The presence of blaIMP genes on plasmids DNA isolated from multidrug-resistant Pseudomonas aeruginosa strains at University Hospital in Bialystok (Poland)--first report

Resistance to carbapenems is emerging, and it is a great problem to therapeutics. Seven multidrug-resistant (MDR) of Pseudomonas aeruginosa strains were isolated from urine and bronchial specimens. All isolates showed resistance to imipenem and meropenem (MIC; > or =16 mg/L). The resistance to carbapenems in two of seven strains was associated with the production of a metallo-beta-lactamases. Plasmids DNA probes were used to investigate the presence of genes coding for IMP-type enzymes. PCR experiments revealed that bla(IMP) genes were present in two isolates of Pseudomonas aeruginosa (MIC >32 microg/mL for both carbapenems).     

CzcR-CzcS, a two-component system involved in heavy metal and carbapenem resistance in Pseudomonas aeruginosa

Pseudomonas aeruginosa is an environmental bacterium involved in mineralization of organic matter. It is also an opportunistic pathogen able to cause serious infections in immunocompromised hosts. As such, it is exposed to xenobiotics including solvents, heavy metals, and antimicrobials. We studied the response of P. aeruginosa upon exposure to heavy metals or antibiotics to investigate whether common regulatory mechanisms govern resistance to both types of compounds. We showed that sublethal zinc concentrations induced resistance to zinc, cadmium, and cobalt, while lethal zinc concentrations selected mutants constitutively resistant to these heavy metals. Both zinc-induced and stable zinc-resistant strains were also resistant to the carbapenem antibiotic imipenem. On the other hand, only 20% of clones selected on imipenem were also resistant to zinc. Heavy metal resistance in the mutants could be correlated by quantitative real time PCR with increased expression of the heavy metal efflux pump CzcCBA and its cognate two-component regulator genes czcR-czcS. Western blot analysis revealed reduced expression of the basic amino acid and carbapenem-specific OprD porin in all imipenem-resistant mutants. Sequencing of the czcR-czcS DNA region in eight independent zinc- and imipenem-resistant mutants revealed the presence of the same V194L mutation in the CzcS sensor protein. Overexpression in a susceptible wild type strain of the mutated CzsS protein, but not of the wild type form, resulted in decreased oprD and increased czcC expression. We further show that zinc is released from latex urinary catheters into urine in amounts sufficient to induce carbapenem resistance in P. aeruginosa, possibly compromising treatment of urinary tract infections by this class of antibiotics.     

Metallo-beta-lactamase-producing gram-negative rods isolated from inpatients and outpatients, detected using Etest MBL

The aim of presented study was to detect MBL-positive strains in a group of clinical carbapenem-resistant strains isolated from inpatients and outpatients during last four years. From the beginning of November 2001 to the end of October 2005, one hundred and four strains resistant to carbapenem antibiotics--imipenem and meropenem were cultured from clinical samples obtained from patients of the Infant Jesus Clinical Hospital Centre for Trauma Treatment in Warsaw and from patients of outpatient clinics. Strains were identified and their susceptibility to antibacterial agents was determined in the automatic ATB Expression system (bioMérieux). Resistance to imipenem and meropenem was confirmed with a disc diffusion method. Production of metallo-beta-lactamases (MBL) was examined with the use of Etest MBL (AB Biodisk, Sweden), and extended-spectrum beta-lactamases (ESBL) by means of following procedures: DDST and / or DD (four variants) (Oxoid Ltd., England). MBL-positive strains (36) were cultured in cases of infections in adult patients (35 strains) and in a child (1 strain). Majority of strains belonged to the species P. aeruginosa (27), several strains - to the species P. putida (6) and remaining strains--to P. stutzeri, A. xylosoxidans, and E. cloacae (1 strain of each species). Four strains were producers of MBL-type and ESBL-type beta-lactamases. According to our knowledge and accessible literature described strains (except one paediatric strain) are the first MBL-positive strains isolated from adult hospitalized patients and adult ambulatory patients in Poland. Additionally, MBL-positive E. cloacae strain is probably the first MBL producer isolated in Poland, which belongs to the group of enteric rods. MBL-producing strains of Gram-negative rods, detected by phenotypic Etest MBL method, will be verified with genetic procedures.     

Classification of OprD sequence and correlation with antimicrobial activity of carbapenem agents in Pseudomonas aeruginosa clinical isolates collected in Japan

A total of 99 clinical isolates of metallo-ß-lactamase-negative Pseudomonas aeruginosa collected in Japan between 1998 and 2001 were studied for their susceptibilities to carbapenem agents and corresponding oprD gene mutations. The OprD sequence of each strain was grouped into two major classes, based on the pattern of alterations. Eighty strains (80.8%) were so-called 'full length type', whose OprD proteins were fully encoded. The remaining 19 strains (19.2%) were so-called 'defective type', which possessed deletions or major alterations that might cause conformational changes in the OprD porin protein. The changes in 'defective type' strains led to 15-, 17- and 23-fold increases in the geometric mean MIC for imipenem, meropenem and biapenem compared with 'full length type' strains, respectively. 'Full length type' strains were further classified into six carbapenem susceptible types with the exception of four carbapenem-resistant subtypes with additional amino acid substitutions at D43, G183, R154, G314, G316. However, 'defective type' strains were classified into four types as follows: 10 strains which contained a stop codon within the coding region; six strains which contained IS; one strain with a short deletion near the C-terminal domain; and two strains without a stop codon in the sequenced region. Western blot analysis using OprD antibody showed that binding abilities of OprD proteins against 'full length type' strains were normal, whereas those against 'defective type' strains were lost without exception. These results indicate that OprD structure and antimicrobial activities for carbapenem agents proved to be highly correlated in P. aeruginosa     

Antibiotic susceptibility of Bacteroides fragilis group strains in Hungary

Resistance rates to different antibiotics of 495 Bacteroides fragilis group strains were followed between 1987 and 1994 in Hungary. In 1992 the strains were collected in three different laboratories, whereas during the other periods strains were isolated in one centre. Metronidazole, chloramphenicol, imipenem and amoxicillin/clavulanic acid were the most active drugs. A high level of resistance was observed in 1987 for ampicillin (88% at > 4 mg/L), erythromycin (51% at > 4 mg/L), tetracyclin (53% at > 8 mg/L) and clindamycin (27% at > 4 mg/L). The same level of resistance was seen during the further years for clindamycin and ampicillin. Resistance to cefoxitin increased from 6% to 11% between 1987 and 1993/1994. No differences in resistance rates were observed between the strains collected in the three centers. For 100 strains, the results of the E test were compared with those of the micro-broth dilution test, both being used routinely for testing the antibiotic susceptibility of Bacteroides fragilis group strains in this period.     

Neuroinfections and antimicrobials: do we need new molecules or new strategies?

Within last 15 years, analyzing patterns of etiology and resistance in organisms causing neuroinfections, emergence of resistance has been observed in Slovakia in S. haemolyticus to teicoplanin (11%), Ps. aeruginosa and A. baumannii to meropenem (20%) and Candida spp. (non-albicans Candida spp.) to fluconazol (20%). There are no new antibiotics against carbapenem resistant Ps. aeruginosa and Acinetobacter baumannii.     

Simultaneous determination of three carbapenem antibiotics in plasma by HPLC with ultraviolet detection

A simple, precise and accurate high-performance liquid chromatography (HPLC) method using ultraviolet (UV) detection has been developed for simultaneous determination of carbapenem antibiotics: imipenem, meropenem and ertapenem in human plasma. Samples were spiked with ceftazidime as internal standard and proteins were precipitated by acetonitrile. Separation was achieved on a C8 column with a mobile phase composed of phosphate buffer 0.1M (pH 6.8) and methanol in gradient elution mode. Detection was performed at 298 nm. Calibration curves were linear from 0.5 to 80 mg/L for each compound, with correlation coefficients over 0.997. Intra- and inter-day validation studies showed accuracy between -4.5 and 8.1% and precision below 10.4%. Mean recoveries were 82.2, 90.8 and 87.7% for imipenem, meropenem and ertapenem, respectively. This method provides a useful tool for the therapeutic drug monitoring of carbapenems.     

Antibiotic Susceptibility of Bacteroides fragilis Group Strains in Hungary

Resistance rates to different antibiotics of 495 Bacteroides fragilis group strains were followed between 1987 and 1994 in Hungary. In 1992 the strains were collected in three different laboratories, whereas during the other periods strains were isolated in one centre. Metronidazole, chloramphenicol, imipenem and amoxicillin/clavulanic acid were the most active drugs. A high level of resistance was observed in 1987 for ampicillin (88% at > 4 mg/L), erythromycin (51% at > 4 mg/L), tetracyclin (53% at > 8 mg/L) and clindamycin (27% at > 4 mg/L). The same level of resistance was seen during the further years for clindamycin and ampicillin. Resistance to cefoxitin increased from 6% to 11% between 1987 and 1993/1994. No differences in resistance rates were observed between the strains collected in the three centers. For 100 strains, the results of the E test were compared with those of the micro-broth dilution test, both being used routinely for testing the antibiotic susceptibility of Bacteroides fragilis group strains in this period.     

Drug susceptibility of Pseudomonas aeruginosa strains isolated from patients of a hospital and specialistic outpatients clinics of the SP ZOZ in Nidzica

The aim of this study was to evaluate a frequency of isolation and antimicrobial susceptibility testing (AST) of Pseudomonas aeruginosa strains cultured from clinical specimens collected from patients hospitalized in wards and specialistic outpatients clinics of a hospital in Nidzica (01. 09. 2000 -31. 12. 2003). During over three years 392 Pseudomonas aeruginosa strains were cultured from 16346 clinical samples provided to bacteriological laboratory. P. aeruginosa strains were isolated from 2.5% of examined specimens. Susceptibility of Pseudomonas aeruginosa strains to antimicrobial agents was tested. The highest in vitro activity against clinical P. aeruginosa strains demonstrated imipenem. One strain was resistant to imipenem. This strain was isolated from a patient of a surgical department. Metalo-beta-lactamase was not detected (MBL-negative strain).Twenty nine strains were ESBL producer (7.4% of all strains). The contribution of Pseudomonas aeruginosa strains to the etiology of nosoconial and ambulatory infections increases. In vitro activity of antibacterial agents against P. aeruginosa strains should be monitored during therapy of infections. Resistance to antibiotics/chemothe-rapeutics may be acquired during treatment with antibacterial agent to which P. aeruginosa strain was susceptible according to the antibiogram.     

Comparative clinical and epidemiological evaluation of beta-lactam antibiotics in the treatment of intraabdominal infections]

We performed a retrospective, comparative study to evaluate efficacy, safety and economic outcomes of empiric cefoperazone/sulbactam monotherapy compared with the meropenem, imipenem/cilastatine and combination of cefepime plus metroindazol in patients with intra-abdominal infection. A total of 468 patients diagnosed with intra-abdominal abscess, peritonitis, pancreatitis were included in the study (the severity of infection according to scale APACHE II was less than 15). Patients were randomized to be treated with either 500 mg meropemen i.v. every 8 hours or 500 mg imipenem/cilastatine i.v. every 8 hours or 2 g cefepime i.v. every 12 hours plus 500 mg metronidazol twice daily or cefoperazone/sulbactam 2 g daily administered every 12 hours. Overall positive clinical responses (cure or improvement) were achieved at the end of treatment for 87.5 patients in meropenem group, 86.6% in the imipenem/cilastatin group, 85.3% in the cefepime group and 86.8% in cefoperazone/sulbactam group. Total cost of the treatment per 100 patients with intra-abdominal infections for cefoperazone/sulbactam was 1957031 roubles, for combinations of cefepime with metronidazol--2497815 roubles. For carbapenem group cost achieved for meropenem--3085291 rub., for imipenem/cilastatin--2653388 roubles. Rate "cost-effectiveness" in total: 784.47$ for cefepime, and 834.39$ for imipenem/cilastatine, 970.21$ for meropenem and 615.4$ for cefoperazone/sulbactam. The most expensive treatment was considered to be with meropenem and imipenem/cilastatine, main share is determined by initial cost of preparations. Less expensive was treatment by cefoperazone/sulbactam with cefepime and by metronidazol.     

Antibiotic susceptibility and occurrence of ESBL in Pseudomonas aeruginosa strains isolated from different clinical specimens

The aim of this study was to evaluate the drug susceptibility of 132 P. aeruginosa strains isolated from patients hospitalized in SPSK University Hospital in Bialystok. The isolates were obtained from clinical specimens over an 11-month period in 2001 and 2002. All the strains were identified in automatic ATB system using API 20 NE strips, and their susceptibility to antibiotics was tested by standard disc-diffusion method and agar dilution method. The minimal inhibitory concentration (MIC) was determined for five antibiotics: piperacillin, amikacin, ceftazidime, imipenem and ciprofloxacin. The majority of strains were susceptible to ceftazidime (91.7%), piperacillin combined with tazobactam (85.6%), amikacin (80.3%), meropenem and imipenem (81.8%). Many of our strains were resistant to cefotaxime (73.5%), ticarcillin (53%) and ciprofloxacin (48.5%). Also, the trial was undertaken to detect strains producing extended-spectrum beta-lactamases (ESBL) and inducible beta-lactamases (IBL) among P. aeruginosa rods isolated from different specimens. ESBL-producing strains were detected with double disc test (DDST) and combination double disc (CD) test. Clavulanate was applied as the inhibitor of these beta-lactamases. Strains producing ESBL were not found. On the other hand, as many as 127 P. aeruginosa strains (96.2%) produced inducible beta-lactamases (IBL).     

Single or in Combination Antimicrobial Resistance Mechanisms of Klebsiella pneumoniae Contribute to Varied Susceptibility to Different Carbapenems

Resistance to carbapenems has been documented by the production of carbapenemase or the loss of porins combined with extended-spectrum β-lactamases or AmpC β-lactamases. However, no complete comparisons have been made regarding the contributions of each resistance mechanism towards carbapenem resistance. In this study, we genetically engineered mutants of Klebsiella pneumoniae with individual and combined resistance mechanisms, and then compared each resistance mechanism in response to ertapenem, imipenem, meropenem, doripenem and other antibiotics. Among the four studied carbapenems, ertapenem was the least active against the loss of porins, cephalosporinases and carbapenemases. In addition to the production of KPC-2 or NDM-1 alone, resistance to all four carbapenems could also be conferred by the loss of two major porins, OmpK35 and OmpK36, combined with CTX-M-15 or DHA-1 with its regulator AmpR. Because the loss of OmpK35/36 alone or the loss of a single porin combined with bla _CTX-M-15 or bla _DHA-1-ampR expression was only sufficient for ertapenem resistance, our results suggest that carbapenems other than ertapenem should still be effective against these strains and laboratory testing for non-susceptibility to other carbapenems should improve the accurate identification of these isolates.     

非免疫抑制患者医院获得性肺炎及呼吸机相关肺炎病原菌组成及耐药性分析

目的分析非免疫抑制患者医院获得性肺炎及呼吸机相关肺炎临床特征、病原菌组成及耐药性,指导临床诊断及合理使用抗菌素。方法回顾性分析46例前瞻性观察诊断的非免疫抑制患者医院获得性肺炎及呼吸机相关肺炎临床及微生物学资料。结果平均起病时间为人院后（14．3±13．2）d,最常见基础疾病依次为脑血管意外（16／46）,慢性肺部疾病（13／46）和糖尿病（5／46）。培养阳性率58．7％,最常见细菌依次为鲍曼不动杆菌（8／27）、铜绿假单胞菌（6／27）、阴沟肠杆菌（3／29）及金黄色葡萄球菌（3／29）。80．4％患者入院72h内使用过抗生素,初始经验性治疗最常使用的抗菌素依次为头孢菌素（29／46）、碳青霉烯类（9／46）、糖肽类（5／46）。8株鲍曼不动杆菌对头孢哌酮／舒巴坦中介MIC〉32mg／L,对其他抗生素耐药;2株铜绿假单胞菌对美罗培南高度耐药MIC=128mg／L。结论非免疫抑制患者医院获得性肺炎及呼吸机相关肺炎多发生在有脑血管疾病及慢性肺疾病老年患者,我院非免疫抑制患者医院获得性肺炎及呼吸机相关肺炎最常见的病原菌多药耐药鲍曼不动杆菌,铜绿假单胞菌对碳青霉烯类耐药率较高。应优化医院获得性肺炎及呼吸机相关肺炎初始抗生素使用。     

奇异变形杆菌耐药性的4年监测及碳青霉烯类耐药株的耐药机制研究

目的了解本地区2007年到2010年奇异变形杆菌的临床分布与常用抗菌药物的耐药情况,了解碳青霉烯类耐药菌株可能存在的机制。方法回顾分析2007年到2010年临床分离奇异变形杆菌的资料及整体耐药情况;对保存的耐亚胺培南（IPM）、美罗培南（MEM）或厄他培南（ETP）的菌株进行复苏,并做Hoage试验进行产碳青霉烯酶的确认,同时对试验菌株进行耐药基因的PCR扩增检测。结果2007年到2010年,奇异变形杆菌在临床各送检样本中以痰液分离率最高：51．1％、34．4％、22．1％和35．4％,其次为尿液：14．3％、28．O％、34．9％和33．6％;耐药监测分析显示,4年间对喹诺酮类、青霉素类、头孢菌素类及氨基糖苷类耐药率相对较高且较为稳定;对碳青霉烯类耐药最低但增加明显,亚胺培南从2007年的1．8％升到2010年的16．1％,美罗培南从2007年的1．7％升到2010年的16．8％。15株耐碳青霉烯类菌株中,Hoage试验阳性7株,6fn。基因阳性11株,blaCTX-M基因阳性13株。结论本地区奇异变形杆菌对临床常用的抗菌药物均有较高的耐药性,对碳青霉烯类药物的耐药率最低,但增加明显。位于质粒上的blaKPc基因所产生的碳青霉烯酶和6如cTx-M基因所产生的超广谱β-内酰胺酶是本菌对β-内酰胺类抗菌药物耐药的主要原因,临床应引起高度重视。     

Genetic definition of the substrate selectivity of outer membrane porin protein OprD of Pseudomonas aeruginosa.

Earlier studies proved that Pseudomonas aeruginosa OprD is a specific porin for basic amino acids and imipenem. It was also considered to function as a nonspecific porin that allowed the size-dependent uptake of monosaccharides and facilitation of the uptake of quinolone and other antibiotics. In the present study, we utilized P. aeruginosa strains with genetically defined levels of OprD to characterize the in vivo substrate selectivity of this porin. An oprD::omega interposon mutant was constructed by gene replacement utilizing an in vitro mutagenized cloned oprD gene. In addition, OprD was overexpressed from the lac promoter by cloning the oprD gene into the broad-host-range plasmid pUCP19. To test the substrate selectivity, strains were grown in minimal medium with limiting concentrations of the carbon sources glucose, gluconate, or pyruvate. In minimal medium with 0.5 mM gluconate, the growth rates of the parent strain H103 and its oprD::omega mutant H729 were only 60 and 20%, respectively, of that of the OprD-overexpressing strain H103(pXH2). In contrast, no significant differences were observed in the growth rates of these three strains on glucose or pyruvate, indicating that OprD selectively facilitated the transport of gluconate. To determine the role of OprD in antibiotic uptake, nine strains representing different levels of OprD and OprF were used to determine the MICs of different antibiotics. The results clearly demonstrated that OprD could be utilized by imipenem and meropenem but that, even when substantially overexpressed, it could not be significantly utilized by other beta-lactams, quinolones, or aminoglycosides. In addition, competition experiments confirmed that imipenem had common binding sites with basic amino acids in the OprD channel, but not with gluconate or glucose.     

Molecular and computational approaches to understand resistance of New Delhi metallo β-lactamase variants (NDM-1, NDM-4, NDM-5, NDM-6, NDM-7)-producing strains against carbapenems

The discovery of NDM-1 and its variants has caused the emergence of antibiotic resistance in the community and hospital setting, causing major concern for health care across the globe. New Delhi Metallo-β-lactamase is known to hydrolyse almost all β-lactam antibiotics. Studies have shown the hydrolytic activates of NDM-1 and some of its variants, however a comparative study of these NDM variants has not been explored in detail. Hence, we proposed to check their catalytic activity by performing a comparative study between NDM-1 and its variants. The study was initiated to clone NDM variants (NDM-1, NDM-4, NDM-5, NDM-6 and NDM-7) followed by overexpression of the recombinant proteins to check their hydrolytic properties against β-lactam antibiotics. The minimum inhibitory concentration of carbapenems antibiotics for bla_NDM-5 clone was found four fold increased, whereas no change was observed in the clones having other variants. The hydrolytic activity of carbapenem with NDM-5 variant was found to be augmented as per the kinetics parameter where Km was decreased and kcat, kcat/Km values increased as compared to the NDM-1. Molecular docking studies were employed to identify the variations in the binding ability among all NDM variants with imipenem or meropenem. Simulation studies at 100?ns showed a good stability of NDM-5 with imipenem and meropenem as compared to NDM-1. CD spectroscopy data revealed significant changes in the secondary structure of NDM variants. We conclude that NDM-5 showed higher hydrolytic activity as compared to other variants. This study provides a comparative analysis of the severity of NDM producing strains.     

In vitro effect of subinhibitory concentrations of ceftazidime and meropenem on the serum sensitivity of Pseudomonas aeruginosa strains

The aim of this study was to determine the effect of subminimal inhibitory concentrations (subMICs) of ceftazidime, meropenem and gentamicin on the in vitro serum sensitivity of Pseudomonas aeruginosa strains isolated from a variety of isolation sites at two medical wards and an intensive care unit in a government university hospital in Croatia. A total of 20 serum-resistant P aeruginosa strains isolated from different clinical specimens were selected. Bacteria were exposed to 1/2, 1/4, 1/8, 1/16, and 1/32 x MIC of each antibiotic tested. Sensitivity of P. aeruginosa strains to bactericidal activity of normal human serum before and after bacterial exposure to subMICs was determined. Significant difference in serum sensitivity of the strains was observed after the bacteria were exposed to subMICs of ceftazidime and meropenem (p < 0.01), while the exposure to subMICs of gentamicin did not affect significantly the resistance of tested strains to the serum bactericidal activity. Comparing the number of serum-resistant strains before and after exposure to subMICs of antibiotics, statistically significant differences were determined (p < 0.01) after exposure of the strains to 1/2, 1/4, 1/8 and 1/16 x MIC of meropenem, and after exposure to 1/2, 1/4 and 1/8 x MIC of ceftazidime. SubMICs of ceftazidime and meropenem affected not only the resistance to serum bactericidal activity of bacteria, but also their morphology. The alterations in bacterial morphology caused by subMICs of ceftazidime and meropenem could be connected with consecutive bacterial serum sensitivity.     

Antimicrobial sensitive of Morganella morganii

The aim of this study was the evaluation of the antimicrobial sensitive of Morganella morganii rods isolated from clinical samples. This study included 50 of M. morganii strains isolated in the Clinical Microbiology Department of dr. A. Jurasz University Hospital in 2008-2009. All of strains were sensitive to carbapenems (imipenem, meropenem, ertapenem, doripenem) and piperacillin/tazobactam and most of them to beta-lactam antibiotics, aminoglycosides and fluorochinolons. Resistance to tetracyclines demonstrated 38,0% strains and to doxycycline - 40,0%. One out of 6 strains isolated from urine samples were sensitive to nitrofurantoin. Extended Spectrum Beta-Lactamases were produced by 5 (10,0%) strains.     

近三年产气肠杆菌临床感染情况分析

目的监测嵊州市人民医院近三年产气肠杆菌对常用抗生素耐药情况。方法对嵊州市人民医院2013年至2015年间收集的产气肠杆菌临床科室分布情况进行统计,并做临床常用抗生素耐药性分析,用WHONET 5.4软件进行统计学分析。结果分离得到的产气肠杆菌主要来源于神经外科、重症医学科和呼吸内科的痰液和尿液标本。药敏结果显示其对第一、二代头孢类抗生素耐药率普遍较高:对头孢唑啉几乎全耐药,对头孢呋辛耐药率约为55%;第四代的头孢吡肟能明显抑制产气肠杆菌生长,2013年到2015年对头孢吡肟耐药率分别为8.18%、9.14%和10.74%;对氨基糖苷类抗生素庆大霉素和丁胺卡那,以及碳青霉烯类抗生素亚胺培南和美罗培南具有很高的敏感性。结论嵊州市人民医院院感科近年间通过对产气肠杆菌临床用药的合理监测,及时向临床医生反馈微生物实验室的耐药结果,避免了抗生素滥用,使得其对抗生素耐药率未出现明显提高。氨基糖苷类抗生素和碳青霉烯类抗生素对其仍具有很高的疗效,应继续通过药敏试验加强对其耐药性监测,指导临床合理用药。     

铜绿假单胞菌对碳青霉烯类抗生素耐药相关基因的筛选

为了在基因组水平筛选获得铜绿假单胞菌PAO1对碳青霉烯类抗生素耐药相关基因,本研究通过构建PAO1转座突变体文库、筛选对亚胺培南、美罗培南和比亚培南耐药及敏感突变株;通过随机PCR、核苷酸测序及序列比对的手段,确定了突变体中转座子的插入位点及其破坏的基因,获得了48个PAO1中对碳青霉烯类抗生素耐药和敏感相关的基因,其中27个基因突变后表现为耐药性增强,21个基因突变后表现为药物敏感性增强.本实验筛选获得的48个基因中有5个与Alvarez-Ortega和Dotsch等人筛选的基因重复.通过对PA0011,PA0667及PA3901进行基因敲除及遗传互补,进一步确证这3个基因均与PAO1对碳青霉烯类的耐药性相关.本次筛选发现了38个新的与碳青霉烯类耐药相关的基因,其中包括13个class4类基因.对这些新基因的进一步研究,不仅有利于全面了解铜绿假单胞菌的耐药机制及其调控网络,而且有利于药物作用靶点的发现,为有效治疗铜绿假单胞菌的感染提供新思路.