|Year : 2010 | Volume
| Issue : 4 | Page : 332-336
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|
|Date of Acceptance||20-Aug-2010|
|Date of Web Publication||20-Oct-2010|
M S Alfaresi
Department of Pathology & Laboratory Medicine, Zayed Military Hospital, PO Box 3740, Abu Dhabi
United Arab Emirates
Source of Support: None, Conflict of Interest: None
Purpose: This study aimed to develop an improved method for the detection of bacterial SHV-type extended-spectrum β-lactamases (ESBLs). Materials and Methods: Our method was based on real-time polymerase chain reaction (PCR) in which the amplification of the product was monitored with a ﬂuorescent probe. This method enabled the detection of bla SHV genes with high degrees of sensitivity and speciﬁcity. Results: Based on ESBL phenotyping methods and bla gene DNA sequencing, we identified 240 bla genes from 662 Enterobacteriaceae isolated from clinical culture specimens. Of these 240 isolates, 26 had the bla SHV-28 genotype and three had the bla SHV-1 genotype. With our new real-time PCR assay, we detected 29 out of 29 bla SHV genes in ESBL-producing isolates. Conclusion: This method represents a powerful tool for epidemiological studies of SHV ESBLs. Furthermore, it has potential for use in diagnostic microbiology.
Keywords: Real-time PCR, SHV, extended-spectrum β-lactamase
|How to cite this article:|
Alfaresi M S, Elkoush A A. Real-time polymerase chain reaction for rapid detection of genes encoding SHV extended-spectrum β-lactamases. Indian J Med Microbiol 2010;28:332-6
|How to cite this URL:|
Alfaresi M S, Elkoush A A. Real-time polymerase chain reaction for rapid detection of genes encoding SHV extended-spectrum β-lactamases. Indian J Med Microbiol [serial online] 2010 [cited 2020 Feb 23];28:332-6. Available from: http://www.ijmm.org/text.asp?2010/28/4/332/71827
| ~ 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. , ESBL-producing organisms have achieved notoriety for causing nosocomial infections. ,
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 More Details coli, and Pseudomonas aeruginosa, among others.  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. ,, 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.  However, despite considerable effort for over a decade, laboratory detection of ESBL production remains problematic. ,,,,,, 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). 
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, , oligotyping,  PCR single-strand conformational polymorphism, , and ligase chain reaction. , 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.,
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].
|Table 1 :Sequences of the primer pair and the probe for PCR amplification of the SHV gene from bacterial isolates |
Click here to view
|Figure 1 :Alignment of the nucleotide sequences of genes that encode β-lactamases SHV-1 (top; EU418908) and SHV-5 (bottom; EU441171) based on the Lahey website. PCR amplification primers and probes are indicated with coloured arrows. Double slashes between the primer sequences indicate sequence interruptions|
Click here to view
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.
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  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.
|Figure 2 :Real-time PCR detection of amplified SHV ESBL in eight clinical isolates. Negative samples are indicated by flat lines. The positive isolates were blaSHV-1 (violet, brown, green, and dark blue lines), blaSHV-28 (blue line), and the reference strain, blaSHV-18 (black line)|
Click here to view
| ~ 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. 
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. , 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|| |
|1.||Bush K, Jacoby GA, Medeiros AA. A functional classiﬁcation scheme for beta-lactamases and its correlation with molecular structure. Antimicrob Agents Chemother 1995;39:1211-33. |
|2.||Paterson DL, Bonomo RA. Extended-spectrum Beta-lactamases: A clinical update. Clin Microbiol Rev 2005;18:657-86. |
|3.||Bradford PA. Extended-spectrum Beta-lactamases in the 21st century: Characterization, epidemiology, and detection of this important resistance threat. Clin Microbiol Rev 2001;14:933-51. |
|4.||Gniadkowski M. Evolution of extended-spectrum Beta-lactamases by mutation. Clin Microbiol Infect 2008;14:11-32. |
|5.||Huletsky A, Knox JR, Levesque RC. Role of Ser-238 and Lys-240 in the hydrolysis of third-generation cephalosporins by SHV-type beta-lactamases probed by site-directed mutagenesis and three-dimensional modeling. J Biol Chem 1993;268:3690-7. |
|6.||Page MG. Extended-spectrum Beta-lactamases: Structure and kinetic mechanism. Clin Microbiol Infect 2008;14:63-74. |
|7.||Paterson DL, Ko WC, Von Gottberg A, Mohapatra S, Casellas JM, Goossens H, et al. International prospective study of Klebsiella pneumoniae bacteraemia: Implications of extended-spectrum Beta-lactamase production in nosocomial infections. Ann Intern Med 2004;140:26-32. |
|8.||Livermore DM. Beta-Lactamases in laboratory and clinical resistance. Clin Microbiol Rev 1995;8:557-84. |
|9.||Moland ES, Sanders CC, Thomson KS. Can results obtained with commercially available MicroScan microdilution panels serve as an indicator of Beta-lactamase production among Escherichia coli and Klebsiella isolates with hidden resistance to expanded-spectrum cephalosporins and aztreonam? J Clin Microbiol 1998;36:2575-9. |
|10.||Pfaller MA, Segreti J. Overview of the epidemiological proﬁle and laboratory detection of extended-spectrum Beta-lactamases. Clin Infect Dis 2006;42:S153-63. |
|11.||Tenover FC, Raney PM, Williams PP, Rasheed JK, Biddle JW, Oliver A, et al. Evaluation of the NCCLS extended-spectrum beta-lactamase conﬁrmation methods for Escherichia coli with isolates collected during Project ICARE. J Clin Microbiol 2003;41:3142-6. |
|12.||Thomson KS. Controversies about extended-spectrum and AmpC beta-lactamases. Emerg Infect Dis 2001;7:333-6. |
|13.||Babini GS, Livermore DM. Antimicrobial resistance amongst Klebsiella spp collected from intensive care units in Southern and Western Europe in 1997-1998. J Antimicrob Chemother 2000;45:183-9. |
|14.||Chanawong A, M'Zali FH, Heritage J, Lulitanond A, Hawkey PM. Characterisation of extended-spectrum beta-lactamases of the SHV family using a combination of PCR-single strand conformational polymorphism (PCR-SSCP) and PCR-restriction fragment length polymorphism(PCR-RFLP). FEMS Microbiol Lett 2000;184:85-9. |
|15.||Lee SH, Kim JY, Shin SH, Lee SK, Choi MM, Lee IY, et al. Restriction fragment length dimorphism-PCR method for the detection of extended-spectrum beta-lactamases unrelated to TEM- and SHV-types. FEMS Microbiol Lett 2001;200:157-61. |
|16.||Mabilat C, Courvalin P. Development of "oligotyping" for characterization and molecular epidemiology of TEM beta-lactamases in members of the family Enterobacteriaceae. Antimicrob Agents Chemother 1990;34:2210-6. |
|17.||M'Zali FH, Heritage J, Gascoyne-Binzi DM, Snelling AM, Hawkey PM. PCR single strand conformational polymorphism can be used to detect the gene encoding SHV-7 extended-spectru-beta-lactamase and to identify different SHVgenes with in the same strain. J Antimicrob Chemother 1998;41:123-5. |
|18.||Kim J, Lee HJ. Rapid discriminatory detection of genes coding for SHV beta-lactamases by ligase chain reaction. Antimicrob Agents Chemother 2000;44:1860-4. |
|19.||Niederhauser C, Kaempf L, Heinzer I. Use of the ligase detection reaction-polymerase chain reaction to identify point mutations in extended-spectrum Beta-lactamases. Eur J Clin Microbiol Infect Dis 2000;19:477-80. |
|20.||Chia JH, Chu C, Su LH, Chiu CH, Kuo AJ, Sun CF, et al. Development of a multiplex PCR and SHV melting-curve mutation detection system for detection of some SHV and CTX-M beta-lactamases of Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae in Taiwan. J Clin Microbiol 2005;43:4486-91. |
|21.||Pitout JD, Hossain A, Hanson ND. Phenotypic and molecular detection of CTX-M-beta-lactamases produced by Escherichia coli and Klebsiella spp. J Clin Microbiol 2004;42:5715-21. |
|22.||Tofteland S, Haldorsen B, Dahl KH, Simonsen GS, Steinbakk M, Walsh TR, et al. Effects of phenotype and genotype on methods for detection of extended-spectrum-beta-lactamase-producing clinical isolates of Escherichia coli and Klebsiella pneumoniae in Norway. J Clin Microbiol 2007;45:199-205. |
|23.||Livermore DM, Yuan M. Antibiotic resistance and production of extended-spectrum beta-lactamases amongst Klebsiella spp from intensive care units in Europe. J Antimicrob Chemother 1996;38:409-24. |
|24.||Fluit AC, Visser MR, Schmitz FJ. Molecular detection of antimicrobial resistance. Clin Microbiol Rev 2001;14:836-71. |
[Figure 1], [Figure 2]
|This article has been cited by|
||Diagnosis and antimicrobial treatment of invasive infections due to multidrug-resistant Enterobacteriaceae. Guidelines of the Spanish Society of Infectious Diseases and Clinical Microbiology
| ||Jesús Rodríguez-Baño,José Miguel Cisneros,Nazaret Cobos-Trigueros,Gema Fresco,Carolina Navarro-San Francisco,Carlota Gudiol,Juan Pablo Horcajada,Lorena López-Cerero,José Antonio Martínez,José Molina,Milagro Montero,José R. Paño-Pardo,Alvaro Pascual,Carmen Peña,Vicente Pintado,Pilar Retamar,María Tomás,Marcio Borges-Sa,José Garnacho-Montero,Germán Bou |
| ||Enfermedades Infecciosas y Microbiología Clínica. 2015; |
|[Pubmed] | [DOI]|
||Extended-spectrum -lactamase/AmpC-producing uropathogenic Escherichia coli from HIV patients: do they have a low virulence score?
| ||K. Padmavathy,K. Padma,S. Rajasekaran |
| ||Journal of Medical Microbiology. 2013; 62(Pt_3): 345 |
|[Pubmed] | [DOI]|