Molecular Identification of Extended-Spectrum β-lactamase and Integron Genes in Klebsiella pneumonia

Introduction: Infections caused by Gram negative bacteria, producing extended-spectrum β-lactamase, including Klebsiella pneumoniae are increasing all over the world with high morbidity and mortality. The aim of the present study was determined antimicrobial profile susceptibility and the prevalence of antibiotic resistance genes by multiplex PCR. Methods: In the present study, we obtained one-hundred isolates of K. pneumoniae from different clinical samples. The antibiotic susceptibility testing was done in thirteen antibiotic and, therefore, M-PCRs were conducted using the DNA amplification for detection of ESBLs (blaTEM, blaCTX-M, blaSHV) and int (I, II, III) genes. results: The results of resistance to amoxicillin/clavulanate, ciprofloxacin, amikacin, trimethoprimsulfamethoxazole, cefotaxime, ampicillin, aztreonam, imipenem, gentamicin, ceftazidime, Cefepime, ceftriaxone and levofloxacin were obtained 37%, 37%, 93%, 84%, 52%, 87%, 59%, 8%, 24%, 67%, 52%, 43% and 26%, respectively. The frequency of the extended-spectrum β-lactamase K. pneumoniae was obtained 37%. The prevalence of resistance genes of ESBLs in the M-PCR method showed that the blaTEM, blaCTX and blaSHV were 38%, 24% and 19%, respectively, however, only 8 (8%) out of 100 isolates were found to have positive outcomes for the existence of class 1 integrons and there were no detected class 2 or class 3 integrons. conclusions: Our results recommend the likely co-carriage of some ESBLs genes and antibiotic resistance integrons on the same plasmids harboring the MDR genes. _______________________________________________________________________________________

capability against infection from MDR pathogens. 2 The release of extended-spectrum β-lactamases (ESBLs) are unique of the significant mechanisms of antimicrobial agents resistance. 3 The appearance of ESBLs in Enterobacteriaceae groups has been a problem of medical concern. ESBLs producing K. pneumonia can degrade a wide-ranging of beta-lactam groups such as penicillins, cephalosporins, carbapenems and cephamycins. 2,4 In general, ESBLs are distributed into four classes A, B, C and D. Temoneira (TEM), cefotaximase (CTX-M) and sulfhydryl variable (SHV) are present in class A ESBLs. 5 Some researchers have described various occurrences of ESBLs between 6% and 88% in difference health care locations particularly between K. pneumoniae and E. coli. 6 SHV type β-lactamases relate to high level resistance to ceftazidime, but not to Cefazolin and cefotaxime, while CTX-M β-lactamases are more an effective against cefotaxime. In contrast, TEM β-lactamases confer resistance to oxyimino-β-lactams groups, such as ceftazidime, cefotaxime, and aztreonam. In addition to ESBLs, the transportable genetic elements, such as integrons, provide for the evolution and distribution of MDR genes (blaCTX-M, blaIMP, and blaGES) in K. pneumoniae by vertical or horizontal transmission. 7 According to the variation aminoacid sequence of the IntI protein, 5 classes of integrons have been identified. Three classes of antibiotic resistance integrons (ARIs) (classes 1, 2, and 3) have been generally complexed in MDR phenotypes criteria and are recognized depending on their particular integrase genes. 8 The transportable class 1 integrons are related to transposon Tn21 and have been commonly occur in ESBL producing clinical isolates of K. pneumoniae. However, Class 2 integrons found less frequently in ESBL-producing bacteria like K. pneumoniae and Escherichia coli, and class 3 integrons are infrequently in ESBL-producing K. pneumoniae. 10 In previous reports have confirmed the relations among ESBL producing bacteria and resistance to several groups of antibiotics, as well bla ESBL with ARI gene carriage in clinical isolates of K. pneumoniae. 11 Unfortunately, in the last decades, the incidence of ESBL-producing K. pneumoniae and their resistance is raising. 12 The identification of ESBLs genes, involving CTX-M ,TEM and SHV, by molecular methods in bacteria that producing ESBL and their antimicrobial susceptibility profile can provide appropriate evidence about their high risk factors and epidemiology related to their infections. 13,14 A few studies have been conducted to identify the types of ESBLs producing Enterobacteriaceae in Iranian hospitals. 15,16 The aim of the present study was detection of bla TEM , bla CTX-M, bla SHV , and int genes (I,II,III) in the clinical K. pneumonia strains isolated from two large urban teaching general hospitals in Tehran, Iran by multiplex PCR (M-PCR) and their antibiotic resistance profile.

MEtHODs
This cross-sectional study was directed during a one year period of time from April 2014 till March, 2015, in two teaching hospitals in Tehran, Iran. Generally, 100 non-repetitive K. pneumoniae strains were obtained from different clinical specimen including blood, skin lesions, broncho-alveolar lavage (BAL), urine, sputum, cerebrospinal fluid (CSF), Pus/swap, pleural effusion, ascites and catheter. Each sample was cultured on the Mac Conkey agar (Merck Co., Germany) and incubated in 37 °C for 24 h. Then, all suspected grown colonies were recognized as K. pneumoniae by standard biochemical and microbiological tests such as, urease, Gram staining, oxidase, motility, citrate utilization, urease production, TSI, KIA, MR-VP, SIM and confirmed API 20E system. (Analytab, Inc., New York).
Antibiotic susceptibility test was achieved by disc diffusion method on the Mueller-Hinton Agar (Merck Co., Germany) plates agreeing with Clinical and Laboratory Standards Institute (CLSI) guideline for the following antibiotics: amoxicillin/clavulanate (Aug; 20/10 μg), ciprofloxacin (CIP: 5 μg), amikacin (AK: M-PCRs were performed using the DNA amplification instrument master cycler gradient (Eppendorf Co., Germany) for detection of ESBLs genes (bla TEM , bla CTX-M and bla SHV ) and int genes (I,II,III). Genomic DNA was acquired from K. pneumoniae colonies grown overnight on blood agar (Merck Co., Germany) plates by the boiling lysis method. 23 Briefly, a loopful of bacterial colonies was suspended in the 700 μl sterile distilled water and boiled for 10 min and centrifuged at 7000×g for 4 min at 4°C and then cooling in ice for 10 minutes and centrifugation for 3 min at 8000×g. The concentration and the quality of the extracted cellular DNA were assessed using a Nanodrop spectrophotometer (ND-1000; Thermo Scientific; Wilmington, DE, USA). The genes encoding carbapenemases were amplified using the primer sequences, which were presented in Table 1, for m-PCR. M-PCR was done for amplification of ESBLs genes (bla TEM , bla CTX-M , bla SHV ) and in a volume of 1.5 μl of extracted genomic DNA was added to a total volume of 25 μl PCR reaction mixture including 2.5 μl of 10× PCR buffer, 1.5 μl MgCl 2 (50 mM), 0.5 μl dNTPs (10 mM), 1.25 μl of each primer, 0.5 μl of Taq DNA polymerase (5 U/μl) (Amplicon Co., Denmark) and 8.5 μl sterile distilled water. The reaction mixture was performed with the following PCR procedure: Denaturation at 94°C for 1 min, 35 cycles with denaturation at 94°C for 30 s, annealing at 59°C for 30 s, extension at 72°C for 1min and final extension at 72°C for 6 min. So, amplification of int genes (I,II,III), the reaction mixture was completed in a thermal gradient cycler (Eppendorf Co., Germany) with the following PCR protocol: The cycling conditions were one cycle of 5 min at 95 °C; 30 cycles of 1 min at 95°C, 1min at 65°C, and 1min at 72°C; and one cycle of 10min at 72°C (11).
ESBLs genes amplification test showed that the prevalence of bla TEM , bla CTX and bla SHV were 38%, 24% and 19%, respectively. M-PCR simultaneously amplified and identified the existence of bla TEM (445 bp), bla CTX-M (593 bp) and bla SHV (747 bp), respectively (Figure 1). Molecular distribution analysis of integrons genes showed that, only 8 (8%) out of 100 isolates were found to have positive outcomes for the existence of class 1 integrons and no class 2 or class 3 integrons were identified between isolates (Figure 2).

cONcLUsIONs
In conclusion, this project approves that a high level of bla TEM and class 1 integrons-positive ESBL K. pneumoniae is circulating in two hospitals of Tehran, Iran. The trend of MDR profiles related with the recovery of the bla TEM and lass 1 integron genes is worrying. This highpoints values is require for establish an antibiotic susceptibility surveillance network for monitoring of Enterobacteriaceae spp., infection in Iran.

AcKNOwLEDgMENt
This study was financially supported by the Islamic Azad University, Science and Research Branch (SRBIAU). We thank the following hospitals for referring isolates and epidemiological and demographic data for use in this study: Rasool Akram, Ali Asghar Children's Hospital.