| ObjectivePseudomonas aeruginosa (PA), an opportunistic pathogenic bacterium, was an important nosocomial pathogen. Pseudomonas aeruginosa develops congenital and acquired multi-drug resistance mechanisms and its resistance rate to various antibiotics was increasing, which leads to higher difficulty in treatments of nosocomial infections. In this study, drug resistance in clinically isolated P. aeruginosa was analyzed. The presence and function of active efflux pumps were detected and discussed. Furthermore, thirty-two multi-drug resistant P. aeruginosa strains were isolated from clinical samples and analyzed. The presence of oprD2,β-lactamase genes, aminoglycoside-resistant gene, integrase gene and drug-resistant gene cassettes carried by integrons were detected. This study aims to reveal the drug-resistant mechanisms and spread of drug resistance genes in P. aeruginosa, which may provide guidance in clinical antibiotic treatments.Methods1. Detection and analysis of active efflux pumps in clinically isolated P. aeruginosa. Sixty strains of P. aeruginosa were collected from clinical specimens in The First Affiliated Hospital of Guangzhou Medical College from September 2008 to September 2009. K-B test was used to detect the distribution of resistance in P. aeruginosa to nine commonly used antibiotics (gentamicin, aztreonam, ceftazidime, imipenem, amikacin, piperacllin/tazobactam, ciprofloxacin, levofloxacin and polymyxin B). Agar dilution methods were used to detect resistant phenotypes of four efflux pumps (MexAB-OprM, MexCD-OprJ, MexEF-OprN, MexXY-OprM) against carbenicillin, erythromycin, imipenem and gentamicin in the presence of cyanide chorophenyl hydrazone(CCCP), a efflux pump inhibitor. Furthermore, PCR was used to detect the variations of the regulatory gene (mexR) of MexAB-OprM.2. Detection and analysis of type I integrons and drug-resistant gene cassettes carried by integrons in multi-drug resistant P. aeruginosa.Thirty-two strains of multi-drug-resistant P. aeruginosa were collected from clinical specimens in The First Affiliated Hospital of Guangzhou Medical College from 2008 to 2010. Integrase gene and qacEΔ1-sull gene which was a genetic marker for type I integrons were detected by PCR and their integration types were analyzed. Integron resistance gene cassettes in its variable regions were detected and PCR products from the variable regions were analyzed by restriction fragment length polymorphism to detect the drug-resistance genes.3. Detection and analysis of outer membrane protein oprD2 gene,β-lactamase gene and aminoglycoside- resistant genes in multi-drug resistant P. aeruginosa. Resistance to 8 commonly used antibiotics (aztreonam, ciprofloxacin, cefepime, gentamicin, imipenem, ampicillin, cotrimoxazole, piperacillin / tazobactam) was detected by K-B test in 32 multi-drug resistant strains. Deletions in the outer membrane porin oprD2 gene were detected by PCR. Metallo-β-lactamase genes including NDM-1, IMP, GIM, VIM, SPM, GES and KPC were detected by PCR to evaluate B class metallo-β-lactamase production. Aminoglycoside resistant genes were detected by PCR to analyze the resistant mechanism in multi-drug resistant P. aeruginosa . OXA-typeβ-lactamase genes and plasmid-type AmpCβ-lactamase genes were also detected by multiplex PCR.Results1.Resistant rates to 9 commonly used antibiotics in 60 clinical isolated P. aeruginosa were listed as follows: gentamicin 36.67% (22/60), aztreonam 33.33% (20/60), ceftazidime 31.67% (19 / 60), imipenem 28.33% (17/60), amikacin 26.67% (16/60), piperacillin / tazobactam 23.33% (14/60), ciprofloxacin 21.67% (13/60), levofloxacin 11.67% (7 / 60), polymyxin B 0. Strains with positive efflux pump phenotypes were listed as follows: 35 strains (58.33%) positive for MexAB-OprM, 13 strains (21.67%) positive for MexXY–OprM, 11 strains (18.33%) positive for MexCD-OprJ, and 10 strains (16.67%) positive for MexEF-OprN. The mexR gene could be amplified in 28 of 35 MexAB-OprM efflux phenotype-positive strains, with a detection rate of 80.0% (28/35).2. Integrase gene could be amplified in 5 of 32 multi-drug-resistant strains, and qacEΔ1-sull gene could be amplified in 4 of 5 integrase gene positive strains. Resistant genes could be amplified in the variable region of drug-resistant gene cassettes in 3 of 4 type I integron positive strains. Two combinations of resistance genes were found by BLAST analysis of resistance gene cassettes: (1) aadB-blaPSE-1, in which aadB encoded resistance to aminoglycoside such as gentamicin, kanamycin and tobramycin, and in which blaPSE-1 belonged toβ-lactamase resistance gene encoding resistance to cephalosporins, and (2) aadB—aac(6ˊ)-Ⅱa—blaPSE-1, in which aac(6ˊ) II encoded resistance to amikacin, netilmicin and tobramycin.3. Resistant rates to 8 commonly used antibiotics in 32 multi-drug resistant strains were listed as follows: cotrimoxazole 100%, ampicillin 96.88%, aztreonam 87.50%, cefepime 62.50%, ciprofloxacin 62.50%, imipenem 59.38%, piperacillin / tazobactam 56.25% and gentamicin 31.25% respectively.Twenty-six of 32 multi-drug resistant strains were missing oprD2 gene, and the deletion rate is 81.25% (26/32).NDM-1, IMP, GIM, VIM, SPM, GES, KPC genes in these strains were not detectable by PCR.The results for aminoglycoside- resistant gene detection by PCR were listed as follows: aac(6′)-Ⅱgene could be amplified in 4 multi-drug resistant strains and ant(2′′)-Ⅰgene could be amplified in 4 multi-drug resistant strains. Seven strains harbored the aminoglycoside-resistant genes with a detection rate of 21.86% ,of which 1 strain had both gene aac(6′)-Ⅱand ant(2′′)-Ⅰ. BLAST results showed that the sequences of PCR products with aac(6′)-Ⅱgene shared 100% homology with the sequences of HQ880255.1 and HQ880250.1 in P. aeruginosa, and the sequences of PCR products with ant(2'')-I also sheared 100% homology with the sequences of JF742758.1.The results for OXA-typeβ-lactamase gene and plasmid-mediated AmpC gene detection by multiplex PCR were listed as follows: OXA23 was amplified from No. 21 specimens, while No. 29 was positive for OXA51. BLAST results showed that, PCR products of No. 21 specimens with the OXA23 gene shared 99% homology with sequences JF421124.1, CP002522.1 and HQ008358.1 in Acinetobacter baumannii and PCR products in No.29 specimens with class Dβ-lactamase gene shared 100% homology with the sequence CP002522.1 and 99% homology with the sequence HQ222987.1 in Acinetobacter baumannii Plasmid-mediated AmpC gene could not be amplified by multiplex PCR.Conclusions1.In clinical commonly used antibiotics, the resistant rate of clinical isolated strains of P. aeruginosa to gentamicin was the highest, and the resistant rate to polymyxin B was the lowest. In 4 kinds of efflux pump phenotypes the MexAB-OprM was the most common type and its presence may be an important mechanism of multi-drug resistance in P. aeruginosa.2.The major type of integrons was type I integron in multi-drug resistant P. aeruginosa. The existence of aminoglycoside modifying enzyme gene andβ-lactamase gene in integrons with multi-drug resistance in multi-drug resistant P. aeruginosa was closely related.3. All of the multi-drug P. aeruginosa strains were resistant to sulfonamides, and the resistant rate ofβ-lactams was also high (over 50%). But the resistant rate to aminoglycosides was relatively low. Aminoglycosides can be considered as an option for combined-medication on antimicrobial therapy after the patients had received high dose broad-spectrum antibiotics. The deletion of outer membrane porin OprD2, the existence ofβ-lactamase gene and aminoglycoside-resistant gene with multi-drug resistance was closely related in P. aeruginosa multi-drug resistance. |
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