Detection and characterization of multidrug resistance and extended-spectrum-beta-lactamase-producing ( ESBL S ) Pseudomonas aeruginosa isolates in teaching hospital

Pseudomonas aeruginosa is the most common pathogen causing nosocomial infections. The objective of this study was to investigate the extended-spectrum-beta-lactamase (ESBLs) producing and multidrug resistance of hospital isolates of P. aeruginosa and to determine the presence of several resistance genes. A total of 86 isolates of P. aeruginosa were collected from teaching hospital in Kashan, Iran. Susceptibility to eight antimicrobial agents was performed by disk diffusion method. ESBL-phenotypic detection was carried out by double-disk synergy test; and the presence of the genes encoding of bla(TEM), bla(SHV), bla(CTX-M), bla(OXA) and bla(GES)-like genes was studied by polymerase chain reaction. The prevalence of ESBLs was 8.1%. The presence of genes encoding ESBLs was confirmed in seven isolates, comprising seven bla(GES-2), one bla(SHV-1), one bla(SHV-5) and one bla(CTX-M-1) genes. P. aeruginosa demonstrated the highest resistance rate to piperacillin (38.4%), 67.5% of isolates were sensitive to imipenem whereas 32.5% were MDR (resistant to three or more classes of antibiotics). A multidrug-resistant (MDR) phenotype occurred frequently in P. aeruginosa. bla(GES-2), which compromises the efficacy of imipenem detected in all of seven ESBLproducing P. aeruginosa strains. Proper infection control practices and barriers are essential to prevent spreading and outbreaks of ESBL-producing and MDR P. aeruginosa in our teaching hospital.


INTRODUCTION
Pseudomonas aeruginosa is widely known as an opportunistic organism, frequently involved in infections of immunosuppressed patients, and also causes outbreaks of hospital-acquired infections (Wirth et al., 2009).P. aeruginosa is intrinsically resistant to many antimicrobial agents, including most β-lactams, the older quinolones, chloramphenicol, tetracycline, macrolides, trimethoprimsulfamethoxazole and rifampin (Dundar and Otkun, 2010;Rossolini and Mantengoli, 2005).Multidrug resistance can be relatively common amongst nosocomial isolates of P. aeruginosa, which represent the large majority of clinical isolates (Rossolini and Mantengoli, 2005).Resistance in P. aeruginosa may be mediated via several distinct mechanisms including the production of βlactamases, efflux pumps, and target-site or outer membrane modifi-cations (Tam et al., 2010).The production of extended-spectrum beta-lactamases (ESBLs) confers resistance at various levels to expandedspectrum cephalosporins, such as cefotaxime and ceftazidime, and to aztreonam, but not normally to the cephamycins and carbapenems (Mirsalehian et al., 2010).These enzymes are encoded by different genes located on either chromosomes or plasmids (Chuang and Rasmussen, 2006).Two major classification schemes exist for categorizing β-lactamase enzymes: Ambler classes A through D, based on amino acid sequence homology, and Bush-Jacoby-Medeiros groups 1 through 4, based on substrate and inhibitor profile (Drawz et al., 2010).The activity of Class A enzymes is inhibited in vitro by β-lactamase inhibitors such as clavulanic acid, sulbactam and tazobactam but those belonging to class D are not (Bradford, 2001).Recently, however, several class A, B and D extended spectrum β-lactamases have been reported in P. aeruginosa (Park et al., 2005).Clavulanic acid-inhibited ESBLs conferring resistance to expanded spectrum cephalosporins have been reported, first in Enterobacteriaceae and then in Pseudomonas aeruginosa (Nordmann and Guibert, 1998).The TEMtype ESBLs are derivatives of TEM-1 and TEM-2.TEM-1 is able to hydrolyze ampicillin at a greater rate than carbenicillin, oxacillin, or cephalothin, and has negligible activity against extended-spectrum cephalosporins (Paterson and Bonomo, 2005).The SHV-type ESBLs may be more frequently found in clinical isolates than any other type of ESBLs (Jacoby, 1997).SHV refers to sulfhydryl variable.SHV-1 is a β-lactamase with activity against penicillins and narrow spectrum cephalosporins such as cephalothin and cephaloridine (Harada et al., 2008).
SHV-5 and SHV-12 are among the most common members of this family (Paterson et al., 2003).The most common group of ESBLs not belonging to the TEM or SHV families was termed CTX-M to highlight their greater activity against cefotaxime than against ceftazidime (Bonnet, 2004) but some of them, such as CTX-M-15 and -19, also hydrolyse ceftazidime efficiently (Poirel et al., 2001) which may complicate their phenotypic recognition (Pitout et al., 2005).CTX-M ESBLs are the most commonly isolated ESBLs in many parts of the world, particularly Europe (Drawz et al., 2010).The OXA-type βlactamases are so named because of their oxacillinhydrolyzing abilities.These β-lactamases are characterized by hydrolysis rates for cloxacillin and oxacillin greater than 50% that for benzylpenicillin (Paterson and Bonomo, 2005).GES-1 possesses hydrolytic activity similar to the classic class A ESBLs, is inhibited by β-lactamase inhibitors, and is generally classified into ESBLs (Poirel et al., 2000).However, some of the GES variants, such as GES-2 and 4, also have hydrolytic activity against carbapenems (Poirel and Weldhagen et al., 2001;Wachino et al., 2004).The occurrence and detection of ESBL in P. aeruginosa are undefined in Kashan.This study aimed to investigate the presence and prevalence of bla(TEM), bla(SHV), bla(CTX-M), bla(OXA) and bla(GES)-like genes, responsible for extended spectrum beta-lactamases (ESBLs) production, antibiotic susceptibilities and multidrug resistance (MDR) of P. aeruginosa, isolated from clinical specimens of patients of hospital.

MATERIALS AND METHODS
This descriptive study was carried out in Beheshti hospital from 2010 to 2011 (Kashan city, Isfahan province, Iran).86 isolates of P. aeruginosa were isolated from patients enrolled in this study.Conventional biochemical tests were used for identification at the species level in 86 g negative, short rods was based on colonial morphology, oxidase positivity, and the presence of characteristic pigments.The organisms were isolated from urine (n = 23), the trachea (n = 18), stool (n = 17), wound (n = 11), blood (n = 5), bronchoscopy fluid (n = 4), sputum (n = 3), vaginal discharge (n = 2), pleural fluid (n = 1), gastrointestinal fluid (n = 1) and pus (n = 1).All isolates were cultured in Luria-Bertani (LB) broth and grown at 37°C for genomic DNA isolation.The isolates were stored in vials containing LB broth with 50% glycerol at -20°C.The purity of the strains was checked before of analysis.The susceptiblities of the bacterial isolates to eight antimicrobial agents (imipenem 10 µg, ciprofloxacin 5 µg, ceftazidime 30 µg, ceftriaxone 30 µg, cefotaxime 30 µg, aztreonam 30 µg, piperacillin 100 µg and gentamicin 10 µg; [Mast Group Ltd., Merseyside, U.K.] were determined by the disk diffusion method according to the criteria published by the Clinical and Laboratory Standards Institute(CLSI).P. aeruginosa ATCC 27853 was used as quality control in each susceptibility determination.Multidrug-resistant isolates were defined as those resistant to three or more classes of antipseudomonal agents (carbapenems, penicillins, cephalosporins, monobactams, fluoroquinolones and aminoglycosides).ESBL production in all the isolates was detected by double-disc synergy test as described by Jarlier et al. (1998).Briefly of 30 µg each ceftazidime, ceftriaxone, cefotaxime and aztreonam disks were placed at a 20 mm (center-to-center) from an amoxicillin-clavulanate 20/10 µg disk (Mast Group Ltd., Merseyside, U.K.) on Mueller-Hinton agar(Merck, Germany) and incubated at 37°C for 16 h.Enhancement of the zone of inhibition of the oxyimino ß-lactam due to synergy of the clavulanate in the amoxicillin-clavulanate disk was considered as evidence of ESBLproduction.This criterion also fulfills the CLSI guidelines.This increase occurs because the clavulanic acid present in the amoxyclav disc inactivates the ESBL produced by the test organism.
Bacterial DNA was prepared by suspending one or two fresh colonies in 100 ml of sterile distilled water and heating at 95°C for 10 min.After centrifugation, the supernatant was stored at 4°C before analysis.The detection of β-lactamase genes were performed under standard PCR conditions using published set of primers (Table 1).Amplicons were revealed by electrophoresis on a 1.2% agarose gel and a subsequent exposure to UV light in the presence of ethidium bromide.

DISCUSSION
The occurrence of multidrug-resistant P. aeruginosa strains is increasing worldwide and limiting our therapeutic options (Bonomo and Szabo, 2006).Our study reported the highest resistance rate to piperacillin, cefotaxime, imipenem, ceftriaxone, gentamicin, ceftazidime, aztreonam, ciprofloxacin respectively.In our study, gentamicin resistance value was higher than values reported in study conducted by Zhanel et al. (2010).In this study, ciprofloxacin exhibited nearly high susceptibility pattern (71%) and Zhanel et al. (2010) reported that 19.0 to 24.1% isolates were resistant to fluoroquinolones.
Cephalosporins, are known anti-pseudomonal drugs, especially the third-genetration ceftadizime, that has demonstrated high susceptibility pattern (72.1%) with P. aeruginosa isolates, however cefotaxime and ceftriaxone were the two of the cephalosporin drug tested in this study, with susceptibility value of 14 and 60.5% respectively.These low resistance value observed were comparable with the report from Malaysia of 40 and 31% (Jombo et al., 2008).In our study, prevalence of MDR P. aeruginosa isolates, was 32.5% which lower than values reported in other studies in Iran (Mirsalehian et al., 2010), Malaysia (Lim et al., 2009) and Pakistan (Ullah et al., 2009) and this rate is lower than study was performed in this hospital five years ago (Moniri et al., 2005).In contrast, Tam et al. (2010) reported that 14.0% of P. aeruginosa isolates were MDR.The multidrugresistant phenotype in P. aeruginosa could be mediated by several mechanisms including multidrug efflux systems, enzyme production, outer membrane protein (porin) loss and target mutations.Inappropriate empirical therapy has been associated with increased mortality in P. aeruginosa infections; delays in starting appropriate therapy may contribute to increased length of hospital stay and persistence of infection.In addition, worse clinical outcomes may be associated with MDR infections owing to limited effective antimicrobial options.Our study reported 8.1% ESBL production among P. aeruginosa isolates.The studies conducted by others depicted low rates, 3.7% (Woodford et al., 2008), 4.2% (Lim et al., 2009), 7.7% (Jacobson et al., 1995) respectively, of ESBL production in P. aeruginosa.
In our study, the bla GES-2 gene was detected in all ESBL producing isolate.This was not accordance with other results in Middle East and other study in Iran.Recent reports from Brazil showed that bla GES-1 are present in P. aeruginosa strains (Castanheira et al., 2004).In this study, we describe the presence of CTX-M-1 ‫-ك‬ lactamase in 1 clinical bacterial strains of P. aeruginosa.The first description of the presence of CTX-M ESBLs in P. aeruginosa and S. maltophili reported by Al Naiemi et al. (2006).The CTX-M ESBLs provide P. aeruginosa with an additional powerful resistance mechanism with potential serious clinical implications, including limitation of the therapeutic options.ESBLs producing organisms pose unique challenges to clinical microbiologists, clinicians, infection control professionals and scientists engaged in finding new antibacterial agents.The incidence of ESBLs varies with geographic location and time.ESBL producing strains are usually found in those areas of hospitals where antibiotic use is frequent and the patient's condition is critical.These resistant organisms are clinically important because they result in increased morbidity and mortality.In addition, ESBL producing bacteria are frequently resistant to many classes of antibiotics, resulting in difficult-to-treat infections.Currently, carbapenems are regarded as the drugs of choice for treatment of infections caused by ESBL-producing organisms.Unfortunately, use of carbapenems has been associated with the emergence of carbapenem-resistant Pseudomonas spp.The development and spread of ESBLs is most likely caused by the overuse of expandedspectrum cephalosporins in the hospital setting.Proper infection control practices and barriers are essential to prevent spreading and outbreaks of ESBL-producing bacteria.
In conclusion, gentamicin still shows the highest potency against P. aeruginosa in our hospital.However, emerging of resistance to carbapenems, in particular resistance to imipenem, may limit their prescription in the future.The prevalence of ESBL-producing P. aeruginosa occurred in 8.1% of isolates and MDR phenotype was common in P. aeruginosa.The most prevalent genes among ESBL producers were bla GES-2 , which compromises the efficacy of imipenem, tend to be geographically restricted.

Table 1 .
Primers used in this study.

Table 2 .
Antimicrobial susceptibility pattern on P. aeruginosa isolated from patients of Shahid Beheshti hospital.

Table 3 .
Resistance to one or more antimicrobials among P. aeruginosa isolated from patients of Shahid Beheshti hospital in Kashan.