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Indian Journal of Medical Microbiology, Vol. 28, No. 4, October-December, 2010, pp. 332-336 Original Article Real-time polymerase chain reaction for rapid detection of genes encoding SHV extended-spectrum β-lactamases MS Alfaresi, AA Elkoush Department of Pathology & Laboratory Medicine, Zayed Military Hospital, PO Box 3740, Abu Dhabi, United Arab Emirates Date of Submission: 08-May-2010 Code Number: mb10100 PMID: 20966564 Abstract Purpose: This study aimed to develop an improved method for the detection of bacterial SHV-type extended-spectrum β-lactamases (ESBLs). Keywords: Real-time PCR, SHV, extended-spectrum β-lactamase Introduction Extended-spectrum β-lactamases (ESBLs) have spread threateningly in many regions of the world and presently comprise over 300 variants ( http://www.lahey.org/Studies ). A typical characteristic of ESBLs is their ability to hydrolyse oxyimino cephalosporins and aztreonam, while being inhibited by β-lactamase inhibitors. [1],[2] ESBL-producing organisms have achieved notoriety for causing nosocomial infections. [2],[3] SHV-type ESBLs evolved from the SHV-1 enzyme common to Klebsiella pneumoniae and are coded by genes found on both the bacterial chromosome and on plasmids. They have migrated into Citrobacter spp., Escherichia coli, and Pseudomonas aeruginosa, among others. [3] SHV-1-derived ESBLs are a less diverse collection of enzymes than the TEM family. The majority of SHV-1-derived ESBLs possess a serine instead of a glycine residue at position 238. The substitution at residue 238 is often paired with a second mutation at position 240 (lysine for glutamic acid); substitutions at residues 156 (glycine to aspartic acid) and 179 (aspartic acid to glycine, asparagine, or alanine) can also lead to the ESBL phenotype. [4],[5],[6] More than 100 SHV derivatives have been reported; however, not all of the SHV derivatives described are ESBLs (http://www.lahey.org/Studies/). Methods for the rapid detection and identification of ESBLs are essential in studying the epidemiology of antimicrobial-resistant bacteria. Delayed treatment of infections caused by organisms that produce ESBL is associated with increased mortality. [7] However, despite considerable effort for over a decade, laboratory detection of ESBL production remains problematic. [2],[3],[8],[9],[10],[11],[12] The presence of these enzymes does not always elevate minimum inhibitory concentrations (MICs) of oxyimino cephalosporins and monobactams to levels indicative of resistance as defined by the Clinical Laboratory Standards Institute (CLSI). [13] For research and epidemiological studies of SHV and TEM derivatives, several molecular methods are available, including polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis, [14],[15] oligotyping, [16] PCR single-strand conformational polymorphism, [14],[17] and ligase chain reaction. [18],[19] Nucleotide sequencing remains the standard method for the determination of specific β-lactamase genes in bacterial isolates. Such methods are not appropriate for routine detection of ESBL production in clinical settings.[20],[21] In contrast, real-time PCR-based detection of ESBL producers offers the potential for faster diagnosis and earlier procurement of epidemiological information for outbreak control. Randegger and Hδchler reported a rapid and sensitive method, termed SHV melting-curve mutation detection (MCMD), for detection of mutations in three codons at positions 179, 238, and 240 of the bla SHV gene in a single reaction. In this study, we describe a real-time PCR method to detect the bla SHV gene in clinical isolates. Materials and Methods Bacterial strains and susceptibility testing We studied 662 unique stains of Enterobacteriaceae isolated from patients. The genera were identified as E. coli and K. pneumoniae. All of the strains were isolated between 1 January 2008 and 31 December 2009 from patients in different units at our hospital, a 260-bed tertiary care government hospital. Identification of bacterial species was performed using an automated identification and microdilution system (VITEK, bioMeriux, Mercy I'Etoile, France) and an overnight ESBL panel. For each isolate, the MICs of the broad-spectrum cephalosporins such as cefotaxime, ceftazidime, and cefepime, the monobactam antibiotic aztreonam, and the ureidopenicillin piperacillin were determined. The results were recorded and interpreted according to the Clinical Laboratory Standards Institute (CLSI) guidelines. Determination of the ESBL-producing phenotype The ESBL-producing phenotypes of 662 isolates were determined using an AST-GN27 VITEK ESBL card (VITEK, bioMerieux) according to the manufacturer's instructions. E. coli ATCC 25922 and K. pneumoniae ATCC 700603 were used as negative and positive controls, respectively, for ESBL production. Design of the consensus primer pair and probe Sequence information and characterisation of most of the known types of bla SHV β-lactamases was retrieved from the literature and associated databases. Based on the overviews of the Lahey Clinic, Burlington, MA (www.lahey.org) and the resources at NCBI (www.ncbi.nlm.nih.gov), the bla SHV sequences were aligned using the Geneious 4.6 software package (Biomatters, Auckland, New Zealand) and the most conserved regions were identified. Primers and a black hole quencher (BHQ) probe were designed based on these conserved domains [Figure - 1]. In principle, this approach was designed to amplify and identify all known types of lactamases that carry the conserved motifs [Table - 1]. DNA extraction and real-time PCR assay A loopful of bacteria harvested from an agar plate was suspended in 50 μl of sterile water. Bacterial DNA extraction was performed using the QIAMP DNA MINI kit (QIAGEN, Dueseeeldorf, Germany). A final DNA concentration of 2-20 ng/μl was used in the PCR reactions. Once optimal reaction conditions had been determined empirically on the Rotor-Gene 3000 apparatus (Corbett research, Sydney, Australia), the PCR assay was run with all ESBL-producing clinical isolates, the ATCC 700603 K. pneumoniae as a positive control, E. coli ATCC 25922 as a non-ESBL-producing control, and water as a non-template control. Each 25 μl reaction tube contained 5 μl of extracted DNA, 12.5 μl of Rotor-Gene Multiplex PCR Master Mix (QIAGEN), 0.5 μM of the forward and reverse primers, 0.2 μM of the BHQ probe, and 6 μl of RNase-free water. Cycling conditions were: 5 minutes PCR initial activation step at 95ºC, 30 cycles of 15 seconds at 95ºC, and 15 seconds at 60ºC for a single fluorescence reading on the ROX channel. Real-time data were analysed using Rotor-Gene software. DNA sequencing All 16S rRNA gene PCR-positive DNA extracts were screened for the presence of bla TEM, bla SHV, and bla CTX-M sequences using consensus PCR primers [22] synthesised by Eurofins MWG, Germany. Standard QIAGEN PCR mixtures were used with the HOTSTART Taq master mix kit (QIAGEN) as previously described (26) in a Rotor-Gene 3000 PCR system. Bidirectional sequencing was performed using a BigDye v. 3.1 cycle sequencing kit and a model 3100 genetic analyzer (Applied Biosystems, CA, USA). Editing and alignment of DNA sequences were performed with the Geneious 4.6 software package (Biomatters). Results Isolation, selection, and antibiotic susceptibility We studied 662 ESBL carrier samples isolated from 662 patients in different medical care units of the hospital. The majority of strains were isolated from urine specimens; others were from blood, tracheal/bronchial aspirate, wound swabs, sputum, and ear discharge. A total of 240 (36%) strains that produced ESBL were identified using phenotyping methods. These 240 ESBL producers included 150 (62%) species of E. coli and 90 (38%) species of K. pneumoniae. All 240 ESBL-producing strains were resistant to the extended-spectrum cephalosporins, cefazolin and cefotaxime (95% of the MICs were >64 mg/l), but they retained susceptibility to imipenem. A total of 50% of the strains were resistant to gentamicin, and 50% were resistant to trimethoprim-sulfamethoxazole. DNA sequencing and distribution of bla genes Of the 150 (94%) ESBL-positive E. coli isolates, 141 carried the bla CTX-M15 gene, while nine carried the bla TEM gene. Of the 90 (97%) ESBL-positive K. pneumoniae isolates, three carried the bla SHV-1 gene, and 26 carried the bla SHV-28 gene. The other 61 strains carried the bla CTX-M15 gene. Real-time PCR detection The new real-time PCR assay correctly detected the positive control and all of the bla SHV ESBL-producing isolates that were previously sequenced and typed. Those isolates included 26 K. pneumoniae isolates that carried the bla SHV-28 gene and three that carried the bla SHV-1 gene. No PCR products were observed in the negative control, water control or the CTX-M or TEM clinical isolates [Figure - 2]. Based on these results, the assay displayed 100% sensitivity and 100% specificity for detection and discrimination of bla SHV ESBL and bla SHV non-ESBL genes in standard and clinical isolates. Additionally, monitoring by gel electrophoresis revealed that the amplification products were of the expected length (402 bp) and were absent from the negative controls and negative isolates. Discussion Among the Enterobacteriaceae, ESBL production is the major emerging mechanism of resistance to expanded-spectrum cephalosporins and monobactams and is a matter of major concern in the field of microbial drug resistance. It is likely that the ESBLs produced by this family of organisms will become increasingly complex and diverse in the future, which will create increasing challenges with respect to the creation of guidelines for the detection of ESBLs in the clinical microbiology laboratory. Indeed, in a recent study, it was estimated that up to 33% of ESBLs in Europe go undetected. [23] In the early studies on ESBLs, isoelectric focusing analysis was often used to characterise β-lactamases. However, many β-lactamases possess identical isoelectric points; further, the determination of isoelectric points can be equivocal and technically demanding. For these reasons, specific identification of ESBLs by isoelectric focusing analysis may not be efficient. This problem prompted the National Committee for Clinical Laboratory Standards to establish a working group to address the problem. Although the activities thus evoked led to improved recommendations, the principal problems of synergy testing, limited sensitivity and a requirement for overnight incubation remained. Recently, a number of molecular biological methods have been proposed for the identification of SHV derivatives. [3],[24] The disadvantages of these molecular biology based methods, which include labour expenditure, cost, and lack of general applicability, have outweighed their advantages and have so far prevented their broad acceptance. Real-time PCR allows the non-laborious, reliable detection and quantification of most nucleic acid target sequences. Here, we present a rapid method for using real-time technology to detect bla SHV ESBL and bla SHV non-ESBL genes in standard and clinical isolates. The method works perfectly with single copy DNA templates obtained from extracts of bacterial colonies on plates. Moreover, this method is performed in a closed system and requires no post-amplification manipulations such as restriction digestion or electrophoresis. Consequently, the results are available within 1 hour. The assay described in this study was validated and optimised in several ways. These included choosing the best primers and probe to detect the target gene, verifying the specificity of the amplicon using the BLAST algorithm, analysing the length of the amplicon by gel electrophoresis, comparing the target sequences of the processed isolates with sequences in GenBank, optimising both primers and probe concentrations to ensure the most sensitive and most efficient assay, and testing known positive controls and isolates. With the limited number of strains processed in the present study, the rapid method reached 100% sensitivity and 100% specificity for detection and discrimination of bla SHV ESBL and bla SHV non-ESBL genes in standard and clinical isolates. We have developed a new real-time PCR method for rapid detection of SHV-producing members of the Enterobacteriaceae. This assay could be useful for investigating local outbreaks and is suitable for use in regional reference facilities. At the local level, use of this method would reduce the workload of reference laboratories. Moreover, the specificity of the assay obviates the need for time-consuming, costly DNA sequencing. The ease, speed, and reliability of this real-time PCR SHV assay make it a powerful tool for epidemiological studies of SHV ESBLs. It should be considered for implementation as a routine diagnostics tool. References
Copyright 2010 - Indian Journal of Medical Microbiology The following images related to this document are available:Photo images[mb10100f2.jpg] [mb10100t1.jpg] [mb10100f1.jpg] |
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