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SHORT COMMUNICATION
Year : 2016  |  Volume : 18  |  Issue : 1  |  Page : 55-59

Molecular characterisation of extended spectrum beta lactamase-producing Escherichia coli and Klebsiella pneumoniae isolates from urinary specimens


1 Department of Microbiology, Government Medical College, Ernakulam, Kerala, India
2 Department of Microbiology, Government Medical College, Thiruvananthapuram, Kerala, India
3 Department of Microbiology, Amrita Institute of Medical Sciences, Kochi, Kerala, India

Date of Web Publication28-Jun-2016

Correspondence Address:
Neethu John
Department of Microbiology, Government Medical College, Ernakulam, Kerala
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0972-1282.184767

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  Abstract 

Extended spectrum beta lactamase (ESBL) production is a major resistance mechanism of Gram-negative bacteria. Phenotypic tests for ESBL detection only confirm whether an ESBL is produced or not, but it cannot detect the ESBL subtype. There are various genotypes of ESBLs. Of these, the most common are the TEM, SHV and CTX-M types. Fifty phenotypically ESBL-producing Escherichia coli and Klebsiella pneumoniae urinary isolates were subjected to polymerase chain reaction to look for the presence of blaTEM, blaSHVand blaCTX-Mgenes. Forty-one isolates were positive for TEM gene, eleven isolates were positive for SHV gene and 10 isolates were positive for CTX-M gene. Both TEM and SHV genes were present in six isolates. Two isolates showed the simultaneous presence of CTX-M and TEM genes. All the three genes were present in two isolates.

Keywords: CTX-M, extended spectrum beta lactamase, polymerase chain reaction, SHV, TEM


How to cite this article:
John N, Lancy J, Girija K R. Molecular characterisation of extended spectrum beta lactamase-producing Escherichia coli and Klebsiella pneumoniae isolates from urinary specimens. J Acad Clin Microbiol 2016;18:55-9

How to cite this URL:
John N, Lancy J, Girija K R. Molecular characterisation of extended spectrum beta lactamase-producing Escherichia coli and Klebsiella pneumoniae isolates from urinary specimens. J Acad Clin Microbiol [serial online] 2016 [cited 2019 Sep 23];18:55-9. Available from: http://www.jacmjournal.org/text.asp?2016/18/1/55/184767




  Introduction Top


The prevalence of extended spectrum beta lactamase (ESBL) producing bacteria varies worldwide. They are reported in almost all parts of the world and are increasing at an alarming rate. The situation in India is not different. The prevalence of ESBLs in India has now reached epidemic proportions, ranging from 62% to 100% in  Escherichia More Details coli and Klebsiella spp. as observed in the 10 Indian medical centre SENTRY study.[1] High rates of ESBL-positive isolates were observed in nine study centres in India, each representing distinct cities in various regions of India: New Delhi, Lucknow, Indore, Mumbai, Hyderabad, Bengaluru, Chennai, Tamil Nadu and Kolkata indicating that ESBL-positive strains in the country are not restricted to any single city or region.[2]

ESBL-producing strains of Enterobacteriaceae have emerged as a challenge in hospitalised as well as community-based patients.[3] There are several genotypes of ESBLs. Of these, the most common genotypes are the TEM, SHV and CTX-M.

In the 1980s and 1990s, TEM- and SHV-ESBLs were the predominant ESBL types whereas the CTX-Ms were less prevalent. CTX-M enzymes, though first discovered in 1989, began to be detected at a higher rate only after 2000. They were confined not only to the hospital setting but also to the community, with E. coli being the most important pathogen-producing CTX-M enzymes.[4]

TEM- and SHV-ESBLs, with over 100 mutations being reported, have become a major cause of hospital-acquired infection, particularly in the Intensive Care Unit. It has been suggested that the naturally occurring TEM-type ESBLs are the result of fluctuating selective pressure from several beta-lactams within a given institution rather than selection with a single agent.[5] There are over 100 variants of CTX-M to date and they have been associated with numerous outbreaks of infections both in hospitals and in the community.

Molecular characterisation of ESBL-producing isolates can be a useful epidemiological tool in the surveillance of these multidrug-resistant (MDR) organisms. Such data can help us know the transmission of these organisms in hospitals, which is useful to make effective and timely interventions. In this background, a study was conducted on fifty ESBL-producing MDR E. coli and Klebsiella pneumoniae urinary isolates to check for the presence of blaTEM, blaSHV and blaCTX-M genes using polymerase chain reaction (PCR).


  Materials and Methods Top


The study was conducted in the Department of Microbiology, Government Medical College, Thrissur, during the period of August 2011 to July 2012.

According to the recent studies in India, the prevalence of ESBL ranges from 40% to 100%.[1],[6],[7],[8] From this data, the sample size of the study was calculated as 150, taking a power of 80% and allowable error as 20% at 5% confidence level. The presence of ESBL production was tested by double disc diffusion test described by Jarlier et al.[9] and phenotypic confirmatory test (PCT) recommended by Clinical and Laboratory Standards Institute (CLSI), 2010.[10]

Molecular characterisation of fifty MDR isolates determined as ESBL producers by PCT was done at the Department of Agricultural Microbiology, College of Horticulture, Kerala Agricultural University. The sample size for doing PCR was restricted to fifty due to financial constraints. A random selection of fifty MDR E. coli (n = 41) and K. pneumoniae (n = 9) was made by lot method from the 150 isolates, on which ESBL production was detected by PCT. PCR was carried out to detect the presence of the most common ESBL genes – blaTEM, blaSHV and blaCTX-M. PCR was done in Eppendorf Mastercycler, Gradient (Eppendorf, Germany).

DNA template preparation was done as follows: Using a sterile loop, a medium-sized colony of a fresh culture of the organism on nutrient agar plate was transferred to PCR vial and suspended in 10 µl distilled water. Denaturation was done at 98°C for two min. The vial was then centrifuged at 11,000 rpm for one min. The supernatant (1 or 2 µl) was taken as the template to do the PCR.

The master mix was prepared by adding 2.5 µl Taq buffer, 1 µl dNTPs, 2 µl Taq polymerase, 16.5 µl distilled water and 1 µl each of forward primer and reverse primer. Finally, after dispensing 24 µl of the master mix in the individual amplification tubes, 1 µl of the template DNA was added in the corresponding tubes to make up the total volume to 25 µl. The PCR primers used (Sigma Aldrich, Bengaluru) and the expected amplicon sizes are shown in [Table 1].
Table 1: Primers used and amplicon sizes

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Thermal cycling conditions used were initial denaturation done at 94°C, two min and followed by thirty cycles of denaturation at 94°C, 45 s; annealing temperature kept at 58°C one min for blaTEM, blaSHV and 50°C one min for blaCTX-M; extension at 72°C, two min and final extension at 72°C, 10 min.

PCR products were analysed by agarose gel electrophoresis with 1% agarose gels in 50X Tris-acetate-EDTA buffer. The gels were stained with ethidium bromide. PCR products were visualised under ultraviolet light [Figure 1],[Figure 2],[Figure 3].
Figure 1: Polymerase chain reaction to detect blaTEM (900 bp) lane 2, 3, 4, 5, 6 and 7 – positive for blaTEM

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Figure 2: Polymerase chain reaction to detect blaSHV (850 bp) lanes 3, 5, 8 and 9 – positive for blaSHV

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Figure 3: Polymerase chain reaction to detect blaCTX-M (550 bp) lanes 3, 8, 9, 10, 12 and 13 – positive for blaCTX-M

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  Results Top


The total number of urinary specimens received during this period was 4550, out of which 812 samples yielded a significant growth of Gram-negative Bacilli. Among this 812 Gram-negative isolates, 451 (55.5%) were MDR, which means they were resistant to three or more antimicrobial classes. MDR E. coli (n = 368) and MDR K. pneumoniae (n = 76) were the predominant MDR Gram-negative urinary isolates.

Based on the sample size calculated, 150 MDR urinary isolates of E. coli (n = 117) and K. pneumoniae (n = 33) were tested for the presence of ESBL production by double disc diffusion test described by Jarlier et al.[9] and PCT recommended by CLSI, 2010.[10]

Out of the 150 MDR isolates, 91% (136/150) were ESBL producers, 9% were negative for ESBL. Out of 117 E. coli studied, 107 (91.5%) were ESBL producers. Out of the 33 K. pneumoniae isolates under study, 29 (87.9%) were ESBL producers.

Out of fifty ESBL-producing MDR E. coli and K. pneumoniae isolates, 41 showed the presence of blaTEM. Eleven isolates were positive for blaSHV and 10 isolates for blaCTX-M. Hence, blaTEM predominated in the isolates, followed by blaSHV and blaCTX-M [Table 2].
Table 2: Distribution of TEM, SHV and CTX-M genes among 50 multidrug-resistant isolate

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  Discussion Top


Phenotypic tests for ESBL detection only confirm whether an ESBL is produced, but it cannot detect the ESBL subtype. The phenotypic tests need to be evaluated periodically as their performance may change with the introduction of new enzyme. There are various genotypes of ESBLs. Of these, the most common genotypes are the TEM, SHV and CTX-M. Other clinically important types include OXA, VEB, PER, BEL-1, BES-1, SFO-1, TLA and IBC. Identification to this level is very difficult and is not available in all routine microbiology laboratories.

CTX-M beta-lactamases are more active against cefotaxime and ceftriaxone than against ceftazidime, but point mutations can increase their activity against ceftazidime as well. CTX-M now accounts for most of the ESBLs found in Enterobacteriaceae. The CTX-M group of ESBLs are diverse, with alleles divided into five distinct phylogenetic groups. In most countries, there are mixtures of CTX-M types, with ESBL isolates from India being completely dominated by the presence of CTX-M-15.[1],[11]

In a study by Sharma et al. from Chandigarh on molecular detection methods of beta-lactamases, 84% of the ESBL producers belonged to TEM- and SHV-ESBL types.[12] The study further concluded that the rest 16% of ESBL producers may belong to other ESBL subgroups. Another study by Jemima and Verghese from Chenna found that 15.83% of ESBL-positive isolates had blaCTX-M gene.[13] They also noted that among those CTX-M-producing isolates, CTX-M-1 gene was positive in 47.3% isolates. Sekar et al. reported that 44.4% of E. coli and K. pneumoniae strains were found to be positive for blaCTX-M gene by PCR.[14]

In another study by Manoharan et al., distribution of TEM, SHV and CTX-M ESBL types among 121 study isolates was noted.[15] They observed that TEM and CTX-M were predominantly seen in E. coli (39.2%), while among Klebsiella spp., TEM, SHV and CTX-M occurred together (42.6%). Another finding made by them was that higher resistance was noted among isolates carrying all the three (TEM, SHV and CTX-M) ESBL types.

The molecular characterisation of randomly selected ESBL-carrying isolates in this study showed a predominance of TEM-ESBLs followed by SHV-ESBLs and CTX-M ESBLs. This finding does not correlate with the current trend that CTX-M ESBLs are emerging as the predominant ESBL type. This may be because all the ESBL-carrying isolates were not studied for ESBL genes. Data from the past 10 years establish CTX-M genotype as the predominant ESBL in Europe and East Asia. Not many studies are available from different parts of our country checking the presence of the three genes – TEM, SHV and CTX-M together among the ESBL-producing pathogens. Therefore, the observations in this study indicate the need for a more detailed surveillance of the MDR isolates in this region, with special attention to molecular level detection. Although molecular characterisation of ESBLs is not very important in deciding the treatment, they provide useful epidemiological information which can make relevant contributions in formulating antibiotic policy of the area.


  Conclusion Top


This study found TEM followed by SHV as the major genotype in a study group of 50 ESBL-producing E. coli and K. pneumoniae urinary isolates. This is in contrast to current global and Indian scenario where CTX-M is the major genotype. The sample size of this study was restricted due to financial constraints, which is a limitation of the study. This fact points to the need for a large scale genotypic study to understand the epidemiologic pattern of drug-resistant isolates. The control measures include strict implementation of antibiotic policy, proper hand hygiene protocol and long-term surveillance of the antibiotic susceptibility pattern of the microbial isolates.

Acknowledgement

Our sincere gratitude to Dr. Girija D, Professor and Head, Department of Agricultural Microbiology, Kerala Agricultural University, for providing the facilities to do polymerase chain reaction. We are also grateful to other staff members of the department for their timely help and support, especially by Ms. Deepa and Mrs. Anjaly.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
  References Top

1.
Matha D, Manoharan A, Vasanthan G. Epidemiology and implications of ESBL. Crit Care Update 2009;14:152-62.  Back to cited text no. 1
    
2.
Hawser SP, Bouchillon SK, Hoban DJ, Badal RE, Hsueh PR, Paterson DL. Emergence of high levels of extended-spectrum-beta-lactamase-producing gram-negative Bacill in the Asia-Pacific region: Data from the study for monitoring antimicrobial resistance trends (SMART) program, 2007. Antimicrob Agents Chemother 2009;53:3280-4.  Back to cited text no. 2
    
3.
Gupta V. An update on newer beta-lactamases. Indian J Med Res 2007;126:417-27.  Back to cited text no. 3
[PUBMED]  Medknow Journal  
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Borg MA, Scicluna E, de Kraker M, van de Sande-Bruinsma N, Tiemersma E, Gür D, Antibiotic resistance in the Southeastern Mediterranean – Preliminary results from the ARMed project. Euro Surveill 2006;11:164-7.  Back to cited text no. 4
    
5.
Paterson DL, Bonomo RA. Extended-spectrum beta-lactamases: A clinical update. Clin Microbiol Rev 2005;18:657-86.  Back to cited text no. 5
    
6.
Pushpa D, Arora U, Kaur K. Prevalence of extended spectrum beta lactamase producing gram negative uropathogens in a tertiary care hospital. J Acad Clin Microbiologists 2009;11:56-65.  Back to cited text no. 6
    
7.
Babypadmin S, Appalaraju B. Extended spectrum-lactamases in urinary isolates of and – Prevalence and susceptibility pattern in a tertiary care hospital. Indian J Med Microbiol 2004;22:172-4.  Back to cited text no. 7
    
8.
Supriya S. Evaluation of ESBL in urinary isolates. Indian J Med Res 2004;22:172-4.  Back to cited text no. 8
    
9.
Jarlier V, Nicolas MH, Fournier G, Philippon A. Extended broad-spectrum beta-lactamases conferring transferable resistance to newer beta-lactam agents in Enterobacteriaceae: Hospital prevalence and susceptibility patterns. Rev Infect Dis 1988;10:867-78.  Back to cited text no. 9
    
10.
Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing; 20th Informational Supplement. CLSI M100-S20. Wayne, PA: Clinical and Laboratory Standards Institute; 2010.  Back to cited text no. 10
    
11.
Hawkey PM. Prevalence and clonality of extended-spectrum beta-lactamases in Asia. Clin Microbiol Infect 2008;14 Suppl 1:159-65.  Back to cited text no. 11
    
12.
Sharma J, Ray P, Sharma M, Ghosh S. Comparison of isoelectric focusing and polymerase chain reaction for the detection of ß-lactamases. Indian J Med Microbiol 2010;28:376-9.  Back to cited text no. 12
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Jemima SA, Verghese S. Molecular characterization of nosocomial CTX-M type beta-lactamase producing Enterobacteriaceae from a tertiary care hospital in South India. Indian J Med Microbiol 2008;26:365-8.  Back to cited text no. 13
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Sekar B, Arunagir K, Menaka K, Lalitha P, Aparna V, Oommen PK. Detection and Characterization of bla-CTX-M Gene by PCR-RFLP Analysis Among Third Generation Cephalosporin Resistant Gram-negative Isolates. XXX National Congress of Indian Association of Medical Microbiologists. Microcon; 2006. p. 27.  Back to cited text no. 14
    
15.
Manoharan A, Premalatha K, Chatterjee S, Matha D; SARI Study Group. Correlation of TEM, SHV and CTX-M extended-spectrum beta lactamases among Enterobacteriaceae with their antimicrobial susceptibility. Indian J Med Microbiol 2011;29:161-4.  Back to cited text no. 15
[PUBMED]  Medknow Journal  


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