Indian Journal of Medical Microbiology Medknow Publications on behalf of Indian Association of Medical Microbiology ISSN: 0255-0857 EISSN: 1998-3646 Vol. 28, Num. 4, 2010, pp. 376-379

Indian Journal of Medical Microbiology, Vol. 28, No. 4, October-December, 2010, pp. 376-379

Brief Communication

Comparison of isoelectric focusing and polymerase chain reaction for the detection of β-lactamases

J Sharma¹, P Ray², M Sharma², S Ghosh³

¹ Department of Microbiology, Dr. HSJ Institute of Dental Sciences and Hospital, Panjab University, Chandigarh, India
² Department of Medical Microbiology, PGIMER, Chandigarh, India
³ Department of Experimental Medicine (SG), PGIMER, Chandigarh, India
Correspondence Address:
J Sharma, Department of Microbiology, Dr. HSJ Institute of Dental Sciences and Hospital, Panjab University, Chandigarh, India, contactjyotisharma@yahoo.co.in

Date of Submission: 21-Dec-2009
Date of Acceptance: 01-Jul-2010

Code Number: mb10110

PMID: 20966574
DOI: 10.4103/0255-0857.71829

Abstract

Keywords: Extended spectrum β-lactamase, isoelectric focusing, isoelectric point

Introduction

Extended spectrum β-lactamases (ESBLs) confer resistance to broad-spectrum β-lactam antibiotics and are of great therapeutic concern for infection caused by many species of the family Enterobacteriaceae. Many of these enzymes confer β-lactam resistance that is not readily detected in routine antibiotic susceptibility tests. ESBL detection is a problem confronting clinical laboratories worldwide. It is clinically important to detect and specifically target ESBL because ESBL producers may be clinically resistant to many β-lactams.^[1] Nevertheless, one has to bear in mind that currently available phenotypic ESBL detection tests only confirm whether an ESBL is produced or not but cannot detect which ESBL subtype is present. Definitive identification is very demanding and only possible by molecular detection methods. Different techniques which are necessary for the task of identifying the exact ESBL subtype, e.g., DNA probing, polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), isoelectric focusing (IEF) are available only in research facilities and not in routine microbiology diagnostic laboratory. ^[2]

Materials and Methods

Bacterial strains

A total of 100 strains were screened to get 25 ESBL producing isolates, each of Klebsiella pneumoniae and Escherichia coli. Bacterial isolates were collected in the Department of Medical Microbiology during May 2002 to Jan 2003, from pus, sputum and blood culture of patients admitted to the tertiary care hospital. Strains showing increased zone of inhibition to third generation cephalosporins, i.e., Ceftazidime (30 μg) and cefotaxime (30 μg), and to fourth generation cephalosporin [cefepime (30 μg)] were screened for ESBL production.

ESBL detection

Extended spectrum β-lactamase detection was carried out following the NCCLS recommended method for screening and confirmation using cefotaxime and ceftazidime as substrates (National Committee for Clinical Laboratory Standards, 2000). ^[3] Cefepime (30 μg) was also tested as a substrate following the same method.

A > 5 mm increase in zone diameter for either of the antimicrobial agents tested in combination with clavulanic acid versus its zone when tested alone was taken as positive result for ESBL production.

Reference strains

Three strains, E. coli J53 R1, E. coli C ₆₀₀ PUD16 and K. pneumoniae ATCC 700603, were used as standard ESBL-positive strains. E. coli J53 R1 harboured TEM-ESBL and rest of the strains carried SHV-ESBL. A non-ESBL-producing organism (E. coli ATCC 25922) was used as a negative control.

Isoelectric focusing of β-lactamases

Crude cell extract containing β-lactamase was prepared from 250 ml Luria-Bertani broth culture (Difco, Maryland, USA). Cells were pelleted by centrifugation, washed in double distilled water and repelleted. Cells were resuspended in 5 ml water and broken with an ultrasonicator (Misonix, Farmingdale, NY, USA), for 3 cycles of 15 seconds each at 4°C. Cell debris was removed by centrifugation and the supernatants of the sonic extracts were frozen at -20°C until tested. The protein concentrations in the sonic extracts of the cells were determined by the Bradford method. ^[4] Detection of β-lactamases and determination of pIs were performed by IEF electrophoresis with Ampholine PAG plate pH gradient 3-9.5 gels (Amersham Biosciences, New Jersey, USA) in a Multiphor II electrophoresis system apparatus (Amersham Biosciences). Reference markers from Amersham Biosciences with known pIs [Table - 1] were electrophoresed in parallel as controls. Gel was run at 1500 V, with 50 mA current and 30 W power for 2 hours. β-lactamase activity was revealed by staining with nitrocefin (Oxoid, Hampshire, UK 50 μg/ml).

Polymerase chain reaction for β-lactamase encoding genes

PCR analysis for β-lactamase genes of the family TEM and SHV was carried out, using primers (Sigma, MO, USA) described in [Table - 2]. For PCR amplifications, ~500 pg of DNA was added to 50 μl mixture containing 200 μM of dNTPs, 0.4 μM of each primer and 2.5 U of Taq polymerase (Roche diagnostics, Indianapolis, IN, USA) in 1× PCR buffer. Amplification was performed in a Techne^; Genius Thermocycler (Techne Ltd., Cambridge, UK) with cycling parameters comprising initial denaturation at 94°C for 3 minutes followed by 35 cycles each of denaturation at 94°C for 30 seconds, annealing at 50°C for 30 seconds, amplification at 72°C for 2 minutes and final extension at 72°C for 10 minutes, for the amplification of bla TEM. For bla SHV amplifications, conditions for thermal cycling remained the same except for a Tm of 55°C. The amplified products were separated in 1.5% agarose gel. The gel was visualised by staining with ethidium bromide (0.5 μg/ml) in a dark room for 30 minutes. A 100 bp ladder molecular weight marker (Roche diagnostics) was used to measure the molecular weights of amplified products. The images of ethidium bromide stained DNA bands were digitised using a gel documentation system (AlphaimagerΤ 3400, Boston, Massachusetts, USA).

In order to evaluate the better method of ESBL detection, results of both the methods were analysed statistically using test of significance.

Results

IEF was carried out for 25 ESBL positive strains of K. pneumoniae and E. coli both. The isoelectric points (pI) of detected bands ranged between 5 and 8.1 for the isolates of K. pneumoniae [Figure - 1] and [Figure - 2] and between 4 and 8.1 for those of E. coli [Figure - 3]. IEF helped in detecting 14 (56%) of ESBL producers amongst E. coli and 18 (72%) in K. pneumoniae [Table - 3]. PCR detected 21/25 (84%) of ESBL producers among E. coli and the same number of ESBL producers among K. pneumoniae [Table - 3]. Statistical analysis revealed PCR as a better method of ESBL detection than IEF (evaluated at 5% level of significance).

Discussion

We evaluated 50 ESBL producers (E. coli and K. pneumoniae; 25 each) using IEF and PCR. IEF could detect 64% of ESBL producers and it failed to detect 44% of ESBL producers among E. coli and 28% of ESBL producers among K. pneumoniae. Though in earlier days IEF was the method of choice for the detection of β-lactamases,^[5],[6] there are certain problems associated with IEF like it is labour intensive, expensive, needs technical expertise and is also lab infrastructure oriented. Moreover, in addition to this, the major drawback of this technique is its low specificity and it requires the use of highly resolved pH gradients. Though this method is efficient in detecting β-lactamases and gives very high sensitivity,^[5] we cannot differentiate between ESBL subtypes and their variants. During the early days of studying ESBLs, determination of the isoelectric point was usually sufficient to identify the ESBLs that were prevalent. The recent scenario of ESBLs reflect numerous ESBL subtypes. List of ESBL variants is also never ending. According to the current data on ESBLs, we have >130 variants of TEM and >50 variants of SHV. ^[7] With so many variants of TEM/SHV type β-lactamases, many of which possess identical isoelectric points, determination of ESBLs becomes more difficult. Several recent studies also highlight the use of this method for ESBL detection^[8],[9],[10] but the method is combined with other molecular detection procedures like PCR, RFLP (Restriction fragment length polymorphism), PFGE (Pulse field gel electrophoresis) and nucleotide sequencing. A study by Padmini et al. has highlighted that as the PhastSystem procedure did not generate any accurate pI, the presence of β-lactamases was further confirmed by PCR screening using specific CTX-M and SHV primers.^[11]

With the help of PCR, 84% of ESBL producers were detected in both the organisms (E. coli and K. pneumoniae). PCR could not detect 16% of ESBL producers. The present study involved the detection of TEM and SHV genes only. So, this figure represents the prevalence of TEM and SHV genes; the rest 16% ESBL producer may belong to other ESBL subgroups, which remained undetected by this technique. Absolute detection by PCR needs designing of primers of various subtypes prevalent in particular geographical location, and with the help of multiplex PCR we can detect different types and more number of ESBL producers. Detection of ESBLs by PCR is supported by various studies. ^[12],[13] But association of PCR with RFLP (PCR-RFLP) helps in detecting specific nucleotide changes, ^[14] and its association with single strand conformational polymorphism (PCR-SSCP) helps in distinguishing between a number of SHV variants. ^[15],[16]

Though there are a number of detection methods for screening and confirmation of ESBLs, still their detection remains a problem for diagnostic microbiological laboratories. Phenotypic tests for ESBL detection only confirm whether an ESBL is produced or not but cannot detect which ESBL subtypes are present. Identification to this level is very difficult and is not available in all routine microbiology laboratories. The techniques which are necessary for the task of identifying the exact ESBL subtype are available only in research facilities. Considering the pros and cons of this technique, we can conclude that IEF is useful in terms of initial/preliminary screening of β-lactamases and gives no confirmation of ESBL subtypes/variants. So, the technique should not be considered as a method of choice for ESBL detection. On the other hand, speed of the procedure, ease of performance and reliability of test render PCR a powerful tool for ESBL detection for epidemiological surveys and makes it a potential candidate for implementation in routine diagnosis of ESBLs.

Acknowledgements

We acknowledge Dr. M. Sitaram Kumar, Dr. Reddy's Laboratories, Hyderabad, for kindly providing us ESBL standard stains.

References

1.	Jacoby GA. Extended-spectrum beta lactamases and other enzymes providing resistance to oxyimino-beta-lactams. Infect Dis Clin North Am 1997;11:875-7.  Back to cited text no. 1
2.	Bradford PA. Extended-spectrum β-lactamases in the 21st century: Characterization, epidemiology and detection of this important resistance threat. Clin Microbiol Rev 2001;14:933-51.  Back to cited text no. 2
3.	National Committee for Clinical Laboratory Standards: Performance Standards for antimicrobial disc susceptibility tests. 7 th ed. Approved standards M2-A6. National Committee for Clinical Laboratory Standards. Wayne Pa; 2000.  Back to cited text no. 3
4.	Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248-54.  Back to cited text no. 4
5.	Mathew M, Harris AM, Marshall M, Ross GW. The use of analytical iso electric focusing for detection and identification of β-lactamases. J Gen Microbiol 1975;88:168-78.  Back to cited text no. 5
6.	Jouvenot M, Deschaseaux ML, Royez M, Mougin C, Cooksey RC, Michael-Briand Y, et al. Molecular Hybridization versus isoelectric focusing to determine TEM-type β-lactamases in Gram Negative Bacteria. Antimicrob Agents Chemother 1987;31:300-5.  Back to cited text no. 6
7.	Jacoby G, Bush K. Amino acid sequences for TEM, SHV and OXA extended-spectrum and inhibitor resistant β-lactamases. Available from: http://www.lahey.org/studies/webt.htm [last accessed on 2008 May 9].  Back to cited text no. 7
8.	Hanson ND, Thomson KS, Moland ES, Sanders CC, Berthold G, Penn RG. Molecular characterization of a multiply resistant Klebsiella pneumoniae encoding ESBLs and plasmid-mediated AmpC. J Antimicrob Chemother 1999;44:377-80.  Back to cited text no. 8
9.	Yamasaki K, Komatsu M, Yamashita T, Shimakawa K, Ura T, Nishio H, et al. Production of CTX-M-3 extended-spectrum β-lactamase and IMP-1 metallo β-lactamase by five Gram-negative bacilli: Survey of clinical isolates from seven laboratories collected in 1998 and 2000, in the Kinki region of Japan. J Antimicrob Chemother 2003;51:631-8.  Back to cited text no. 9
10.	Garza-Ramos U, Martínez-Romero E, Silva-Sánchez J. SHV-type Extended-spectrum ß-lactamase (ESBL) are encoded in related plasmids from enterobacteria clinical isolates from Mexico. Salud Publica Mex 2007;49:415-21.  Back to cited text no. 10
11.	Padmini SB, Appala Raju B, Mani KR. Detection of Enterobacteriaceae producing CTX-M extended spectrum β-lactamases from a tertiary care hospital in south India. Indian J Med Microbiol 2008;26:163-6.  Back to cited text no. 11  [PUBMED]
12.	Courvalin P. Genotypic approach to the study of bacterial reisstance to antibiotics. Antimicrob Agents Chemother 1991;35:1019-23.  Back to cited text no. 12
13.	Mabilat C, Courvalin P. Development of oligotyping for characterization and molecular epidemiology of TEM β-lactamases in members of family Enterobacteriaceae. Antimicrob Agents Chemother 1990;34:2210-6.  Back to cited text no. 13
14.	Nόesch-Inderbinen MT, Hächler H, Kayser FH. Detection of genes coding for extended-spectrum SHV beta-lactamases in clinical isolates by a molecular genetic method, and comparison with the Etest. Eur J Clin Microbiol Infect Dis 1996;15:398-402.  Back to cited text no. 14
15.	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-spectrum β-lactamase and to identify different SHV genes within the same strain. J Antimicrob Chemother 1998;41:123-5.  Back to cited text no. 15
16.	M'Zali FH, Gascoyne-Binzi DM, Heritage J, Hawkey PM. Detection of mutations conferring extended-spectrum activity on SHV β-lactamases using polymerase chain reaction single strand conformational polymorphism (PCR-SSCP). J Antimicrob Chemother 1996;37:797-802.  Back to cited text no. 16

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