Frequency of extended-spectrum beta-lactamase (ESBLs)-multidrug resistance produced by Pseudomonas aeruginosa isolated from clinical specimens in Khorramabad city, Iran.

Background: Pseudomonas aeruginosa is one of the most important pathogenic bacteria causing hospital infections, which has intrinsic resistance to many antibiotics. One of the reasons for the emergence of drug resistance in P. aeruginosa isolates is the production of ESBLs (extendedspectrum beta-lactamase) enzymes. Resistance rate is increasing due to the production of these enzymes in P. aeruginosa. The goal of this study was to determine the prevalence of ESBL senzymes in P. aeruginosa isolates from different samples of patients in Khorramabad city by CDT (combined disk test) phenotypic method. Methods: This study was an investigation on 70 P. aeruginosa samples isolated from patients in Original Research Article Azizi et al.; BMRJ, 5(6): 490-495, 2015; Article no.BMRJ.2015.052 491 medical centers of Khorramabad city during one year (2013). The bacteria were identified by routine biochemical tests. Antibiotic sensitivity of the isolates was evaluated by double disk diffusion method. Phenotypic investigation of ESBLs production among the tested isolates using cefotaxime and ceftazidime disks alone and in combination with clavulanic acid (combined disk test) was performed. Results: Results of combined disk test showed that, out of 70 isolated pseudomonas, 100% of the samples had multi-drug resistance. Maximum resistance to cefixime (79.1%) and minimum resistance to meropenem (25%) were observed. Out of 70 isolates, 25 (35.7%) were phenotypic beta-lactamase-producing enzymes. That highest percentage was related to wounds, equal to 12 (17%). Conclusion: Considering the increasing prevalence of P. aeruginosa strains which produce extended-spectrum beta-lactamase, it is recommended to use an appropriate treatment protocol based on determining antibiogram pattern of strains. This means that all isolated before treating with antibiotics were examined by antibiotic susceptibility test. Also antibiotics should be used according to CLSI (Clinical and Laboratory Standards Institute). This issue is of serious concern for applying infection-control criteria with the purpose of preventing spread of this microbe.

medical centers of Khorramabad city during one year (2013). The bacteria were identified by routine biochemical tests. Antibiotic sensitivity of the isolates was evaluated by double disk diffusion method. Phenotypic investigation of ESBLs production among the tested isolates using cefotaxime and ceftazidime disks alone and in combination with clavulanic acid (combined disk test) was performed. Results: Results of combined disk test showed that, out of 70 isolated pseudomonas, 100% of the samples had multi-drug resistance. Maximum resistance to cefixime (79.1%) and minimum resistance to meropenem (25%) were observed. Out of 70 isolates, 25 (35.7%) were phenotypic beta-lactamase-producing enzymes. That highest percentage was related to wounds, equal to 12 (17%). Conclusion: Considering the increasing prevalence of P. aeruginosa strains which produce extended-spectrum beta-lactamase, it is recommended to use an appropriate treatment protocol based on determining antibiogram pattern of strains. This means that all isolated before treating with antibiotics were examined by antibiotic susceptibility test. Also antibiotics should be used according to CLSI (Clinical and Laboratory Standards Institute). This issue is of serious concern for applying infection-control criteria with the purpose of preventing spread of this microbe.

INTRODUCTION
P. aeruginosa is one of the most important pathogenic bacteria, which causes hospital infections like pneumonia, bacteremia, and urethritis infections [1]. This bacterium is one of the causative agents of chronic pulmonary infections and death in children and adults with cystic fibrosis disease [1]. Also, it is an opportunistic pathogenic bacterium which is a major cause of mortality in patients with an impaired immune system [1]. As a result of clinical consumption of antibiotics, the prevalence of hospital P. aeruginosa strains which are multi-drug resistant (MDR) has been increased worldwide and turned into a serious problem in hospital management [1,2]. Intrinsic resistance to antimicrobial agents in this bacterium exacerbates treatment conditions of its infections [1,2]. Resistance mechanisms of P. aeruginosa include producing beta-lactamase, secretion pumps, and external membrane changes [3]. Resistance to various drugs is usually the result of a combination of different mechanisms in one strain or the function of a specific mechanism. Kind of resistance due to the production of beta-lactamase enzymes is the resistance factor to beta-lactamase [3]. Betalactamase has been an appropriate drug for treating P. aeruginosa; but, after a while, some of the bacteria show resistance to these antibiotics by producing beta-lactamase [3]. Resistance of P. aeruginosa strains to the second and third generation (extended-spectrum) cephalosporins and monobactams is iscused by extended-spectrum beta-lactamase enzymes (ESBLs) [4,5]. These enzymes are coded by either genes of chromosomal or plasmid origin [4,5]. ESBLs are type A beta-lactamases [5,6] and include a number of evident ESBL specifications which inhibit their activity in the presence of clavulanic acid (beta-lactamase inhibitor). This specification is used as a confirmatory test in tracing ESBLs in laboratory [6]. Therefore, bacterial strains resistant to one of the third generation cephalosporins (cefotaxime, ceftriaxone, cefixime, and ceftazidime) and its beta-lactamase is stopped in the presence of clavulanic acid is. Thus, the treatment of infections caused by this bacterium fails despite its easy laboratory detection [6,7]. The most common ESBLs reported from Western and Asian countries are ESBLs that were derived from TEM and SHV. These ESBLs are located on large plasmids and often transfer to various strains of the genus or other genera of the family enterobacteriaceae with indicators of resistance [7]. Nowadays, indiscriminate use of antibiotics in Iran has led the bacteria to gain resistance mechanisms and show resistance to drugs [2]. Considering the mentioned points and due to the role and importance of strains in uncontrollable hospital infections, the aim of this research was survey of P. aeruginosa isolated from clinical samples in Khorramabad city, Lorestan province, Iran.

Screening Beta-lactamase Producer Samples (ESBLs)
This test was done by the disk diffusion method (Kirby-Bauer) based on CLSI standards. At first standard microbe suspension was prepared with half McFarland concentration, and cultured on Mueller-Hinton agar medium [1,2,3,4]. After complete dispersion of the microbial suspension on the mentioned medium, ceftazidime, cefotaxime, and piperacillin antibiotic disks along with their three combined disks with clavulanic acid (ceftazidime/clavulanic acid, cefotaxime /clavulanic acid, piperacillin/tazobactam) were placed on the medium at the distance of 25 mm (Fig. 1). After 24 h incubation of the plates at 37ºC, the diameter of the inhibition zone around the disk was measured. An Increased inhibition zone of equal to or more than 5 mm in the plate containing clavulanic acid disk was determined as ESBLs producer strain [1,2].

Microbial Sensitivity Pattern for MDR Strains
The Microbial sensitivity pattern for MDR strains was as listed in Table 1.According to the obtained results, 100% of the isolates showed multi-drug resistance (Table 1). Maximum resistance to ciprofloxacin was observed in pulmonary secretions. The highest resistance was observed for cefiximein.
Most beta-lactamase production in clinical specimen was found in wounds. That 35% of the strains about 40% of the wounds were betalactamase producers. The ESBL frequency in different clinical samples in this study are listed in Table 2.

DISCUSSION AND CONCLUSION
MDR P. aeruginosa is the most important risk factor in hospital centers. Nowadays, the problem of resistance to beta-lactamase drugs and extended-spectrum cephalosporins is becoming a serious problem and we envisage that in the near future, the world will need new antibiotics to replace the existing ones  (Table 1). Among the strains, the maximum percentage of P. aeruginosa isolates existed in wound and urine samples; consequently, the maximum rate of resistance was observed in these samples. Maximum resistance to ciprofloxacin was observed in pulmonary secretions. Wound infections were most resistant to cefixime. This issue demonstrated that maximum resistance was in the samples isolated from hospitalized patients, which indicated increased antibiotic resistance in hospital environments. Increased acquisition of ESBL genes through horizontal gene transfer, which influences long-term hospitalization, can be associated with extended-spectrum betalactamase and nosocomial infection could lead to considerable dispersion of ESBL producer bacteria [9]. The notable point is about the frequency of sample; in the present work, the maximum number of isolates and resistance was found in wounds, which was in agreement with the study at Shahid Beheshti Hospital, Kashan, Iran [1]. In this research, maximum rate of resistance was obtained for cefixime (79.1%) and meropenem (25%) showed minimum resistance, which was almost in agreement with previous studies [1,5]. A study in Saudi Arabia, showed a prevalence of ESBL pseudomonas of 41.6%, which was in agreement with percentage of ESBL in the present study [5]. In studies conducted by Woodford et al. [10] in the UK, the prevalence of ESBL producer strains of P. aeroginosa was 3.8%. The percentage of 4.2% in Malaysian and 7.2% in UK individuals, respectively, has been reported [11]. Meanwhile, other studies indicated high levels of ESBLs in strains of P. aeruginosa in Thailand (28%), India (20.3%) and burn hospital Tehran (39.4%) [12][13][14][15][16][17]. The frequency distribution of ESBL producers among P. aeruginosa strains isolated from clinical specimens in Khorramabad, Iran were 17.1%, 8.5%, 4.2%, 2.8%, 1.4%, 1.4% for wound, urine, pulmonary secretions, blood, tissue and body fluid respectively, while the prevalence rate was 35.7% (Table 2). It should be noted that appearance of different types of antibiotic-resistant and ESBL producer strains has been associated with excessive and longterm consumption of broad spectrum cephalosporins, according to most of the reports [1,5,10, 18,19]. The prevalence of these organisms can cause endemic and epidemic nosocomial infections that subject to certain conditions such as underlying disease, use of invasive devices, and previous use of antibiotics [20]. Nosocomial infection scan be reduced by the use of infection control methods, such as communicable disease control, sterilization, disinfection, proper use of equipment, application of sanitation, hand washing and limiting the use of oxyimino-cephalosporins [9].

ACKNOWLEDGMENT
Hereby, a respected management and colleagues Razi Herbal Medicines Research Center, Lorestan University of Medical Sciences for cooperation is sincerely appreciated. Also, of all the people who helped us in this project comes to appreciate.