Plasmid mediated amikacin resistance in clinical isolates of Pseudomonas aeruginosa

Ten multidrug resistant (MDR) isolates of Pseudomonas aeruginosa, obtained from hospitalized burn patients, were selected for plasmid detection, curing and transformation experiments. These isolates were also studied for plasmid mediated resistance. All the isolates were found to harbour R plasmid. Curing and transformation experiments showed that resistance to amikacin was plasmid mediated. -lactamase production was also tested. It is suggested that plasmids should be characterised in all MDR P. aeruginosa strains and a nation wide antibiotic policy should be made to minimise the emergence of drug resistance.     

Investigation of plasmid-mediated quinolone resistance in Pseudomonas aeruginosa clinical isolates

Aims: To investigate plasmid-mediated quinolone resistance in clinical isolates of Pseudomonas aeruginosa with the polymerase chain reaction (PCR). The plasmid-mediated quinolone resistance genes have been identified in many bacteria within the Enterobactericeae family, they have not been detected in P. aeruginosa isolates. Subjects and Methods: Identification of the isolates and testing of antibiotic susceptibility was performed in Vitek2 Compact (Biomeriux, France) and Phoinex (BD, USA) automated systems. Screening for the qnrA, qnrB, qnrS, qnrC, aac (6′)-Ib-cr and qepA genes was carried out by PCR amplification and aac (6′)-Ib-cr DNA sequencing. Results: The qnr and the qepA genes were not detected in any of P. aeruginosa isolates. The aac (6’)-Ib gene was detected in six of the isolates and positive isolates for aac (6’)-Ib were sequenced for detection of the aac (6’)-Ib-cr variant but aac (6’)-Ib-cr was not detected in any isolates. Conclusions: Plasmid-mediated quinolone resistance genes have so far not been identified in P. aeruginosa isolates. However, qnrB have detected in P. florescens and P. putida isolates. This is the first study conducted on the qnrA, qnrB, qnrS and qnrC genes as well as the qepA and aac (6’)-Ib-cr genes in P. aeruginosa clinical isolates.     

Evaluation of fluoroquinolone resistance mechanisms in Pseudomonas aeruginosa multidrug resistance clinical isolates

Efflux transporters have a considerable role in the multidrug resistance (MDR) of Pseudomonas aeruginosa, an important nosocomial pathogen. In this study, 45 P. aeruginosa clinical strains, with an MDR phenotype, have been isolated in a hospital of Northern Italy and characterized to identify the mechanisms responsible for their fluoroquinolone (FQ) resistance. These isolates were analyzed for clonal similarity, mutations in genes encoding the FQ targets, overexpression of specific Resistance Nodulation-cell Division efflux pumps, and search for mutations in their regulatory genes. The achieved results suggested that the mutations in genes encoding ciprofloxacin targets represented the main mechanism of FQ resistance of these strains; 97.8% of these isolates showed mutations in gyrA, 28.9% in gyrB, 88.9% in parC, and 6.7% in parE. Another mechanism of resistance was overexpression of the efflux pumps in some representative strains. In particular, overexpression of MexXY-OprM drug transporter was found in five isolates, whereas overexpression of MexCD-OprJ was detected in two isolates; surprisingly, in one of these last two isolates, also overexpression of MexAB-OprM pump was identified.     

Beta-lactamase-mediated resistance to extended spectrum cephalosporins among clinical isolates of Pseudomonas aeruginosa.

The role of chromosomal cephalosporinases and secondary beta-lactamases in resistance to extended spectrum cephalosporins in clinical isolates of Pseudomonas aeruginosa was investigated. Strains 687, 59, and 58 expressed an inducible chromosomal cephalosporinase, efficiently enhanced with cefoxitin and imipenem. The inducible activity in strain 802 was produced at a moderately elevated basal level and may be involved in resistance to extended spectrum cephalosporins and aztreonam. All strains produced secondary beta-lactamases inhibited by clavulanate: strains 687, 59, and 58 had carbenicillinases with pIs of 5.7 and 5.3. Strain 802 expressed a secondary beta-lactamase of pI 7.6 which may be a novel extended spectrum beta-lactamase different from known enzymes of P. aeruginosa.     

Resistance to antibiotics in clinical isolates of Pseudomonas aeruginosa

OBJECTIVES: To analyse the global resistance to some antibiotics used to treat nosocomial infections by Pseudomonas aeruginosa, specially to carbapenems, and its relationship with the presence of carbapenemases, OXA, VIM and IMP. METHODS: The study included 229 P. aeruginosa isolates from a Hospital in Northern Spain (year 2002). Susceptibility to antimicrobial agents was determined by the analysis of the MIC. Genetic typing was carried out by RAPD-PCR fingerprinting with primer ERIC-2. Genetic experiments to detect class-1 integrons were performed by PCR with primers 5'CS and 3'CS. Detection of carbapenemases was done by phenotypic (Hodge test and DDST) and genotypic methods (PCR with primers for imp, vim1, vim2 and oxa40 genes). RESULTS: 23.9% of isolates were resistant to ceftazidime, 35.9% to cefotaxime, 5.3% to amikacin, 54.9% to gentamicin, 14.6% to imipenem and 6.6% to meropenem. Isolates resistant to imipenem (33) were furtherly tested. Genetic typing didn't show clonal relatedness among the most of the isolates. Class-1 integrons were present in most isolates (sizes 600-1700 bp). Phenotypic methods for carbapenemases showed 5 positive isolates. Genotypic methods showed the presence of two isolates with the oxa40 gene. CONCLUSIONS: Meropenem, amikacin and imipenem were the most active agents to treat infections caused by Pseudomonas aeruginosa. In our study, the presence of carbapenemase enzymes wasn't high. Phenotypic tests cannot be considered as accurate screening tool to detect carbapenemases. This is the fist report of the oxa40 gene in Pseudomonas aeruginosa isolates.     

Low-level resistance to fluoroquinolones conferred by efflux overproduction in Pseudomonas aeruginosa: clinical significance and routine detection

The aim of this study was (i) to assess the impact of stable overproduction of efflux systems MexAB-OprM and MexXY-OprM on the bacteriostatic activities of fluoroquinolones in clinical Pseudomonas aeruginosa strains and (ii) to find a convenient test for screening isolates with a low level resistance to fluoroquinolones. The minimal inhibitory concentrations (MICs) of ciprofloxacin and levofloxacin were determined for clinical isolates of P. aeruginosa overexpressing MexAB-OprM or MexXY-OprM. Efflux pumps derepression was associated with a modest two- to fourfold increase in resistance to the tested fluoroquinolones. Clinical significance of low level resistance conferred by the efflux mechanism was evaluated with a Monte Carlo simulation with various fluoroquinolone regimens. With this model, low levels of resistance to ciprofloxacin (MIC > or =0.25 mg/L) or levofloxacin (MIC > or =1 mg/L) such as those due to overproduced MexAB-OprM or MexXY-OprM were predicted to result in poor clinical outcomes. Altogether these data strongly suggest that when derepressed MexAB-OprM or MexXY-OprM provides P. aeruginosa with a resistance that may be sufficient to impair the efficacy of single therapy with highly potent fluoroquinolones such as ciprofloxacin and levofloxacin. Routine detection of clinical strains that displayed low-level resistance to fluoroquinolones with a Mueller Hinton agar containing 0.20 mg/L of ciprofloxacin will help clinician in his therapeutical choice.     

Pathogenic potential and genetic diversity of environmental and clinical isolates of Pseudomonas aeruginosa

The aim of this study was to investigate the occurrence of virulence genes among clinical and environmental isolates of Pseudomonas aeruginosa and to establish their genetic relationships by Enterobacterial Repetitive Intergenic Consensus PCR (ERIC‐PCR). A total of 60 P. aeruginosa isolates from environmental and clinical sources were studied. Of these, 20 bacterial isolates were from soil, 20 from water, and 20 from patients with cystic fibrosis. Analysis of ERIC‐PCR demonstrated that the isolates of P. aeruginosa showed a considerable genetic variability, regardless of their habitat. Numerous virulence genes were detected in both clinical and environmental isolates, reinforcing the possible pathogenic potential of soil and water isolates. The results showed that the environmental P. aeruginosa has all the apparatus needed to cause disease in humans and animals.     

Mechanisms of resistance in clinical isolates of Pseudomonas aeruginosa less susceptible to cefepime than to ceftazidime

The MIC of cefepime determined with the MicroScan WalkAway system was ≥2 times higher than that of ceftazidime for 105 clinical isolates of Pseudomonas aeruginosa. This phenotype was confirmed by reference microdilution in 68 (64.8%) isolates, corresponding to 48 different rep-PCR patterns. The PSE-1 blactamase was identified in only 13.2% isolates, while oxacillinases were not identified in any of the 68 isolates. The level of expression of mexB, mexD and mexY was determined by real-time RT-PCR in eight clinical isolates representative of the different clones and patterns of susceptibility to cefepime and ceftazidime and in strain PAO1. All clinical strains overexpressed the mexY gene (18.3- to 152.7-fold in comparison with PAO1), although there was not a linear relationship between MIC of cefepime and level of mexY expression. Five of these strains contained mutations in the regulatory gene mexZ. mexD and mexB were also overexpressed in three and two isolates, respectively. Different mutations were observed in the regulatory genes nalD, mexR, nfxB and nalC. In conclusion, we have documented in our institution a polyclonal spread of P. aeruginosa with higher MICs of cefepime than of ceftazidime, related to overexpression of Mex-XY-OprM, coincident in some isolates with the production of PSE-1, Mex-CD-OprJ or MexAB-OprM.     

Physiological diversity in clinical isolates of Pseudomonas aeruginosa

The results of 91 characteristics selected from 165 tests performed on 127 clinical and other isolates of Pseudomonas aeruginosa were subjected to numerical analysis. Only one cluster, representing a single biotype of the species, was revealed at a similarity (SSM) level of 95% and above. Ten melanogenic and six of the non-melanogenic isolates studied gave diverse atypical results in many of the tests; these strain appear to be aberrant forms of the species.     

Outer membranes of environmental isolates of Pseudomonas aeruginosa.

The outer membrane composition of 30 environmental isolates of Pseudomonas aeruginosa was examined. Other than variations in the amounts of lipoprotein H2, there were no major differences in the outer membrane protein or lipopolysaccharide patterns when compared with those of previously studied clinical isolates.     

Virulence attributes in Brazilian clinical isolates of Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic human pathogen responsible for causing a huge variety of acute and chronic infections with significant levels of morbidity and mortality. Its success as a pathogen comes from its genetic/metabolic plasticity, intrinsic/acquired antimicrobial resistance, capacity to form biofilm and expression of numerous virulence factors. Herein, we have analyzed the genetic variability, antimicrobial susceptibility as well as the production of metallo-β-lactamases (MBLs) and virulence attributes (elastase, pyocyanin and biofilm) in 96 strains of P. aeruginosa isolated from different anatomical sites of patients attended at Brazilian hospitals. Our results revealed a great genetic variability, in which 86 distinct RAPD types (89.6% of polymorphisms) were detected. Regarding the susceptibility profile, 48 strains (50%) were resistant to the antimicrobials, as follows: 22.92% to the three tested antibiotics, 12.5% to both imipenem and meropenem, 11.46% to ceftazidime only, 2.08% to imipenem only and 1.04% to both ceftazidime and meropenem. Out of the 34 clinical strains of P. aeruginosa resistant to both imipenem and meropenem, 25 (73.53%) were MBL producers by phenotypic method while 12 (35.29%) were PCR positive for the MBL gene SPM-1. All P. aeruginosa strains produced pyocyanin, elastase and biofilm, although in different levels. Some associations were demonstrated among the susceptibility and/or production of these virulence traits with the anatomical site of strain isolation. For instance, almost all strains isolated from urine (85.71%) were resistant to the three antibiotics, while the vast majority of strains isolated from rectum (95%) and mouth (66.67%) were susceptible to all tested antibiotics. Urine isolates produced the highest pyocyanin concentration (20.15 ± 5.65 μg/ml), while strains isolated from pleural secretion and mouth produced elevated elastase activity (1441.43 ± 303.08 FAU) and biofilm formation (OD₅₉₀ 0.676 ± 0.32), respectively. Also, MBL-positive strains produced robust biofilm compared to MBL-negative strains. Collectively, the production of site-dependent virulence factors can be highlighted as potential therapeutic targets for the treatment of infections caused by heterogeneous and resistant strains of P. aeruginosa.     

Study of metallo-beta-lactamase production in clinical isolates of Pseudomonas aeruginosa

A study of metallo-beta-lactamase (MBL) production was done in clinical isolates of Pseudomonas aeruginosa. Isolates resistant to ceftazidime and imipenem were screened for MBL production by double disc synergy test (DDST) and minimum inhibitory concentration reduction test. There was complete correlation between two methods for imipenem. For ceftazidime, there was correlation between the two methods in all except four strains. In the screening test for MBL, ceftazidime-EDTA combination was better than imipenem-EDTA combination. 8.05% strains were MBL producers. Presence of MBL producer P. aeruginosa is a cause of concern. Simple DDST can be helpful for monitoring of these emerging resistant determinants.     

The relative contribution of efflux and target gene mutations to fluoroquinolone resistance in recent clinical isolates of Pseudomonas aeruginosa

The clinical utility of fluoroquinolones (FQs) for the treatment of Pseudomonas aeruginosa (PA) and other serious Gram-negative infections is currently decreasing due to the rapid emergence of resistance. Because previous studies have shown that efflux is a common mechanism contributing to FQ resistance in PA, one suggested approach to extend the longevity of this class of drugs is combination therapy with an efflux pump inhibitor (EPI). In order to determine the viability of this approach, it is necessary to understand the relative contribution of efflux- vs. target-mediated mechanisms of FQ resistance in the clinic. A set of 26 recent PA clinical isolates were characterized for antibiotic resistance profiles, efflux pump expression, topoisomerase mutations, and FQ susceptibility with and without an EPI. The contribution of OprM to the overall antibiotic resistance was assessed in a subset of these strains. Our results suggest that the co-administration of an EPI with FQs or other antibiotics currently in use would not be sufficient to combat the complexity of resistance mechanisms now present in many clinical isolates.     

Characterization of imipenem resistance mechanisms in Pseudomonas aeruginosa isolates from Turkey

The emergence of carbapenem resistance in Pseudomonas aeruginosa threatens the efficacy of this important anti-pseudomonal antibiotic class. Between 2003 and 2006, an increase in the number of carbapenem-resistant P. aeruginosa isolates at the Zonguldak Karaelmas University Hospital was observed (Zongul-dak, Turkey). To assess the imipenem resistance mechanisms emerging in these P. aeruginosa isolates, they were characterized by amplified fragment length polymorphism typing, which revealed diversity among imipenem-resistant isolates as well as two clonally related outbreak groups. The molecular mechanism of carbapenem resistance was characterized in a representative isolate from each clonal group. Mutational disruption of oprD was the most frequently encountered resistance mechanism (23/27 isolates).     

铜绿假单胞菌临床分离株主动外排泵的检测

目的了解铜绿假单胞菌临床分离株的耐药特点及其外排泵存在的状况。方法采用K-B法检测铜绿假单胞菌临床分离株对9种常用抗生素（庆大霉素、氨曲南、头孢他啶、亚胺培南、阿米卡星、哌拉西林/他唑巴坦、环丙沙星、左氧氟沙星、多粘菌素B）的耐药情况;通过外排泵抑制剂氰氯苯腙（CCCP）对四种药物（羧苄青霉素、红霉素、亚胺培南、庆大霉素）的琼脂稀释法抑制试验分别检测铜绿假单胞菌的四种外排泵（MexAB-OprM、MexCD-OprJ、MexEF-OprN、MexXY-OprM）的表型;应用PCR检测MexAB-OprM外排泵调节基因mexR的存在情况。结果铜绿假单胞菌对9种常用抗生素的耐药率依次为：庆大霉素36.67%（22/60）,氨曲南33.33%（20/60）,头孢他啶31.67%（19/60）,亚胺培南28.33%（17/60）,阿米卡星26.67%（16/60）,哌拉西林/他唑巴坦23.33%（14/60）,环丙沙星21.67%（13/60）,左氧氟沙星11.67%（7/60）,多粘菌素B为0。外排泵表型阳性株依次为：MexAB-OprM35株,阳性率58.33%（35/60）;MexXY-OprM13株,阳性率21.67%（13/60）;MexCD-OprJ11株,阳性率18.33%（11/60）;MexEF-OprN10株,阳性率16.67%（10/60）。35株MexAB-OprM外排泵表型阳性株中有28株扩增出mexR基因片段,检出率为80.0%（28/35）。结论铜绿假单胞菌临床分离株对庆大霉素耐药率最高,对多粘菌素B耐药率最低;MexAB-OprM外排泵的存在是铜绿假单胞菌临床分离株的重要耐药机制。     

V-antigen genotype and phenotype analyses of clinical isolates of Pseudomonas aeruginosa

The pcrV genotype was analyzed in clinical isolates of Pseudomonas aeruginosa which showed a negative phenotype for secretion of V-antigen PcrV. The suppression of PcrV secretion in these isolates was due not to a lack of the pcrV gene but rather to suppression of PcrV expression.     

Metallo-beta-lactamase VIM-2 in clinical isolates of Pseudomonas aeruginosa from Portugal

Resistance to carbapenems is emerging, and it is a great problem to therapeutics. Three isolates of Pseudomonas aeruginosa from a Portuguese hospital identified in urine and sputum, in 1995, presented a high-level resistance to imipenem (> 32 mg/L). Afterward, one isolate of P. aeruginosa recovered from urine of an ambulatory patient in 1998 showed high resistance to imipenem and meropenem. The resistance to carbapenems in these strains was associated with the production of a class B beta-lactamase, as was demonstrated by imipenem hydrolysis and inhibition by EDTA. Using primers described for bla(IMP) and bla(VIM), the amplification of the latter was observed in all isolates and a VIM-2 metallo-enzyme was identified. The pulsed-field gel electrophoresis (PFGE) patterns of these isolates were indistinguishable, suggesting dissemination to the community of this VIM-2 producer.     

Production of elastase and other exoproducts by environmental isolates of Pseudomonas aeruginosa.

Pseudomonas aeruginosa isolates from environmental sources and bacteremic patients were compared for their levels of elastolytic activity. No significant differences were found. The incidence of production of toxin A, phospholipase C, alkaline protease, and elastase among the environmental strains was also as high as that previously reported for clinical isolates.