Antimicrobial Resistance Patterns and Prevalence of blaPER-1 and blaVEB-1 Genes Among ESBL-producing Pseudomonas aeruginosa Isolates in West of Iran

Background: Pseudomonas aeruginosa is a leading cause of nosocomial infections worldwide. Resistance of P. aeruginosa strains to the broad-spectrum cephalosporins may be caused by extended-spectrum β-lactamases (ESBLs). Objectives: The aim of this study was to determine the antimicrobial resistance patterns and prevalence of PER-1 and VEB-1 type genes among ESBL producing strains of P. aeruginosa. Material and Methods: A total of 106 P. aeruginosa isolates were collected from two university hospitals in Hamadan, Iran, during a7-month study (2009). The antimicrobial susceptibility of isolates was determined by disc diffusion method and interpreted according to the clinical and laboratory standards institute (CLSI) recommendations. Production of ESBL was determined by combined disk test and presence of PER-1 and VEB-1 type ESBL genes was identified by PCR. Results: The resistance against broad-spectrum cephalosporins and monobactames were: cefepime (97%), cefotaxime (92.5%) ceftazidime (51%), and aztreonam (27%). Ciprofloxacin (91.5%), imipenem (84.9%) and meropenem (82.1%) were the most effective anti-pseudomonas agents in this study. The results revealed that 88.7% of the isolates were multidrug resistant, 58.25% of those were ESBL positive. Sixteen (26.6%), 9 (15%) and 3 (5%) strains among ESBL-producing strains contained blaPER-1, blaVEB and blaPER-1-blaVEB, respectively. Conclusions: This study highlighted the need to establish antimicrobial resistance surveillance networks for P. aeruginosa to determine the appropriate empirical treatment regimens. The high prevalence of multidrug resistance and production of ESBLs in P. aeruginosa isolates conﬁrms the necessity of protocols considering these issues in the hospitals.


Background
Pseudomonas aeruginosa is an important cause of nosocomial infections, including pneumonia, burn infection, urinary tract infections, meningitis and bacteremia. The infections can be particularly develop to a severe form in immune deficient patients (1). Antibiotics have been used successfully for several decades, but resistance genes have emerged and disseminated particularly in the last few years (2).
Extended-spectrum β-lactamases (ESBLs) mediate resistance to various broad-spectrum cephalosporins, including cefotaxime, ceftriaxone, ceftazidime, and aztreonam (3). These enzymes originally collected from Klebsiella pneumoniae and Escherichia coli and recently from P. aeruginosa (4)(5)(6)(7). Most of the methods for detection of ESBLs are used in bacterial species such as Klebsiella and E. coli lacking chromosomal β-lactamase activity (8,9), However, the detection of ESBL production in P. aeruginosa has some difficulties, because this bacterium not only has an inducible AmpC enzyme but also has an efflux-mediated resistance and a higher degree of impermeability than Enterobacteriaceae (10,11). The PER-1 and VEB-1 type ESBLs belong to class A of β-lactamases and is associated with high level of resistance to cephems, monobactams and ceftazidime (12,13).

Objectives
The aim of this study was to determine the antibacterial resistance patterns and prevalence of PER-1 and VEB type ESBLs among P. aeruginosa isolated from patients in west of Iran.

Phenotypic Detection of Beta-Lactamase
The isolates were tested for the ESBLs production by using combine disk test (CDT) as CLSI recommendations. CDT were performed on ceftazidime, cefotaxime, cefepime and aztreonam resistant strains by placing disks of ceftazidime, and cefotaxime (30 µg each) at a 20 mm distance from a disk containing ceftazidime-clavulanic acid (30/10μg), cefotaxime-clavulanic acid (30/10µg) and cefepime-clavulanic acid (30/10 µg) (16). ESBL production was inferred when the cephalosporin inhibitory zones were expanded by the clavulanate.

Discussion
P. aeruginosa has a high resistance to antibiotics and is a common cause of morbidity and mortality in hospitalized and immunocompromised patients (18). Treatment of P. aeruginosa infections is complicated by the inhered and acquired resistance to the most of commonly used antimicrobial agents (19). The results from this study showed the high resistance of P. aeruginosa to most of used antimicrobial agents. It was also demonstrated that the prevalence of antibiotic resistance of the isolates was very high in comparison to other studies and most of P. aeruginosa isolates (88.7%) were multi-drug resistant (resistant to ≥ 3 different antibiotic classes) (20)(21)(22)(23)(24)(25).
The prevalence of ESBL-producing P. aeruginosa isolates in this study was also higher than other investigations (5,20,26,27). Among 60 ESBL-positive strains, 16 (26.6%), 9 (15%) and 3 (5%) contained PER-1, VEB-1 and PER-1-VEB-1 genes, respectively. These results indicated that the prevalence of VEB-1 gene in our area, is higher than Turkey and Korea, but the prevalence of VEB-1 and PER-1 genes, is lower than in Thailand (94.44% blaVEB-1) and Italy (34.61% blaPER-1) (5,13,26). Data on the prevalence of ESBL-producing P. aeruginosa strains in our area is limited. In the study performed by Shahcheraghi and colleges on P. aeruginosa isolates in Tehran, the rate of blaVEB and blaPER ESBLs were reported 24% and 17%, respectively, that was similar to our results (28). The high prevalence of PER-1 and VEB-1 indicated the high resistance to penicillins, ceftazidime and cefotaxime, as reported by other studies (26,28). This is the first report about the presence of these enzymes in P. aeruginosa isolates from west of Iran. It has shown that ESBL production in strains of P. aeruginosa can greatly complicate the clinical management of infection if advanced care is not taken. However, further studies are needed to determine other ESBL-types in P.aeruginosa strains in this area and their role in resistance to other antibiotic classes. The results of this study emphasizes on the need for a surveillance network to monitor the trends and emerge of new resistance mechanism in P. aeruginosa from different geographic regions. Therefore, the improvement in antibiotic prescription policies and infection control programs are of high necessity to prevent the spread of such resistant infectious agents.