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Indian Journal of Medical Microbiology, Vol. 27, No. 1, January-March, 2009, pp. 51-54 Brief Communication SHV-28, an extended-spectrum β-lactamase produced by a clinical isolate of Klebsiella pneumoniae in south India Jemima SA, Verghese S Department of Microbiology, International Centre for Cardio Thoracic and Vascular Diseases, A Unit of Frontier Lifeline, Chennai-600 101, Tamil Nadu Date of Submission: 15-Feb-2008 Code Number: mb09012 Abstract SHV-28, an extended spectrum β-lactamase from a clinical isolate of Klebsiella pneumoniae , had an isoelectric point of 7.6 and a substrate profile showing preferential hydrolysis for cefotaxime over ceftazidime. It differed from SHV-1 by one amino acid substitution. The conserved S-T-F-K and K-T-G motifs were identified by SHV-28 protein sequencing.Keywords: Extended-spectrum β-lactamase, SHV-28, K. pneumoniae Extended-spectrum β-lactamases (ESBLs) are enzymes that confer resistance to oxyimino-β-lactams such as cefotaxime (CTX), ceftazidime (CTZ), ceftriaxone and the monobactam, aztreonam but not to cephamycins or carbapenems.[1] ESBLs, which are predominantly derivatives of plasmid-mediated TEM or SHV β-lactamases, arise through mutations that introduce one or more amino acid substitutions that alter the configuration or binding properties of the active site, resulting in an expansion of the substrate range of the enzymes. [2] ESBL-producing clinical isolates are frequently associated with nosocomial outbreaks, with production detected most commonly in Klebsiella pneumoniae in addition to other members of the Enterobacteriaceae family and Pseudomonas aeruginosa . [3] The prevalence of chromosomally encoded SHV β-lactamase was reported worldwide during 2000 and 2001.[4],[5] The product of the first sequenced chromosomal β-lactamase gene from K. pneumoniae was designated LEN-1. Recent data suggest that chromosomally encoded SHV-1 may be more prevalent than LEN-1 in clinical K. pneumoniae isolates. [5] SHV-type ESBLs currently predominate in surveys of resistant clinical isolates in Europe and America. [6] Here, we describe an ESBL, designated SHV-28, produced by a clinical isolate of K. pneumoniae obtained from the urine of a patient in Chennai, South India, in 2006. It differed from SHV-1 β-lactamase by one amino acid substitution. Materials and Methods Case history The patient was a 65-year-old male patient who had undergone coronary artery bypass graft surgery. An isolate of K. pneumoniae was obtained the from urine sample of the patient. The organism was found to be highly resistant to penicillins, aminoglycosides, quinolones and cephalosporins, including third-generation cephalosporins such as CTX, CTZ, cefaperazone and certrioxoine. The organism was susceptible to carbapenems, both imipenem and meropenem. MIC testing and confirmation of ESBL activity Organisms were tested by agar dilution method as described in the CLSI standard M100-S15. [7] Escherichia coli ATCC 25922 and K. pneumoniae ATCC 700603 were used as quality control strains. The double-disk synergy test for the detection of ESBL activity was performed essentially as recommended by the CLSI M100-S15 by screening for synergism between clavulanate (represented by a disk of amoxicillin-clavulanic acid) and CTZ and CTX. [7] The disks were placed at distance of 15-20mm (centre to centre). A potentiation of the inhibitory zones of any of the expanded spectrum β-lactams by clavulanate was considered suggestive of ESBL production. β-lactamase assays Bacteria exponentially growing at 37°C in Luria-Bertanii medium were harvested and cell-free lysate was prepared by the lysozyme and EDTA treatment method. [8] Analytical isoelectric focusing was performed using broad range soluble ampholytes (Biorad, Hercules, CA, USA) covering a pH range of 3.5-10. β-lactamases were visualized by overlaying gels with filter paper moistened with a 0.25mg/mL solution of nitrocefin (Oxoid, UK) in 0.1 M sodium phosphate buffer (pH 7.0) containing dimethylsulphoxide (1%v/v). The intact substrate molecule is yellow, but becomes pink when the β-lactam bond is broken such that the focused bands with β-lactamase activity appear pink on a yellow background. Molecular analysis The presence of bla SHV resistance genes was detected by PCR. A 1052-bp PCR product that included the bla SHV structural gene was amplified from K. pneumoniae with oligonucleotide primers using the template DNA. [9] Cycling parameters for the amplification of the bla SHV gene included a 5-min denaturation at 96°C followed by 35 cycles of denaturation (96°C for 1 min), annealing (55°C for 1 min) and extension (72°C for 1 min) and ending with a final extension period of 72°C for 10 min. Direct sequencing of PCR products PCR products were used as templates for nucleotide sequence determination. A fragment of 1052 bp within bla SHV was amplified. The amplification products were purified with the Ultra Clean PCR purification kit (Mo bio Lab, CA, USA) and their nucleotide sequences were sequenced in an automated sequencing machine at the Medical Research Foundation, Tamil Nadu, India. Nucleotide sequence determinations were performed on both DNA strands and on two independently generated amplimers. Nucleotide sequence accession number The DNA sequence and deduced amino acid sequence of SHV-28 has been deposited in GenBank and assigned the accession number EU441172. Results Antimicrobial susceptibility In the disc agar diffusion test, the isolate producing ESBL was resistant to all β-lactams tested, which included cefazolin, cefuroxime, CTX, CTZ, cefoparazone and certrioxoine. The isolate showed a very high MIC to CTZ of > 1024mg/mL and the MIC to CTX was 1024 mg/mL. There was a strong synergistic effect observed when the combination drugs were used, with CTZ-CLA showing an MIC of 256 μg/mL and CTX-CLA showing an MIC of 128 μg/mL. When the discs were placed 15-20 mm apart, a synergistic effect was observed due to resistance of the isolate to β-lactam antibiotics alone and in combination with β-lactam inhibitors like clavulanic acid. Isoelectric focusing The isolate producing an SHV-28 ESBL was analysed for its β-lactamase content. The pink band was produced at 7.6. No additional band of activity was detected with chromogenic cephalosporin as a substrate. Identification of the SHV-encoding gene by PCR and DNA sequencing By using SHV-specific primers, a DNA fragment of 1052 bp was amplified from the sample of the isolate using colony PCR. Sequence analysis revealed that the SHV gene showed a 100% similarity [Figure - 1] with SHV-28 (GenBank AF299299). Sequence analysis of the SHV-28 β-lactamase gene in K. pneumoniae The SHV-28 gene comprises an initiation codon ATG (positions 57-59) and the stop codon TAA (positions 915-917), which translates into a protein of 286 amino acids [Figure - 1] Within this protein, two highly conserved motifs, serine-threonine-phenyalanine-lysine tetrad (S-T-F-K) at positions 66-69 and lysine-threonine-arginine (K-T-G) at positions 230-232 were found. This included the conserved serine and lysine residues characteristic of β-lactamases possessing a serine active site. The amino acid sequence of SHV-28 differed from the amino acid sequence of SHV-1 by one amino acid substitution: tyrosine to phenylalanine at position 3. Discussion SHV-28 was reported at the Southwest Hospital of the Third Military Medical College in China in 2002 (GenBank AF538324), and it was the first report of SHV-28. Kim et al. reported the presence of SHV-28 in two strains in Korea. [10] SHV-28 was also demonstrated by Tofteland et al. in Norway in 2006. [11] Ndugulile et al. reported the presence of SHV-28 in Africa for the first time in 2005. [12] In the present study, the patient had undergone a catheterization and had an urinary tract infection subsequently.The urine sample was collected on the 4th post-operative day during his stay in the intensive care unit. On Gram′s staining, the urine showed > 25 pus cells/low-power field, with numerous Gram-negative bacilli and moderate budding yeasts with pseudohyphae. It showed a significant growth of K. pneumoniae and Candida sps, both in excess of 1,00,000 colonies/mL. The K. pneumoniae was identified as an ESBL producer and it was only susceptible to carbapenems. The patient was treated with Imipenem 0.5 g thrice a day for 5 days and fluconazole 200 mg once daily for 5 days. Many of these patients are particularly vulnerable to infection as they are on multiple invasive lines that prove to be an access to bacteria. SHV-28 has not been previously reported from India. To the best of our knowledge, this is the first report of the detection of SHV-28 from India. The DNA sequence of the gene showed 100% homology to that of the gene detected from K. pneumoniae that was isolated at China (GenBank AF299299). The gene, encoding β-lactamase SHV-28, detected in this study is a genotype that differs from SHV-1 only by a single amino acid substitution (tyrosine to phenylalanine) at position 3 of SHV-1. It is very important to prevent resistant bacteria by correctly identifying ESBL-producing K. pneumoniae and treating infected patients with appropriate antibiotics. The intensive use of penicillin-inhibitor combinations in the hospital settings and in the treatment of community-acquired infections may facilitate the sporadic appearance of enzymes like the one described here. Studies from other parts of the world reported that the SHV-5 gene was common in K. pneumoniae i solates. [13] Similar studies need to be performed in different parts of our country to know the genotypes of ESBL enzymes in a particular geographical area for epidemiological purpose References
Copyright 2009 - Indian Journal of Medical Microbiology The following images related to this document are available:Photo images[mb09012f1.jpg] |
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