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Abstract
SUMMARY Pseudomonas aeruginosa isolates show steady escalation in resistance to an increasing number of antimicrobial agents and the emergence of multidrug resistant Pseudomonas aeruginosa infections is increasingly recognized, therefore, the current study was undertaken to investigate the prevalence and mechanisms of antimicrobial resistance among 89 clinical isolates of Pseudomonas aeruginosa collected from patients admitted to a university-affiliated hospital and with clinically diagnosed infections of urinary tract, respiratory tract, burns and surgical wounds, ear, or septicemia between March 2002 and May 2003. All the isolates were identified as Pseudomonas aeruginosa by their morphological, cultural and biochemical characteristics according to traditional methods. The antimicrobial susceptibility profiling of the isolates to several antimicrobial agents representing different groups was performed by the agar dilution method. The isolates were found to be resistant to ampicillin, amoxicillin, carbenicillin, streptomycin, nalidixic acid, tetracycline, doxycycline, erythromycin, azithromycin and chloramphenicol. All isolates were susceptible to polymyxin B, amikacin, meropenem and pipracillin while resistance rates of 1.1%, 12.4%, 19.1%, , 29.2%, 31.5%, 32.6%, 56.2%, 79.8%, 86.5% and 89.9% were found for imipenem, ceftazidime, ticarcillin, ceftriaxone, cefepime, ciprofloxacin, cefotaxime, gentamicin, norfloxacin and aztreonam, respectively.
The isolates showed high MIC values to chemical antimicrobial agents, and the MIC values of ethidium bromide, proflavine, methyl paraben and propyl paraben were >1024, 1024, 250 and >50 µg/ml, respectively. The MIC values ranged from 3.12 to 12.5 µg/ml for thiomersal, from 20 to 40 µg/ml for phenyl mercuric nitrate, from 400 to 800 µg/ml for chlorcresol, and from 100 to 150 µg/ml for chlorhexidine. The MIC values for benzalkonium chloride were 200 µg/ml. Multidrug resistant Pseudomonas aeruginosa accounted for 47.1% of the isolates. The possible mechanisms of resistance to antimicrobial agents were investigated. The production of β-lactamases in the tested isolates was detected by nitrocefin test in the isolates. In order to determine the possible types of β-lactamases, crude cell extracts were prepared from some representative selected isolates. The β-lactamase activity was determined in presence and absence of some β-lactamase inhibitors.
The hydrolysis of some β-lactams by crude cell extracts of isolates 33, 35 and 70 and the effect of some β-lactamase inhibitors were followed up spectrophotometrically. The crude cell extract of the tested isolates hydrolysed ampicillin and this effect was inhibited by pCMB but not with clavulanic acid, tazobactam or oxacillin. This pattern may suggest a class C or group 1 β-lactamases which is more likely to be AmpC β-lactamase (cephalosporinase).
The double disk synergy test for detection of extended-spectrum β-lactamases excluded the presence of ESBLs in the isolates. Polymerase chain reaction analysis using specific primer supported the absence of class 1 integron, which is likely to carry class A ESBLs in the isolates. However, other class A, B and D ESBLs require further investigation before exclusion.
To determine the possible mechanism of resistance of isolate No. 33 to imipenem, the effect of crude cell extract of this isolate on imipenem was followed up spectrophotometrically and it was observed that imipenem was hydrolyzed and this effect was inhibited by EDTA and pCMB. The β-lactamase activity of crude cell lysate of isolate No. 33 was followed spectrophotometrically using nitrocefin in presence and absence of EDTA and pCMB. The accelerated hydrolysis of nitrocefin would suggest the existence of over-expression of AmpC β-lactamases.
Also, to investigate the presence of carbapenemases in the imipenem-resistant isolate No. 33, disc diffusion synergy test using EDTA was performed. An increase in the inhibition zone diameter was observed with EDTA which might suggest the existence of carbapenemases. To determine the type of carbapenemase, PCR analysis for amplification of three different genes was performed using specific primers for two metallo-β-lactamases (IMP-1 and VIM-1 type metallo-β-lactamases) and one OXA-type carbapenemase (OXA-50). The absence of amplification products excluded the presence of IMP-1, VIM-1 or OXA-50 β-lactamases in this isolate.
The conflict between the results of the EDTA-disc diffusion synergy test, spectrophotometric assay of imipenem and the PCR amplification might be explained by the presence of carbapenemases other than IMP-1- or VIM-1 MBLs and OXA-50 carbapenemases.