Molecular detection of β-lactamase and integron genes in clinical strains of Klebsiella pneumoniae by multiplex polymerase chain reaction

Introduction: Infections caused by β-lactamase-producing gram-negative bacteria, such as Klebsiella pneumoniae, are increasing globally with high morbidity and mortality. The aim of the current study was to determine antimicrobial susceptibility patterns and the prevalence of antibiotic resistance genes (β-lactamase and integron genes) using multiplex PCR. Methods: One-hundred K. pneumoniae isolates were collected from different clinical samples. Antibiotic susceptibility testing was performed with thirteen different antibiotics. Multiplex-PCR was used to detect β-lactamase (blaTEM, blaCTX-M, blaSHV, blaVEB, blaPER, blaGES, blaVIM, blaIMP, blaOXA, and blaKPC) and integron genes (int I, int II, and int III). Results: The highest and lowest rate of resistance was exhibited against amikacin (93%) and imipenem (8%), respectively. The frequency of β-lactamase-positive K. pneumoniae was 37%, and the prevalence of the blaTEM, blaCTX-M, blaSHV, blaVEB, blaPER, blaGES, blaVIM, blaIMP, blaOXA, and blaKPC genes was 38%, 24%, 19%, 12%, 6%, 11%, 33%, 0%, 28%, and 23%, respectively. Of the 100 isolates, eight (8%) were positive for class I integrons; however, class II and III integrons were not detected in any of the strains. Conclusions: These results indicate co-carriage of a number of β-lactamase genes and antibiotic resistance integrons on the same plasmids harboring multi-drug resistance genes. It seems that these properties help to decrease treatment complications due to resistant bacterial infections by rapid detection, infection-control programs and prevention of transmission of drug resistance.


INTRODUCTION
Klebsiella pneumoniae is an important causative agent of both hospital-acquired and community-acquired infections such as pneumonia, urinary tract infections, meningitis, and septicemia 1 .Multi-drug resistant (MDR) strains can be quite problematic, especially for elderly or immunocompromised patients and infants with an immature physiology 2 .Release of β-lactamases is a significant resistance mechanism against antimicrobial agents 3 .β-lactamase-producing K. pneumonia can degrade a wide range of β-lactam antibiotics such as penicillins, carbapenems, cephalosporins, and cephamycins 2,4 .These enzymes can be divided into four classes (A, B, C, and D) based on the Ambler classification.The temoneira (TEM), cefotaximase (CTX-M), sulfhydryl variable (SHV), Vietnam extended-spectrum β-lactamase (VEB), Pseudomonas extendedresistant (PER), and Guiana extended-spectrum (GES) enzymes belong to class A; the Verona integron-encoded metalloβ-lactamase (VIM), imipenem (IMP), and K. pneumoniae carbapenemase (KPC) enzymes belong to class B; and oxacillin hydrolyzing enzyme (OXA) is classified as class D according to the Ambler classification [5][6][7] .Researchers have reported that the incidence of β-lactamase-producing K. pneumonia ranges from 6 to 88% at different health care locations 8 .Bla SHV β-lactamases are related to high level ceftazidime resistance, but not to cefazolin or cefotaxime resistance, while bla CTX-M β-lactamases are more effective against cefotaxime.In contrast, TEM β-lactamases confer resistance against oxyimino-βlactams groups such as ceftazidime, cefotaxime, and aztreonam.In addition to β-lactamase encoding plasmids, transportable genetic elements such as integrons can also contribute to the evolution and distribution of MDR genes (bla TEM , bla CTX-M, bla SHV, bla VEB , bla PER , bla GES , bla VIM, bla IMP, bla OXA , and bla KPC ) in K. pneumoniae by vertical or horizontal transmission 9,10 .Five classes of integrons have been proposed based on the amino acid sequences of Int I proteins.Three classes of antibiotic resistance integrons (ARIs; I, II, and III), identified based on particular integrase genes 11 , are usually associated with MDR phenotypes.The transportable class I integron is related to transposon Tn21 and is commonly observed in β-lactamaseproducing clinical isolates of K. pneumonia 12 .Class II integrons are detected less frequently in bla KPC -producing bacteria, such as K. pneumoniae and Escherichia coli, and class III integrons are detected quite infrequently in β-lactamase-producing K. pneumoniae 13 .Previous reports have demonstrated the production of various β-lactamases, such as bla-ESBL, and resistance to several antibiotics groups via ARI gene carriage in clinical isolates of K. pneumonia 14 .Unfortunately, the incidence of β-lactamase-producing K. pneumoniae is on the rise 15 .The detection of different β-lactamase genes in resistant bacteria and characterization of their antimicrobial susceptibility profiles could provide important data regarding high risk factors and infection epidemiology [16][17][18] .To date, only a few studies have investigated the types of β-lactamase-producing Enterobacteriaceae and strains possessing integrons present in Iranian hospitals 19,20 .Thus, the aim of the present study was to determine the prevalence of bla TEM , bla CTX-M, bla SHV, bla VEB , bla PER , bla GES , bla VIM, bla IMP, bla OXA , and bla KPC , as well as int genes (I, II and III) in clinical K. pneumonia strains isolated from two large urban university general hospitals in Tehran, Iran using multiplex-polymerase chain reaction (M-PCR).

METHODS
This cross-sectional study was conducted from April 2014 to March 2015, at two teaching hospitals in Tehran, Iran.One hundred non-repetitive K. pneumonia isolates were obtained from different clinical specimens including blood, skin lesions, broncho-alveolar lavage (BAL), urine, sputum, cerebrospinal fluid (CSF), pus, pleural effusion, ascites, and catheter specimens.Each sample was cultured on MacConkey agar (Merck, Darmstadt, Germany) and incubated at 37°C for 24h.Resulting colonies were identified as K. pneumonia using standard biochemical and microbiological tests, including urease, oxidase, motility, citrate utilization, Triple sugar iron agar (TSI), Methyl Red-Voges Proskauer (MR-VP), and Sulfide Indole Motility (SIM), and were further confirmed with the API 20E system (Analytab, Inc., New York).
Multiplex-PCRs were performed to detect β-lactamase genes (bla TEM , bla CTX-M, bla SHV, bla VEB , bla PER , bla GES , bla VIM, bla IMP, bla OXA , and bla KPC ) and int genes (I, II, and III) using a master cycler gradient (Eppendorf Co., Germany).Genomic deoxyribonucleic acid (DNA) was extracted from K. pneumoniae colonies grown overnight on blood agar (Merck Co., Germany) plates using the boiling method 14 .Briefly, a loopful of bacteria from a colony was suspended in 700µl sterile distilled water, boiled for 10 min, centrifuged at 7,000×g for 4 min at 4°C, cooled on ice for 10 min, and then centrifuged for 3 min at 8,000×g.The concentration and quality of the extracted cellular DNA were assessed using a Nanodrop spectrophotometer (ND-1,000; Thermo Scientific; Wilmington, DE, USA).The β-lactamase and integron genes were amplified by M-PCR using specific primers detailed in Table 1.M-PCR was carried using 1.5µl of extracted genomic DNA in a 25µl PCR reaction mixture consisting of 2.5µl 10× PCR buffer, 1.5µl MgCl 2 (50mM), 0.5µl dNTPs (10mM), 1.5µl of each primer, 0.5µl of Taq DNA polymerase (5U/µl; Amplicon Co., Denmark), and 15.5µl sterile distilled water.M-PCR was performed under the following conditions: denaturation at 94°C for 1 min; 35 cycles of denaturation at 94°C for 30s, annealing at 59°C for 30s, and extension at 72°C for 1 min; and a final extension at 72°C for 6 min.For amplification of the int genes (I, II, and III), the reaction mixture was amplified using a thermal gradient cycler (Eppendorf Co., Germany) with the following PCR protocol: one cycle of 5 min at 95°C; 30 cycles of 1 min at 95°C, 1 min at 65°C, and 1 min at 72°C; and one cycle of 10 min at 72°C 14 .

DISCUSSION
β-lactamase-producing K. pneumoniae was first identified in 1983 21 .Most infections caused by K. pneumoniae are due to multi-drug resistant strains such as β-lactamase producing isolates 22 .Recent studies have shown that the incidence of β-lactamase-producing K. pneumoniae is increasing in several countries such as Iran 22,23 , India 24,25 , and Italy 26 .Resistance to various antibiotics is related to the existence of transmissible plasmids and integrons, which can be integrated into plasmids or the chromosome 27 .These transmissible elements often contain resistance factors that can be transferred to other microorganisms.In this study, we examined the susceptibility of 100 clinical K. pneumonia strains against thirteen antibiotics; high resistance was observed for AK (93%), TS (84%), AMP (87%), AZT (59%), GEN (67%), and FEP (52%).Amiri et al. 28 reported that the resistance to ampicillin, ceftazidime, ceftriaxone, aztreonam, and cefotaxime was 92%, 67%, 65%, 64%, and 59%, respectively in K. pneumoniae isolates 28 , values similar to the rates reported in this study.Our results indicate that only eight β-lactamase-producing isolates were resistant to imipenem using the disk diffusion method.This high (83%) susceptibility to imipenem is in agreement with the reports of Mansury et al. 29 , Ahmad et al. 30 , Amiri et al. 28 , and Edelstein et al. 31 .Only three K. pneumoniae isolates were resistant to all antibiotics tested.Antibiotic resistance rates of non-β-lactamase-producing and β-lactamase-producing Klebsiella pneumoniae strains.Multi-drug resistant (MDR) strains are defined as strains resistant to three classes of antimicrobial agents 32 ; therefore, 31% of our isolates can be classified as MDR.This finding contrasts those reported by Mansury et al. 29 .A total of 52% and 67% of our isolates were resistant to the third generation cephalosporins ceftazidime and cefotaxime, respectively, which is similar to the rates reported by Ullah et al. 33 , Amiri et al. 28 , and Jalalpoor et al. 34 .Of the MDR isolates, 28 strains were β-lactamase-positive (28%).These results are in agreement with those of Shukla et al. 35 and Sarojamma et al. 36 who reported that 28% and 32% of their strains were β-lactamase producers, respectively 35,36 .The incidence of β-lactamase-producing Klebsiella spp.has been reported to vary from 42-44% (in the USA) [37][38][39] , 4.9% (in Canada) 40 , 20.8% (in Spain) 41 , 28.4% (in Taiwan) 42 , 78.6% (in Turkey) 43 , 20% (in Algeria) 44 , and 51% (in China) 45 .In this study, the highest percentage of β-lactamaseroducing strains was derived from urine samples (14%).
Ahmed et al. 49 reported that the prevalence of bla PER was 22.4%; however Nasehi et al. 20 , detected bla PER in only 7.5% of their isolates, which is similar to the results of this study.Borges-Cabral et al. 50, reported that bla KPC was present in 41.7% of their isolates; however, Bina et al. 51 did not observed bla KPC (0%) in any of their strains and the rate in the present study was 23%.Limbago et al. 52 observed the bla IMP gene in all of their clinical K. pneumoniae isolates; however, we did not detect this gene in any of our isolates.Udomsantisuk et al. 53 reported that the frequency of the bla VEB gene was 30% among β-lactamase-positive K. pneumoniae strains; however, in the current study, the frequncy of bla VEB gene was 12%.Iraz et al. 54 reported that 86% of the carbapenem-resistant K. pneumoniae strains carried the bla OXA gene; however, Charrouf et al. 55 found that only 6% of their isolates carried this gene.Our results did not match either of these studies; in our study, the prevalence of this gene was 28%.Psichogiou et al. 56 found that the frequency of the bla VIM gene in clinical K. pneumoniae strains was 37.6%, which is consistent with our results (33%).This reflects a significant increase in the prevalence of bla TEM and bla VIM in Iran.In comparison, the major β-lactamase gene found in Arab countries appears to be bla CTX-M 57-59 .
In addition to β-lactamase genes, we also evaluated integron gene prevalence in the 100 K. pneumoniae isolates.Our results indicate that only eight isolates were positive for class I integrons, while class II and class III integrons were not detected in any of the isolates.This finding is comparable to those of Lima et al. 60 and Ashayeri et al. 61 .Class III integrons have been reported only in very few studies 14,62,63 .Mobarak-Qamsari et al. 64 identified 22 (44%) class I integron-carrying K. pneumoniae isolates, but only three (6%) of the isolates had class II integrons and none contained class III integrons 64 .These findings suggest that class I integron genes may play a critical role in the distribution of β-lactamase-encoding genes among clinical β-lactamase-producing K. pneumoniae isolates.The increase in multidrug resistance and the underlying mechanisms require further investigation In conclusions, our study demonstrates that there is a high level of bla TEM, bla VIM , and class I integrons in the β-lactamaseproducing K. pneumoniae strains circulating in hospitals in Tehran, Iran.This trend of MDR profiles associated with the presence of bla TEM, bla VIM , and class I integron genes is worrying.The high prevalence rate of these resistance genes highlights the necessity for establishing a national antibiotic susceptibility surveillance network for monitoring infections due to Enterobacteriaceae spp. in Iran.It seems that these properties help to decrease treatment complications and mortality rate due to resistant bacterial infections by rapid detection of β-lactamases genes, infection-control programs and prevention of transmission of drug resistant-strains.A combination therapy can be useful to prevent resistance during therapy resulting in complete remission of patient and resistant infections control.One of the limitations of the present study was that, other β-lactamases family genes and also other antibiotic resistance mechanisms were not assessed due to the financial constraints of molecular and gene tests.So, further investigations are needed to obtain more accurate and effective results.