|Year : 2010 | Volume
| Issue : 4 | Page : 376-379
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
|Date of Submission||21-Dec-2009|
|Date of Acceptance||01-Jul-2010|
|Date of Web Publication||20-Oct-2010|
Department of Microbiology, Dr. HSJ Institute of Dental Sciences and Hospital, Panjab University, Chandigarh
Source of Support: None, Conflict of Interest: None
Extended spectrum β-lactamases (ESBLs) have been observed in virtually all the species of family Enterobacteriaceae. Threat posed by antibiotic resistance because of ESBLs is more serious as a number of technical problems are associated with the detection of these enzymes. Although a number of detection methods have been designed for ESBLs, every method has its own benefits and shortcomings as well. In earlier days, isoelectric focusing (IEF) was used as the gold standard for ESBL detection. This study was undertaken to compare IEF with polymerase chain reaction, a method which has been extensively used for ESBL detection these days.
Keywords: Extended spectrum β-lactamase, isoelectric focusing, isoelectric point
|How to cite this article:|
Sharma J, Ray P, Sharma M, Ghosh S. Comparison of isoelectric focusing and polymerase chain reaction for the detection of β-lactamases. Indian J Med Microbiol 2010;28:376-9
|How to cite this URL:|
Sharma J, Ray P, Sharma M, Ghosh S. Comparison of isoelectric focusing and polymerase chain reaction for the detection of β-lactamases. Indian J Med Microbiol [serial online] 2010 [cited 2020 Mar 29];28:376-9. Available from: http://www.ijmm.org/text.asp?2010/28/4/376/71829
| ~ 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. 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. 
| ~ Materials and Methods|| |
A total of 100 strains were screened to get 25 ESBL producing isolates, each of Klebsiella pneumoniae and Escherichia More Details 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.
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).  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.
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.  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,, 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, 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.  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,, 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.
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. , But association of PCR with RFLP (PCR-RFLP) helps in detecting specific nucleotide changes,  and its association with single strand conformational polymorphism (PCR-SSCP) helps in distinguishing between a number of SHV variants. ,
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.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3]