An evaluation of four different phenotypic techniques for detection of metallo-beta-lactamase producing Pseudomonas aeruginosa

PURPOSE: The present study was undertaken to detect metallo-beta-lactamase (MBL) in nosocomial isolates of Pseudomonas aeruginosa by four different phenotypic methods. METHODS: Ninety-one consecutive P. aeruginosa isolates were subjected to susceptibility testing by disc-diffusion assay and Vitek 2. Imipenem resistance was determined by three different methods (disc-diffusion, Vitek 2 and E test). Screening for MBL production was done by imipenem-EDTA combined disc test, imipenem-EDTA double-disc synergy test, imipenem-EDTA MBL E test and EDTA disc potentiation using four cephalosporins. RESULTS: Of 63 imipenem resistant isolates, MBL screening could be done in 56 isolates, of which 48 were MBL positive by combined disc test and 36 by the double disc synergy test. For confirmation of MBL production, MBL E test was done in 30 isolates. All the 30 isolates were confirmed to be MBL positive by the MBL E test method. EDTA disc potentiation using four cephalosporins was not very useful for MBL detection. CONCLUSIONS: Imipenem-EDTA combined disc test and imipenem-EDTA MBL E test are equally effective for MBL detection, but given the cost-constraints, combined disc test can be used as a convenient screening method in the clinical microbiology laboratory.     

Pseudomonas aeruginosa: resistance and therapeutic options at the turn of the new millennium

Pseudomonas aeruginosa is a major cause of nosocomial infections. This organism shows a remarkable capacity to resist antibiotics, either intrinsically (because of constitutive expression of beta-lactamases and efflux pumps, combined with low permeability of the outer-membrane) or following acquisition of resistance genes (e.g., genes for beta-lactamases, or enzymes inactivating aminoglycosides or modifying their target), over-expression of efflux pumps, decreased expression of porins, or mutations in quinolone targets. Worryingly, these mechanisms are often present simultaneously, thereby conferring multiresistant phenotypes. Susceptibility testing is therefore crucial in clinical practice. Empirical treatment usually involves combination therapy, selected on the basis of known local epidemiology (usually a beta-lactam plus an aminoglycoside or a fluoroquinolone). However, therapy should be simplified as soon as possible, based on susceptibility data and the patient's clinical evolution. Alternative drugs (e.g., colistin) have proven useful against multiresistant strains, but innovative therapeutic options for the future remain scarce, while attempts to develop vaccines have been unsuccessful to date. Among broad-spectrum antibiotics in development, ceftobiprole, sitafloxacin and doripenem show interesting in-vitro activity, although the first two molecules have been evaluated in clinics only against Gram-positive organisms. Doripenem has received a fast track designation from the US Food and Drug Administration for the treatment of nosocomial pneumonia. Pump inhibitors are undergoing phase I trials in cystic fibrosis patients. Therefore, selecting appropriate antibiotics and optimising their use on the basis of pharmacodynamic concepts currently remains the best way of coping with pseudomonal infections.     

An outbreak of carbapenem-resistant Pseudomonas aeruginosa in a urology ward

Objective  To investigate an outbreak of carbapenem-resistant Pseudomonas aeruginosa (CRPA) in a urology ward.
Methods  Patients infected or colonized with CRPA were prospectively identified by daily laboratory surveillance. Routine infection-control measures were reinforced, disinfection protocols were revised, and a surveillance program was set up, analyzing cross-transmission in the nursing ward and environment cultures from urology wards and the operating theater. CRPA isolates from clinical and environment samples were studied by pulsed-field gel electrophoresis (PFGE), following Xba I and Spe I restriction.
Results  From February 1998 to September 2000, 59 adult urology patients were colonized or infected by CRPA. All patients had been operated on prior to identification of the CRPA isolate and 79% of these procedures were performed in the same cystoscopy room. No patients had received prior carbapenem therapy. No cross-transmission was detected, and environment cultures from the urology ward and theater were negative except for five samples collected in the cystoscopy room. PFGE identified a single clone in the isolates from different patients and the environment samples.
Conclusions  The PFGE analysis indicated that the CRPA outbreak resulted from the contamination of the cystoscopy room via an unsealed drain. The outbreak ended when the drain was sealed.     

铜绿假单胞菌胶体金免疫层析法的研制和应用

为建立一种简单、快速、准确的铜绿假单胞菌检测方法,结合抗重组铜绿假单胞菌外膜蛋白单克隆抗体（mAb）、胶体金标记技术,采用双抗体夹心法,建立了检测铜绿假单胞菌的胶体金免疫层析法,并对该方法的特异性、灵敏度和稳定性进行了评价,对临床痰标本进行了检测。结果显示,该胶体金免疫层析法能在3～15min内完成检测,方法的特异性高、稳定性强,检测灵敏度能达到105CFU/mL。与传统细菌培养方法相比,胶体金免疫层析法的相对灵敏度和特异性分别是80%和90.6%,两种方法的总符合率为86%。结论：利用抗铜绿假单胞菌单克隆抗体建立的胶体金免疫层析法,适合现场快速、特异检测铜绿假单胞菌。     

Multiple virulence factors regulated by quorum sensing may help in establishment and colonisation of urinary tract by Pseudomonas aeruginosa during experimental urinary tract infection

Purpose: Damage caused by an organism during infection is attributed to production of virulence factors. Different virulence factors produced by the organism contribute to its pathogenicity, individually. During infectious conditions, role of virulence factors produced by the pathogen is different, depending upon the site of involvement. Pseudomonas aeruginosa is an opportunistic nosocomial pathogen known to cause infections of the respiratory tract, burn wound, urinary tract and eye. Importance of virulence factors produced by P. Aeruginosa during infections such as keratitis, burn wound and respiratory tract is known. The present study was designed to understand the importance of different virulence factors of P. aeruginosa in urinary tract infection in vivo. Materials and methods: An ascending urinary tract infection model was established in mice using standard parent strain PAO1 and its isogenic mutant, JP2. Mice were sacrificed at different time intervals and renal tissue homogenates were used for estimation of renal bacterial load and virulence factors. Results: Both parent and mutant strains were able to reach the renal tissue. PAO₁ PAO1was isolated from renal tissue till day 5 post-infection. However, the mutant strain was unable to colonise the renal tissue. Failure of mutant strain to colonise was attributed to its inability to produce protease, elastase and rhamnolipid. Conclusion: This study suggests that protease, elastase and rhamnolipid contribute to pathogenesis and survival of P. aeruginosa during urinary tract infection.     

Development of a real-time PCR assay for the specific detection and identification of Streptococcus pseudopneumoniae using the recA gene

We sequenced the evolutionarily conserved genes 16S rRNA, atpD, tuf, and recA from Streptococcus pseudopneumoniae, Streptococcus pneumoniae, Streptococcus mitis, and Streptococcus oralis. Phylogenetic analysis revealed that recA provided good resolution between these species, including discrimination of the novel species S. pseudopneumoniae. By contrast, the more conserved 16S rRNA, tuf and atpD are not sufficiently discriminatory. Therefore, recA sequences were used to develop a real-time PCR assay with a locked nucleic acid-mediated TaqMan probe for the specific detection and identification of S. pseudopneumoniae. The PCR assay showed excellent specificity and a detection limit of <10 genome copies for the detection and identification of S. pseudopneumoniae strains, which makes it a promising tool for molecular identification and epidemiological studies. In conclusion, this article describes for the first time a PCR assay for the specific identification of S. pseudopneumoniae.     

耐亚胺培南铜绿假单胞菌的分布及耐药性分析

目的:了解耐亚胺培南铜绿假单胞菌的感染情况及其耐药特性,为临床合理使用抗生素及防治耐药菌感染提供参考。方法:收集汕头大学医学院第一附属医院2005年4月到2006年4月间分离的92株耐亚胺培南的铜绿假单胞菌,VITEK-60全自动微生物鉴定仪及其配套试剂(GNS-506、GNS-120、GNS-114)进行细菌鉴定及测定其对多种抗生素的耐药性,肉汤二倍稀释法测定其对亚胺培南的MIC值。结果与结论:耐亚胺培南的铜绿假单胞菌感染主要发生神经外科、外科ICU、呼吸科及ICU和神经内科,对亚胺培南的耐药多为中低度耐药(MIC≤32 mg.L-1),对其它多种抗生素的耐药率均达到100%,仅头孢哌酮/舒巴坦复合制剂保持了较高的敏感率,作为经验性用药可考虑选用。     

新型mucA基因突变的铜绿假单胞菌生物被膜的形成和藻酸盐相关基因表达的研究

目的 确定黏液型铜绿假单胞菌PA17的mum基因突变位点，研究藻酸盐合成相关基因在其生物被膜形成过程中的表达，并观察PAl7生物被膜形成过程和形态。方法 PCR方法扩增铜绿假单胞菌PA17的mueA基因全长并测序；改良平板培养法建立PA17的生物被膜模型，半定量RT-PCR测定生物被膜形成24h、3d．6d时藻酸盐合成相关基因,algD、algU和algR的表达，并进行统计学分析；扫描电镜观察不同时间点的生物被膜形态。结果 PA17的mucA基因第166～333位核苷酸片段缺失，第342位A→G；其藻酸盐相关基因algD和algU均在生物被膜形成过程的第6天表达水平最高，algR在24h表达最高，单因素方差分析显示，上述基因在生物被膜形成过程不同时间点表达的差异有统计学意义；PA17于第6天形成成熟生物被膜，形态为薄膜状。结论 PA17是一株含新型mucA突变基因的黏液型铜绿假单胞菌，其藻酸盐相关基因在生物被膜形成的不同时间点的表达差异具有统计学意义，其生物被膜形态为薄膜状。     

阿奇霉素对铜绿假单胞菌生物被膜和毒力因子的干预作用

目的 研究阿奇霉素对铜绿假单胞菌生物被膜形成及毒力因子分泌的干预作用.方法 2倍稀释法测定最低抑菌浓度(MIC);结晶紫染色法测定早期黏附;建立PAO1体外生物被膜模型;扫描电镜观察生物被膜形态;连续稀释法进行活菌计数;弹性蛋白-刚果红法测定弹性蛋白酶活性;偶氮酪蛋白法测定蛋白水解酶活性;氯仿萃取法测定绿脓菌素;苔黑酚法测定鼠李糖脂.结果 PAO1阿奇霉素组对载体的黏附低于PAO1空白对照组(P＜0.05);第3、7天PAO1阿奇霉素组生物被膜少于PAO1空白对照组,且载体活菌计数少于PAO1空白对照组(P＜0.05).PAO1阿奇霉素组弹性蛋白酶活性、蛋白水解酶活性、绿脓菌素浓度、鼠李糖脂浓度均明显低于PAO1空白对照组(P＜0.01),弹性蛋白酶活性及绿脓菌素浓度与PA-JP3株组相当(P＞0.05),而蛋白水解酶活性及鼠李糖脂浓度高于PA-JP3株组(P＜0.05).结论 阿奇霉素可以抑制铜绿假单胞菌的黏附及其生物被膜的形成,并可以抑制其毒力因子的释放.     

某综合医院铜绿假单胞菌2005-2009年耐药性的变迁

目的了解某综合医院2005-2009年铜绿假单胞菌临床分离株的耐药性变迁,为合理应用抗菌药物提供依据。方法应用回顾性调查方法,对广东省汕头市某综合医院2005-2009年住院病人送检标本中分离的铜绿假单胞菌的药敏试验结果进行统计分析。结果 5年间共检出铜绿假单胞菌1809株,总的分离率为16.34%;标本分布以痰液来源为主,占84.9%;几年间铜绿假单胞菌对氨苄西林、复方新诺明、呋喃妥因、头孢唑啉的耐药率变化不大,对阿米卡星、庆大霉素、妥布霉素、亚胺培南、头孢他啶、头孢吡肟、环丙沙星、左旋氧氟沙星等抗菌药物的耐药率均在2007年达到高峰,在2008、2009年两年间呈连续下降趋势。从2009年的情况来看,耐药率最低的是妥布霉素,为28.97%（168/580）,其次为阿米卡星和左旋氧氟沙星,分别为32.07%（186/580）和33.97%（197/580）。结论铜绿假单胞菌对大多数抗菌药物的耐药率有所下降,但总体耐药情况还是比较严重。     

Treatment of infections caused by metallo-β-lactamase-producing Pseudomonas aeruginosa in the Calgary Health Region

This study reviewed 56 patients with significant metallo-beta-lactamase (MBL)-producing Pseudomonas aeruginosa infections between May 2002 and March 2004 to identify features associated with mortality. Immunosuppression (p 0.002), bacteraemia (p 0.08) and inadequate antimicrobial therapy (p <0.001) were associated with death. Among those patients treated with adequate therapy, the use of multiple drug treatment regimens (two or three active agents) was associated with a non-significant two-fold increase in survival (p 0.45). Further prospective studies are warranted to determine the optimal treatment of MBL-producing P. aeruginosa infections.     

铜绿假单胞菌噬菌体的分离鉴定及其控制生物被膜的应用研究

目的 分离鉴定铜绿假单胞菌烈性噬菌体,研究噬菌体控制宿主菌形成牛物被膜的效率.方法 以铜绿假单胞菌临床菌株为指示菌,从不同环境样品中分离噬菌体;采用限制性内切酶图谱分析和宿主范围测定方法,对分离的噬菌体进行分类;利用透射电子显微镜对分离噬菌体进行形态学研究;以TJC729为指示菌,开展噬菌体控制牛物被膜形成的应用研究.结果 分别以14株铜绿假单胞菌临床分离菌株为指示菌,共分离得到13株烈性噬菌体,命名为C1～C13.利用限制性内切酶EcoR Ⅰ分析结果表明,13株噬菌体基因组均为双链DNA,并可被分成8组;宿主谱测定结果显示,c1和C13、C6和C7、C9和C11分别具有相同的宿主范围,其余7株噬菌体的宿主范围各不相同.随机挑选噬菌体C1进行形态学研究,发现噬菌体C1头部具有二十面体结构,尾部较长且无收缩性尾鞘,属于长尾噬菌体科.生物被膜控制实验结果显示,混合噬菌体能够较好地抑制TJC729生物被膜的形成.进一步实验结果显示,噬菌体C1、C10和C12分别与牛物被膜混合培养24 h后,牛物被膜的量分别下降到初始量的32.7%、57.6%、32.8%.结论 分离了13株铜绿假单胞菌烈性噬菌体,它们能够显著抑制宿主菌生物被膜的形成,并对生物被膜造成一定程度的破坏,为控制铜绿假单胞菌引起的感染提供了一个新方法.

Abstract：

Objective To isolate and classigy the bacteriophages specific to Pseudomonas aetuginosa and to investigate biofilm control efficaey of the isolated virulent phages.Methods With P. aeruginosa clinical strains as indicators.bacteriophages were isolated by screening difierent environmental samples.Classification of the isolated phages was done with the methods of restriction fragment analysis of phage genome and host range analysis.Transmission electron microscopy(TEM)was used in phage morphology study.In biogilm control tests,TJC729 was used as the jndicator strain to study the biofilm control efficacy of the isolated phages.Results Total 13 lytic phages specific to P.aeruginosa strains were isolated and named as C1-C13.According to the result of restriction fragment analysis.all 13 phages were double-stranded DNA viruses and could be divided into eight groups.Host range experiments were conducted with 5 laboratory strains and 12 clinical strains of P. aeruginosa.The same infection profiles were observed among phage C1 and C13,C6 and C7,and C9 and C11,respectively.While the remaining 7 phages each had different unique infection profile.Phage C1 was selected randomly to study its morphology.The obtained images showed that phage C1 had an icosahedral head with a non-contractile tail,belonging to the Siphoviridae family.Compared with the single phage,phage cocktail had the best effect on biofilm control.Further experiment results showed that phage C1.C10 and C12 can destroy biofilm after treatment of the biofilm for 24 h.The biofilm amounts were deceased to 32.7%,57.6%and 32.8%of the initial values,respectively.Conclusion Thirteen virulent phages specific to P. aeruginosa had been isolated.The phages could significantly inhibit the biofilm formation and had a certain degree of damage on the biofilm.The results suggested an alternative method for the treatments of P.aeruginosa infections.     

铜绿假单胞菌XN-1随机重组子文库的构建及其在疫苗抗原筛选中的应用研究

目的构建铜绿假单胞菌(Pseudomonas aeruginosa,PA)XN-1的全基因组文库,筛选铜绿假单胞菌新的疫苗候选抗原。方法提取我国分离的临床PA XN-1菌株全基因组,Sau3AⅠ酶切成随机片段后经连接至Bam HⅠ酶切后的pMal-c5x载体,转化至X-blue获得随机重组子。随机重组子经IPTG诱导后使用溶菌酶裂解,收集含有融合的麦芽糖结合蛋白(MBP)的随机重组子的裂解上清,制成含有PA全基因组的蛋白随机文库。通过ELISA方法分别检测单个随机文库蛋白的免疫反应性。将经典的PA强反应抗原PcrV做为参照,分别计算单个随机抗原的相对反应强度并进行比较。提取反应较强的重组子的质粒,测定DNA序列后进行翻译和比对,得到整个强反应性抗原的氨基酸信息。结果成功构建PA XN-1的全基因组文库,筛选到强反应性抗原13个,其中4个抗原的反应强度超过PcrV,分别为Peptidyl-prolyl cis-trans isomerase D、Phosphoenolpyruvate-protein phosphotransferase(PtsP)、Phosphate-starvation-inducible E和Very short patch repair endonuclease。结论基于高效表达系统的PA全基因组文库构建成功,可用于PA及其它病原的疫苗候选抗原的筛选研究。     

河源地区铜绿假单胞菌碳青霉烯耐药机制分析

目的研究本院铜绿假单胞菌耐碳青霉烯类抗生素的耐药现况及主要耐药机制。方法用E-test方法检测铜绿假单胞菌对哌拉西林、头孢他啶、亚胺培南、美洛培南、庆大霉素、妥布霉素、环丙沙星7种抗生素的最小抑菌浓度,用EDTA双纸片扩散法及三维实验分别对金属酶及AmpC、KPC酶表型进行确证。结果从1 068例致病菌中共分离出108例铜绿假单胞菌,18例是对亚胺培南和/或美罗培南不敏感的菌株,耐药率为16.7%,其中有9例金属酶确证实验阳性,3例为AmpC酶持续高产型菌株,KPC酶确证实验尚没有检测出阳性菌株。结论耐碳青霉烯类铜绿假单胞菌多表现为多重耐药,这是多因素共同作用的结果 。     

siHybrids技术沉默铜绿假单胞菌外排泵 mexB基因体外效应的初步研究

目的 初步研究siHybrids技术对铜绿假单胞菌野生菌PAO1外排泵mexB基因体外沉默效应。方法 针对铜绿假单胞菌野生株PAO1外排泵mexB基因设计并合成3条特异性siHybrids分子和1条阴性对照siHybrids分子。在分子浓度为50 nmol/L下,分别以合成的siHybrids分子干扰铜绿假单胞菌野生菌PAO1,并设实验组为铜绿假单胞菌野生菌PAO1空白对照组,阴性对照组scamble(sc)-001,干预组siHybrids( si) -001、siHybrids( si) -002及siHybrids( si) -003,分别在干预12h及24h后采用real-time PCR法检测各实验组中靶基因mexB基因mRNA的表达水平。进一步采用Mueller-Hinton倍比稀释法检测50 nmol/L浓度下,siHy brids分子干预铜绿假单胞菌野生菌PAO1前后氯霉素(CP)、红霉素(EM)、左氧氟沙星(L-OrLX)、头孢他啶(CAZ)、美洛培南(MER)的最小抑菌浓度(MIC)值。结果 不同siHybrids分子干预PAO1 12 h后,mexB基因mRNA表达量无明显差异性;但干预24 h后,mexB基因mRNA表达量：干预组（si-001,si-002,si-003）比空白对照组、阴性对照组（sc-001）有明显下降。对比干预12h、24h后mexB基因mRNA表达量,可以发现空白对照组、阴性对照组（sc-001）mRNA的表达量成上升趋势,而干预组（si-001,si-002,si-003）mexB基因mRNA表达量均呈下降趋势。siHybrids分子在干预铜绿假单胞菌野生菌24h前后的氯霉素(CP)、红霉素(EM)、左氧氟沙星( L-OFLX)、头孢他啶(CAZ)、美洛培南(MER) MIC无明显差异性。结论 在mRNA表达水平上,siHybrids分子能体外干预铜绿假单胞菌PAO1 mexB基因mRNA表达,此种沉默作用呈现时间依赖性,且在24h能有效地发挥干预作用。     

黄芩苷对氟喹诺酮类抗铜绿假单胞菌协同作用的初步报告

目的：研究黄芩苷对氟喹诺酮类药物抗铜绿假单胞菌的协同作用。方法：以二倍稀释法测定药物的体外抗菌活性,筛选耐药菌,然后测定黄芩苷对4种氟喹诺酮类药物（诺氟沙星、环丙沙星、氧氟沙星、左氧氟沙星）抗铜绿假单胞菌的协同作用;以CCCP筛选存在主动外排系统的耐药菌．测定黄芩苷对4种氟喹诺酮类药物抗该类耐药菌的协同作用。结果：临床分离铜绿假单胞菌耐药现象普遍．黄芩苷联合4种氟喹诺酮类药物对耐药铜绿假单饱菌有协同抗菌作用．MIC值下降1-2倍。同时存在主动外排系统的耐药菌也表现出抗菌增敏作用。结论：黄芩苷与氟喹诺酮类药物联合应用对耐药铜绿假单胞菌有协同抗菌,对存在主动外排系统的耐药菌也表现出抗菌增敏作用。     

Development of a novel assay method for colistin sulphomethate

Increased use of colistin therapy for infections caused by Pseudomonas aeruginosa has indicated a need for a more robust microbiological assay technique. This report describes a quick and simple microbiological assay for quantifying levels of colistin sulphomethate in serum and urine samples from cystic fibrosis patients. The technique uses no specialised or costly equipment and is suitable for use in all routine diagnostic microbiology laboratories.     

Eradication of a resistant Pseudomonas aeruginosa strain after a cluster of infections in a hematology/oncology unit

Objectives This report chronicles an outbreak of a multiply resistant strain of Pseudomonas aeruginosa and the measures required to contain this outbreak.
Methods Laboratory-based ward-liaison surveillance allowed the detection of a multiply resistant strain of P. aeruginosa infecting patients in our hematology/oncology unit. Sampling of the immediate environment was carried out. Pulsed field gel electrophoresis was used to compare the patients' organisms with those found in the environment. Extensive dismantling of the drainage system, repeated cleaning and disinfection, and a review of the departmental antibiotic policy were some of the infection control measures instigated.
Results During a period of 11 months, three patients in the hematology department and two patients in the oncology department were infected with multiply resistant P. aeruginosa . There were two cases of pneumonia, one of which was fatal, and two cases of neutropenic septicaemia. Pulsed field gel electrophoresis performed on the isolates showed that the isolates from geographically separate areas could be divided into two strains that were closely related but distinct. Two genotypically identical strains were also isolated from the plumbing systems in the areas of each ward where patients had been treated.
Conclusions The potential for serious nosocomial infections with P. aeruginosa is well recognized. Eradication of the organism from the environment may require the co-ordinated efforts of clinicians, nurses, pharmacy and hospital engineers, working in collaboration with the hospital infection control team. To date, the same strains have not been isolated despite repeated surveillance over the past 18 months and therefore these measures have, in our opinion, successfully removed the potential for nosocomial infection with this resistant organism in our hospital.