracycline. The mexC/oprJ and mexE/oprN gene expression levels were increased 30- to 250-fold and 100- to 760-fold in nfxB and nfxC mutants, respectively. We further found that in defined laboratory strains expression levels of ampC and oprD genes paralleled beta-lactamase activity and OprD protein levels, respectively. Our data support the use of quantitative real-time-PCR chain reaction for the analysis of the antimicrobial resistance gene expression in P. aeruginosa.     

301株铜绿假单胞菌的分布及耐药性分析

目的 通过对临床中分离的铜绿假单胞菌的分布及对临床常用13种抗生素的耐药性进行分析指导临床合理用药.方法 收集大连医科大学附属第二医院2010年1月至12月临床送检的标本,采用全自动细菌和药敏分析仪分离铜绿假单胞菌同时进行药敏试验.结果 铜绿假单胞菌在痰液标本中分离率高达79.06％;对头孢噻肟、头孢西丁、头孢唑啉3种药物的耐药率均大于50％;对头孢他啶,哌拉西林/他唑巴坦耐药率低于20％.结论 铜绿假单胞菌易产生多源耐药,加强耐药性监测,控制医院内感染,对临床医生选用有效的抗生素具有十分重要的意义.     

The influence of flow cell geometry related shear stresses on the distribution,structure and susceptibility of Pseudomonas aeruginosa 01 biofilms

The effects of non-uniform hydrodynamic conditions resulting from flow cell geometry (square and rectangular cross-section) on Pseudomonas aeruginosa 01 (PAO1) biofilm formation, location, and structure were investigated for nominally similar flow conditions using a combination of confocal scanning laser microscope (CSLM) and computational fluid dynamics (CFD). The thickness and surface coverage of PAO1 biofilms were observed to vary depending on the location in the flow cell and thus also the local wall shear stress. The biofilm structure in a 5:1 (width to height) aspect ratio rectangular flow cell was observed to consist mainly of a layer of bacterial cells with thicker biofilm formation observed in the flow cell corners. For square cross-section (1:1 aspect ratio) flow cells, generally thicker and more uniform surface coverage biofilms were observed. Mushroom shaped structures with hollow centers and wall breaks, indicative of ‘seeding’ dispersal structures, were found exclusively in the square cross-section tubes. Exposure of PAO1 biofilms grown in the flow cells to gentamicin revealed a difference in susceptibility. Biofilms grown in the rectangular flow cell overall exhibited a greater susceptibility to gentamicin compared to those grown in square flow cells. However, even within a given flow cell, differences in susceptibility were observed depending on location. This study demonstrates that the spanwise shear stress distribution within the flow cells has an important impact on the location of colonization and structure of the resultant biofilm. These differences in biofilm structure have a significant impact on the susceptibility of the biofilms grown within flow channels. The impact of flow modification due to flow cell geometry should be considered when designing flow cells for laboratory investigation of bacterial biofilms.     

对一株铜绿假单胞菌22种耐药基因的研究

目的研究铜绿假单胞菌多重耐药情况和相关机制。方法采用聚合酶链反应（PCR）法对一株多重耐药铜绿假单胞菌进行β-内酰胺酶基因、氨基糖苷类修饰酶（AMEs）基因、喹诺酮类耐药基因、耐消毒剂基因（qacE△1-sul1）和整合酶基因检测,并对VIM基因进行了测序。结果PCR扩增结果显示该菌株aac（6′）-Ⅰb、blaCARB、gyrA、oprD2、ant（2″）-Ⅰ、qacE△1-sul1、blaIMP-I、blaTEM、blaVEB、aac（3）-Ⅱ、ant（3″）-Ⅰ、intⅠ1、blaVIM基因均为阳性,而aac（3）-Ⅰ、aac（6′）-Ⅱ、blaGES、blaGIM、blaOXA-10群、blaPER、blaSPM、blaSHV、blaDHA基因均为阴性,VIM基因扩增产物测序后经BLAST同源性分析表明为VIM-2型。结论铜绿假单胞菌存在多重耐药基因。管壁涂有季胺类、双胍类消毒剂和磺胺的II代导管的抑菌效果需重新评价。     

ICU患者铜绿假单胞菌获得性肺炎耐药研究

目的监测重症监护病房（ICU）铜绿假单胞菌（Pseudomonas aeruginosa,PA）的感染状况及耐药变迁,指导临床合理使用抗菌药物。方法对2003年至2007年间,我院ICU收治的患者下呼吸道分泌物进行培养及体外耐药试验。结果铜绿假单胞菌是ICU下呼吸道感染的主导致病菌且感染率呈逐年上升的趋势（46．3％-81．0％）,细菌对三代头孢、亚胺培南及氨基糖苷类药物的耐药性也呈逐年上升的趋势,并呈现多重耐药的特性。结论铜绿假单胞菌是ICU院内下呼吸道感染的常见病原菌,其多重耐药性及应引起高度重视。     

126株铜绿假单胞菌的临床分布及耐药性分析

目的调查分析象山县中医医院铜绿假单胞菌的临床分布及药敏情况,为临床合理选用抗生素提供可靠的依据。方法采集疑似患者的标本,进行分离、培养与鉴定。采用自动微生物鉴定／药敏分析仪进行鉴定及药敏试验,对2012年7月至2013年10月分离出的126株铜绿假单胞菌（包括21株黏液型铜绿假单胞菌）的分布及耐药性进行回顾性分析。结果126株铜绿假单胞菌临床主要分布情况：痰占80．2％,尿液占11．1％,脓液占7．1％,以呼吸道感染为主。对铜绿假单胞菌保持活性较强同时耐药率〈20％的抗生素有阿米卡星、妥布霉素、亚胺培南、哌拉西林／他唑巴坦,其中碳青霉烯类耐药率升至5％,原来被认为抗铜绿假单胞菌较为有效的喹诺酮类抗生素的耐药率也有了很大提升,左氧氟沙星耐药率升至33％。黏液型铜绿假单胞菌的体外抗菌药物敏感试验耐药性较弱,且明显弱于非黏液型铜绿假单胞菌的耐药性。结论铜绿假单胞菌为医院呼吸道感染的常见致病菌,对多种抗菌药物呈不同程度耐药。加强动态监测,合理使用抗菌药物,对铜绿假单胞菌感染的预防和药物治疗具有重要指导意义。     

Magnetic fields suppress Pseudomonas aeruginosa biofilms and enhance ciprofloxacin activity

Due to the refractory nature of pathogenic microbial biofilms, innovative biofilm eradication strategies are constantly being sought. Thus, this study addresses a novel approach to eradicate Pseudomonas aeruginosa biofilms. Magnetic nanoparticles (MNP), ciprofloxacin (Cipro), and magnetic fields were systematically evaluated in vitro for their relative anti-biofilm contributions. Twenty-four-hour biofilms exposed to aerosolized MNPs, Cipro, or a combination of both, were assessed in the presence or absence of magnetic fields (Static one-sided, Static switched, Oscillating, Static + oscillating) using changes in bacterial metabolism, biofilm biomass, and biofilm imaging. The biofilms exposed to magnetic fields alone exhibited significant metabolic and biomass reductions (p < 0.05). When biofilms were treated with a MNP/Cipro combination, the most significant metabolic and biomass reductions were observed when exposed to static switched magnetic fields (p < 0.05). The exposure of P. aeruginosa biofilms to a static switched magnetic field alone, or co-administration with MNP/Cipro/MNP + Cipro appears to be a promising approach to eradicate biofilms of this bacterium.     

大蒜素对铜绿假单胞菌生物膜早期黏附及胞外多糖的影响

目的研究大蒜素对铜绿假单胞菌PA01菌株生物膜（biofilm,BF）早期黏附及胞外多糖复合物（Extracellular Polymeric Substances,EPS）的影响。方法利用荧光多功能酶标仪检测各组不同时间点96孔板中pGF-Puv转化PA01菌株的荧光强度,计算黏附率以表示干预对不同时间点细菌黏附的影响,利用荧光显微镜下定性观察细菌的黏附量;应用异硫氰酸标记的刀豆蛋白A（FITC conjugated concanavalin A,FITC-conA）特异性结合细菌EPS,荧光显微镜下定性观察各组EPS的变化;利用硫酸-苯酚法定量各组细菌EPS的产量。结果6h组,大蒜素高浓度干预后细菌的黏附率由0．70±0．03下降至0．50±0．01,t=15．014,P〈0．05,大蒜素低浓度干预后黏附率也有下降,但不及高浓度组明显;除了9h组其他时间组趋势与6h组大致相似,可能与大蒜素含量下降有关。荧光显微镜观察可见生理盐水对照组细菌菌落分布,干预组黏附的细菌稀疏散在分布,以高浓度为甚。FITC-conA可使胞外多糖显色,在荧光显微镜下观察可见大蒜素干预后EPS减少,稀薄;EPS定量实验,EPS总量大蒜素高浓度干预组（181．19±1．59）μg较生理盐水对照组（602．66±21．94）μg有明显降低,t=60．589,P〈0．05。结论大蒜素可显著减少PA01菌株黏附及产EPS的能力。     

Chloraminated drinking water does not generate bacterial resistance to antibiotics in Pseudomonas aeruginosa biofilms

Aim: To determine if exposure of Pseudomonas aeruginosa biofilms to chloraminated drinking water can lead to individual bacteria with resistance to antibiotics. Methods and Results: Biofilms of P. aeruginosa PA14 were grown in drinking water in a Kadouri drip‐fed reactor; the biofilms were treated with either 0·5 mg l^‐1 or 1·0 mg l^‐1 of chloramine for 15 or 21 days; control biofilms were grown in water without chloramine. Fewer isolates with antibiotic resistance were obtained from the chloramine‐treated biofilms as compared to the control. Minimum inhibitory concentrations (MIC) for selected antibiotic‐resistant isolates were determined using ciprofloxacin, tobramycin, gentamicin, rifampicin and chloramphenicol. All of the isolates tested had increased resistance over the wildtype to ciprofloxacin, rifampicin and chloramphenicol, but were not resistant to tobramycin or gentamicin. Conclusions: Under these test conditions, there was no detectable increase in antibiotic resistance in P. aeruginosa exposed as biofilms to disinfectant residues in chloraminated drinking water. Significance and Impact of the study: Chloramine in drinking water, while unable to kill biofilm bacteria, does not increase the potential of P. aeruginosa to become resistant to antibiotics.     

Characterization of antimicrobial resistance of Pseudomonas aeruginosa isolated from canine infections

Aims: To determine the prevalence of Pseudomonas aeruginosa among dogs with suspected soft tissue infections and to characterize these isolates. Methods and Results: Swabs were taken from infected soft tissues of 402 dogs. Pseudomonas aeruginosa strains were confirmed phenotypically and tested for susceptibility to 11 antimicrobial agents and genotyped by SpeI pulsed‐field gel electrophoresis (PFGE). The genetic basis of fluoroquinolone (FQ) resistance and the presence of integrons were also characterized. A total of 27 (6·7%) dogs tested positive for Ps. aeruginosa. Fourteen different SpeI patterns were observed in 25 typeable strains. Among the β‐lactams, three isolates presented resistance to ticarcillin and carbenicillin, while only one isolate exhibited resistance to ceftazidime. Among the aminoglycosides (AGs), three strains showed resistance to amikacin, and four strains exhibited resistance to gentamicin and tobramycin. Four strains with mutations that led to the substitution of Thr at position 83 with Ile in GyrA and the exchange of Ser at position 87 with Leu in ParC displayed resistance to all tested FQs. These strains also carried class 1 integrons and showed resistance to between 6 and 10 antimicrobials. These integrons included four different gene cassettes (aacA4‐aadA1, bla_OXA‐31‐aadA2, aadA1‐arr‐3‐catB3 and cmlA5‐cmlA‐aadA1). Conclusions: A small proportion of infected dogs treated in two animal hospitals in Beijing, China carried Ps. aeruginosa isolates. Low levels of resistance to anti‐pseudomonal agents were observed in these strains. Significance and Impact of the Study: This study is the first report on the antimicrobial resistance profiles of Ps. aeruginosa isolated from infected canine origin in China. Additionally, this is the first report of the oxacillin resistance gene bla_OXA‐31 in a canine Ps. aeruginosa isolate.     

不同标本来源铜绿假单胞菌体外耐药性对比研究

目的比较不同部位培养出的铜绿假单胞菌耐药性,为临床控制和治疗铜绿假单胞菌感染提供依据。方法收集住院患者不同标本中分离的铜绿假单胞菌进行鉴定和药敏试验。结果分离率以痰标本中最高,占57.67%;伤口分泌物次之占30.69%。科室分布以ICU（38.1%）、神经外科（15.34%）和烧伤科（14.81%）最多见。同一种抗菌药物对不同标本分离的铜绿假单胞菌显示出不同敏感性,痰标本中铜绿假单胞菌对常用抗菌药物耐药率较伤口分泌物标本中高,除头孢哌酮/舒巴坦、氨苄西林/舒巴坦和复方新诺明外,对其他常用的12种抗菌药物耐药率比较差异有统计学意义（P〈0.05）。结论不同标本分离的铜绿假单胞菌对抗菌药物耐药性存在差异,临床经验用药时应区别对待不同部位铜绿假单胞菌的感染。     

Regulation of virulence and antibiotic resistance by two-component regulatory systems in Pseudomonas aeruginosa

The Gram-negative opportunistic pathogen Pseudomonas aeruginosa ubiquitously inhabits soil and water habitats and also causes serious, often antibiotic resistant, infections in immunocompromised patients (e.g. cystic fibrosis). This versatility is mediated in part by a large repertoire of two-component regulatory systems that appear instrumental in the regulation of both virulence processes and resistance to antimicrobials. Major two-component regulatory system proteins demonstrated to regulate these diverse processes include PhoP–PhoQ, GacA–GacS, RetS, LadS, and AlgR, among others. Here, we summarize the current body of knowledge of these and other two-component systems that provides insight into the complex regulation of virulence and resistance in P. aeruginosa .     

The cost of multiple drug resistance in Pseudomonas aeruginosa

The spread of bacterial antibiotic resistance mutations is thought to be constrained by their pleiotropic fitness costs. Here we investigate the fitness costs of resistance in the context of the evolution of multiple drug resistance (MDR), by measuring the cost of acquiring streptomycin resistance mutations (StrepR) in independent strains of the bacterium Pseudomonas aeruginosa carrying different rifampicin resistance (RifR) mutations. In the absence of antibiotics, StrepR mutations are associated with similar fitness costs in different RifR genetic backgrounds. The cost of StrepR mutations is greater in a rifampicin‐sensitive (RifS) background, directly demonstrating antagonistic epistasis between resistance mutations. In the presence of rifampicin, StrepR mutations have contrasting effects in different RifR backgrounds: StrepR mutations have no detectable costs in some RifR backgrounds and massive fitness costs in others. Our results clearly demonstrate the importance of epistasis and genotype‐by‐environment interactions for the evolution of MDR.     

铜绿假单胞菌ESBLs和AmpC酶的检测及耐药性分析

目的了解铜绿假单胞菌临床分离株ESBLs和AmpC酶的产生及对常用抗菌药物敏感性,指导临床合理选用抗生素。方法常规培养分离细菌,采用VITEK-60型全自动细菌鉴定仪鉴定细菌;按NCCLS推荐的双纸片确证法和K-B纸片扩散法检测ESBLs和药敏试验;采用头孢西丁纸片扩散法筛选疑产AmpC酶阳性菌株,确诊采用三维试验。结果铜绿假单胞菌产ESBLs和AmpC酶总检出率分别为40.8%和38.2%,其中,单产AmpC酶、单产ESBLs和同产AmpC酶+ESBLs检出率分别为19.7%、26.3%和14.5%。药敏试验显示:产酶株的耐药性明显高于非产酶株,耐药现象在同产AmpC酶和ESBLs菌株中更为严重。结论台州地区临床分离的铜绿假单胞菌产ESBLs和AmpC酶菌株检出率较高。产AmpC酶和ESBLs的菌株呈高度耐药,临床上对产酶菌株引起感染的治疗应根据细菌药敏试验结果,合理选择有效的抗菌药物联合治疗,减少产酶菌株的产生和流行。     

Antimicrobial activity of chlorhexidine diacetate and benzalkonium chloride against Pseudomonas aeruginosa and its response to biocide residues

AIMS: The aims of this study were to evaluate the antimicrobial activity of chlorhexidine diacetate (CHX) and benzalkonium chloride (BZK) for strains of Pseudomonas aeruginosa exhibiting increased minimum inhibitory concentrations (MIC) for CHX, and to determine whether residues of chlorhexidine digluconate (CHG) and Hibiscrub (Hib, a formulation containing CHG) affect the susceptibility of P. aeruginosa to these biocides and a number of antibiotics. METHODS AND RESULTS: The bactericidal activity of CHX and BZK was evaluated for strains of P. aeruginosa exhibiting increased MIC for CHX with established suspension and surface disinfection tests. None of the strains of P. aeruginosa exhibiting raised MIC for CHX was less sensitive than the parent strain to CHX or BZK in either method. A test was designed to investigate the effects of dried CHG and Hib residues on P. aeruginosa cells. Exposure of P. aeruginosa to dried residues of CHG or Hib did not result in the organism becoming less sensitive to either biocide or a number of antibiotics. CONCLUSIONS: Pseudomonas aeruginosa strains with raised MIC to CHX were no less sensitive than the parent strain to CHX and BZK in bactericidal investigations. Exposure to dried residues of CHG and Hib did not render P. aeruginosa less sensitive to either of these agents or a number of antibiotics. SIGNIFICANCE AND IMPACT OF THE STUDY: An increase in the MIC for a biocide in a micro-organism does not necessarily result in a failure of the biocide to effectively kill the organism. The residue that remains after the use of an antimicrobial agent can be at a far lower concentration than that initially applied and this study highlights the necessity for further investigations into the effect of residues, at low concentration, on bacterial populations and their role, if any, in the continued problem of antibiotic resistance.     

临床分离铜绿假单胞菌对氟喹诺酮类抗菌药物的耐药性检测

目的了解临床分离铜绿假单胞菌对喹诺酮类等抗菌药物的耐药性。方法琼脂稀释法测定86株铜绿假单胞菌对5种氟喹诺酮类抗菌药物以及头孢吡肟、美罗培南的耐药性。结果铜绿假单胞菌对诺氟沙星、氧氟沙星、左氧氟沙星、环丙沙星、加替沙星的耐药率分别为50%、61.6%、51.2%、48.8%、51.2%;对头孢吡肟和美罗培南的耐药率分别为30.2%、23.2%。结论铜绿假单胞菌对氟喹诺酮类抗菌药物耐药显著,临床应加强检测和监测。     

Characterization of Pseudomonas aeruginosa isolates from dogs and cats in Japan: current status of antimicrobial resistance and prevailing resistance mechanisms

Seventy-three Pseudomonas aeruginosa isolates were collected from dogs and cats in Japan to investigate antimicrobial susceptibility and resistance mechanisms to anti-pseudomonal agents. Resistance rates against orbifloxacin, enrofloxacin, ciprofloxacin, cefotaxime, aztreonam and gentamicin were 34.2, 31.5, 20.5, 17.8, 12.3 and 4.1%, respectively. The degree of resistance to cefotaxime, orbifloxacin, and enrofloxacin was greatly affected by efflux pump inhibitors, indicating overexpression of efflux pump contributes to these resistances. Notably, orbifloxacin and enrofloxacin resistance was observed even in isolates without mutations in the target sites. This is the first report on cephalosporin- and fluoroquinolone-resistant isolates of P. aeruginosa from Japanese companion animals.     

铜绿假单胞菌的临床分布和耐药性变迁

目的调查温州医学院附属第一医院铜绿假单胞菌的临床分布和耐药性及其变迁情况,指导临床合理用药和控制耐药菌的产生。方法对该院2003年至2005年437株铜绿假单胞菌进行回顾性分析,用17种抗菌药物进行体外药敏试验。结果铜绿假单胞菌分布以呼吸道感染和创口分泌物感染为主。细菌对第3代头孢菌素耐药率高,对头孢他啶、亚胺培南、头孢哌酮/舒巴坦和氟喹诺酮类药物的耐药率均有逐年增加的趋势。耐药率增加与临床广谱抗菌药物用药量增加有关。结论铜绿假单胞菌的耐药性十分突出,应在药敏指导下合理选用抗菌药物,应该适当控制碳青霉烯类和第3代头孢菌素的使用,减缓耐药菌株的产生。