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Indian Journal of Medical Microbiology, Vol. 28, No. 3, July-September, 2010, pp. 207-210 Original Article Observation on integron carriage among clinical isolates of Klebsiella pneumoniae producing extended-spectrum β-lactamases A Bhattacharjee, MR Sen, P Prakash, A Gaur, S Anupurba, G Nath Department of Microbiology, Institute of Medical Sciences (IMS), Banaras Hindu University (BHU), Varanasi - 221 005, India Keywords: Extended-spectrum β-lactamase, integron, Klebsiella Introduction The rising trend of multidrug resistance in microbes is a serious problem worldwide. Conjugational transmission of antibiotic resistance genes across bacterial species and genera has amplified the problem of antibiotic resistance in pathogens. The mobile genetic elements like plasmids and transposons, which are reported to contain genetic units named integrons, contain genes for site-specific recombination systems that are capable of capturing and mobilizing genes called gene cassettes. [1] They are characterized by the presence of an intI gene encoding an integrase, a recombination site (attI), and a promoter. Integrons are divided into two major groups: the resistant integrons (RI) and the super-integrons (SI). Of the three resistant integron classes, class 1 integron is the commonest; this is followed by class 2, while class 3 is rare. [2],[3] Klebsiella pneumoniae is implicated as an important pathogen causing nosocomial and community-acquired infections and is often associated with the production of extended-spectrum β - lactamases (ESBL) belonging to the SHV and CTX-M families. Although there could be independent existence of both the gene cassette and the ESBL gene in a particular organism, ESBLs have been described as a part of complex integrons, which facilitate their horizontal transfer to other related, as well as unrelated, microbes. [4] The present study was undertaken to investigate the occurrence and characterization of integrons among K pneumoniae isolates producing ESBL in a tertiary referral hospital of North India. Materials and Methods Clinical isolates A total of 136 consecutive nonduplicate isolates of K pneumoniae were collected from various clinical specimens of the patients seeking indoor and outdoor services of our tertiary referral hospital during August 2007 to January 2008. All the isolates were confirmed by standard biochemical tests. Antimicrobial susceptibility testing Susceptibility testing was performed against β-lactam and non-β-lactam antibiotics, namely ampicillin (10 μg), amikacin (30 μg), cefepime (30 μg), cefoxitin (30 μg), ceftizoxime (30 μg), cefuroxime (30 μg), cephalexin (30 μg), ciprofloxacin (5 μg), gentamicin (10 μg), imipenem (10 μg), kanamycin (30 μg), netilmicin (30 μg), piperacillin (100 μg), and trimethoprim/sulfamethoxazole (1.25/23.75 μg) (Hi-Media, Mumbai, India). E coli ATCC 25922 was taken as control. Phenotypic detection of ESBL All the isolates were tested by combined disc diffusion and MIC reduction methods after an initial screening following the CLSI guidelines. [5] Characterization of ESBL gene ESBL genes were characterized by performing multiplex polymerase chain reaction (PCR) using primers for blaTEM , blaSHV , blaCTX-M 1, 2, 9 , blaOXA-2 , and blaOXA-10 . PCR conditions and the primers used were as described previously. [6] Detection of class 1 and class 2 integrons by integrase gene PCR PCR was performed using primers Int 1F and Int1R to amplify a 160-bp fragment of class 1 integrase and Int 2F and Int 2R to amplify a 288-bp fragment of class 2 integrase. The primers, PCR conditions, and reaction mixtures used were as described previously. [6] Typing of strains by random amplification of polymorphic DNA Random amplification of polymorphic DNA (RAPD) was performed using primer 7 (GTGGATGCGA) [7] (Metabion, Martinsried, Germany). Each single reaction mixture contained 1 μg of template DNA, 10 mM dNTPs, 2U Taq DNA polymerase (Genei, Bangalore, India), 25 mM MgCl 2 , and 10X reaction buffer supplied by the manufacturer of the enzyme. Biometra thermal cycler (Biometra, Goettingen, Germany) was used and the reactions were run under the following conditions: initial denaturation at 94°C for 5 minutes; 40 cycles at 94°C for 1 minute, 30°C for 1 minute, 72°C for 1 minute; and a final elongation at 72°C for 7 minutes. The isolates were typed according to their band patterns, which were further analyzed by using NTSYS software, and grouped into different clusters. Analysis of plasmid Plasmid was isolated and purified using QIAGEN® Plasmid Mini Kit (Germany) from all the integron-positive isolates (n = 58). An 18-kb plasmid that was common to all isolates was selected and was transformed into the recipient strain E coli JM107 using TransformAid™ (Fermentas, USA). The transformants were selected on Mueller-Hinton Agar containing cefotaxime at 1 μg/mL concentration. The recipient strains were further subjected to PCR detection for the presence of ESBL genes and integrons. The primers and condition for the reactions were same as before. [6] Results Out of 136 K pneumoniae isolates, 63 (46%) were confirmed to be ESBL producers. On characterizing the ESBL gene by multiplex PCR, most of the isolates harboured SHV gene (68%, 43/63), followed by CTX-M (67%, 42/63) and TEM (14%, 9/63), while 8 (13%) of the isolates did not show any amplification with the primers used in this study. A total 48% isolates (30/63) harboured more than one type of gene [Table - 1]. Of the 63 ESBL-positive isolates, 58 (92%) carried class 1 integron, while 6 (9%) isolates harboured class 2 [Table - 2] integron as well class 1 gene cassette [Figure - 1]. Of the 73 non-ESBL producers, only 29 (40%) were positive for integron class 1 and none for class 2. On RAPD analysis of K pneumoniae, eight types were found, which could be arranged in six (A to F) clusters. Type A with 15 and D3 with 14 strains were the groups containing the highest number of strains. On analyzing plasmids of the 58 isolates, integrons were detected in plasmids of 56 isolates (class 1 in 55, class 1 and 2 in 1). Among these we found 46 isolates harbouring the β-lactamase gene in their plasmids. All the TEM enzymes were found to be plasmid mediated and 27 isolates were carrying more than one type of β-lactamase genes in their plasmids. The antibiogram showed that all the ESBL-producing isolates were susceptible to imipenem, 68% were sensitive to amikacin, and 67% to netilmicin; the susceptibility to other antibiotics was moderate or poor [Table - 3]. Discussion Integrons play an important role in the dissemination of antimicrobial resistance through horizontal transmission. Their contribution to the prevalence of transferable extended-spectrum cephalosporin resistance has been demonstrated very recently. [8] In the present study we observed a high frequency of occurrence of integrons among ESBL-positive K pneumoniae (92%), which corroborates well with the previous Asian report. [9] In contrast to this, only 73%, 70%, and 38% of K pneumoniae strains from Australia, the US, and Europe, respectively, have been reported with integron. [8],[10],[11] Indiscriminate use of cephalosporins and quinolones, a weak infection control program, and the absence of a proper antimicrobial policy in the tertiary care setup are the possible factors for the increased detection of integrons in the Asian isolates. In our study, the simultaneous presence of integrons and β-lactamase genes in the same plasmids indicates a possible association between the two. We have reported the presence of class 2 integrons in six K pneumoniae isolates, which is rare and is being described for the first time in this species that were producing ESBL. However, presence of class 2 integrons in ESBL-producing E coli (25%) has been reported from Spain. [3] In this study, the isolates harbouring class 2 integrons were resistant to most of the antibiotics (the exception being imipenem). These isolates belonged to RAPD type A, C, D3, and F [Table - 3]. Further, class 2 integron carriage among the ESBL-producing K pneumoniae were found to be associated with surgical (urology) inpatients of a particular ward. RAPD analysis shows that three β-lactamase gene families were equally prevalent in all the types. Beside this, isolates of a single RAPD type that were carrying same β-lactamase gene differed in their antibiogram profile, which justifies the conjugational transfers of drug-resistant determinants. Examination of the ESBL genotypes and antibiograms of the K pneumoniae isolates indicates that strains with similar antibiograms and genotype are being circulated in the hospital environment; this is evident from the demonstration of class 2 integron-positive ESBL isolates with similar antibiogram patterns [Table - 2]. The implementation of a proper infection control program is necessary to prevent or slow down the transmission of the said genotype. Among non-ESBL-producing K pneumoniae, 40% of the isolates showed integron positivity; a similar result was obtained in an earlier study. [3] However, this could be explained by the possible integration of other drug-resistance determinants in these ESBL-negative isolates. It was evident in the current study that integron positivity was significantly higher among those bacterial strains which were found to be ESBL positive than among those that were ESBL negative, i.e., 58/63 vs 29/73 (z = 6.34, P < 0.001). This finding strongly suggests that integrons have a role in the horizontal transfer of ESBL-mediated resistance among nosocomial isolates of K pneumoniae. References
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