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 ~  Abstract
 ~  Introduction
 ~  Materials and Me...
 ~  Results
 ~  Discussion
 ~  Acknowledgements
 ~  References
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BRIEF COMMUNICATION
Year : 2011  |  Volume : 29  |  Issue : 2  |  Page : 161-164
 

Correlation of TEM, SHV and CTX-M extended-spectrum beta lactamases among Enterobacteriaceae with their in vitro antimicrobial susceptibility


1 Prof. Benjamin M Pulimood Laboratories for Infection, Immunity and Inflammation (BMPLIII), Department of Medicine Unit I and Infectious Disease, Christian Medical College, Vellore - 632 004, Tamil Nadu, India
2 Surveillance of Antimicrobial Resistance in India (SARI) Study Group; Kothari Medical Center, Kolkata - Dr Anuradha Aggarwal, PD Hinduja Hospital, Mumbai - Dr Camilla Rodrigues, Sri Ramachandra Medical College, Chennai - Dr Padma Srikanth, Sir Gangaram Hospital, New Delhi - Dr Chand Wattal, Sundaram Medical Center, Chennai - Dr Vaidehi, and Dr Ram Rajagopal, India

Date of Submission03-Dec-2010
Date of Acceptance23-Mar-2011
Date of Web Publication2-Jun-2011

Correspondence Address:
A Manoharan
Prof. Benjamin M Pulimood Laboratories for Infection, Immunity and Inflammation (BMPLIII), Department of Medicine Unit I and Infectious Disease, Christian Medical College, Vellore - 632 004, Tamil Nadu
India
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Source of Support: Cipla India for funding the SARI study, Conflict of Interest: None


DOI: 10.4103/0255-0857.81799

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 ~ Abstract 

Purpose: The present study was carried out to characterize the ESBL types and evaluated their in vitro activity against a collection of Gram negative bacteria (GNB) from a multicentric Indian surveillance study. Material and Methods: During January 2005 to June 2006, six tertiary care centres in India forwarded 778 non-duplicate GNB to our reference laboratory. Three hundred GNB from this collection were selected based on clinical significance and were used in the present study. Tested isolates included Escherichia coli (167), Klebsiella spp. (122) and Enterobacter spp. (11). ESBL screening and confirmation was performed for all the isolates. Minimum inhibitory concentration of imipenem, meropenem, ertapenem, levofloxacin, amikacin, piperacillin/tazobactam and ceftriaxone was determined by the E-test method. Molecular typing of the ESBLs was performed by polymerase chain reaction among the 121 selected isolates. Results: The study showed excellent susceptibility among the strains to imipenem (100%), meropenem (100%) and ertapenem (98.7%); good susceptibility to amikacin (89.7%) and piperacillin/tazobactam (85.3%) was observed. TEM and CTX-M were predominantly found in E. coli (39.2%) while, among the Klebsiella spp., TEM, SHV and CTX-M occurred together in 42.6% of the isolates. Conclusion: More than one ESBL was produced by many strains, and this was correlated with increased resistance levels. Carbapenems continue to show good in vitro activity and ertapenem is a potential alternative to imipenem and meropenem. Continued antimicrobial resistance surveillance is warranted in light of these findings.


Keywords: Carbapenems, extended-spectrum beta lactamase, resistance, susceptible


How to cite this article:
Manoharan A, Premalatha K, Chatterjee S, Mathai D, SARI Study Group. Correlation of TEM, SHV and CTX-M extended-spectrum beta lactamases among Enterobacteriaceae with their in vitro antimicrobial susceptibility. Indian J Med Microbiol 2011;29:161-4

How to cite this URL:
Manoharan A, Premalatha K, Chatterjee S, Mathai D, SARI Study Group. Correlation of TEM, SHV and CTX-M extended-spectrum beta lactamases among Enterobacteriaceae with their in vitro antimicrobial susceptibility. Indian J Med Microbiol [serial online] 2011 [cited 2019 Feb 18];29:161-4. Available from: http://www.ijmm.org/text.asp?2011/29/2/161/81799



 ~ Introduction Top


Extended-spectrum β-lactamases (ESBLs) are a rapidly evolving group of β-lactamase enzymes produced by the Gram negative bacteria (GNB), which have the ability to hydrolyze all cephalosporins and aztreonam but are inhibited by clavulanic acid. ESBLs are often plasmid mediated, derived from mutations in the classic TEM and SHV genes by one or more amino acid substitutions around the active site. ESBL-producing Enterobacteriaceae were first reported in 1983 from Germany and since then a steady increase of these strains have been reported worldwide. [1]

The prevalence of ESBLs in India has now reached epidemic proportions, ranging from 62% to 100% in  Escherichia More Details coli and Klebsiella spp. isolated from skin and soft tissue infections, blood stream infections and respiratory infections as observed in the 10 Indian medical centre SENTRY study. [2] Apart from TEM and SHV ESBL types, isolates from India additionally produce CTX-M enzymes. [2],[3] CTX-M ί lactamases are more active against cefotaxime and ceftriaxone than against ceftazidime, but point mutations can increase their activity against ceftazidime as well. With this background, it is very important to survey the ESBL type prevalent nationally and determine the resistance levels shown by them to currently available antimicrobial classes.

Imipenem and meropenem are the drugs of choice for the treatment of infections caused by ESBL-producing organisms. Nevertheless, there is concern that the extensive use of these two drugs will place selective pressure on imipenem and meropenem, which are the last good defenses against multiresistant Pseudomonas and Acinetobacter spp. Moreover, the multiple daily dosages required for these antibiotics make them difficult in treatment regimen. [4],[5],[6] Ertapenem is a new carbapenem developed to address the above pharmacokinetic shortcomings of imipenem and meropenem. Ertapenem demonstrates broad-spectrum antimicrobial activity against many Gram positive and negative aerobes and anaerobes and is resistant to nearly all β lactamases, including ESBLs and AmpCs. The extensive protein binding of ertapenem extends the half-life and allows for once-daily dosing.[5],[7] The activity of this drug against ESBL-producing clinically significant Enterobacteriaceae in India needs to be elucidated before it can be considered an alternative to imipenem and meropenem.

Detection of common ESBL genes such as TEM, SHV and CTX-M by molecular methods in ESBL-producing bacteria and their pattern of antimicrobial resistance can provide useful information about its epidemiology and aid in rational antimicrobial therapy. [8] We undertook the study to characterize the ESBL types in clinically significant Enterobacteriaceae collected from multiple geographic sites in India and to determine their pattern of resistance to six antimicrobial agents.


 ~ Materials and Methods Top


Bacterial isolates

A total of 778 isolates were received at the Prof. Benjamin M Pulimood Laboratories for Infection, Immunity and Inflammation (BMPLIII) during 2005-2006 as part of the six-centre Surveillance of Antimicrobial Resistance in India (SARI) study. Three hundred non-duplicate GNB [skin and soft tissue (n = 137), blood (n = 74), respiratory (n = 70), urine (n = 19)] from this collection were selected based on clinical significance and used for the present study. Tested isolates included E. coli (n = 167), Klebsiella spp. (n = 122) and Enterobacter spp. (n = 11).

Antimicrobial susceptibility testing

Minimum inhibitory concentration (MIC) of amikacin, imipenem, levofloxacin, meropenem, piperacillin/tazobactam and ceftriaxone besides ertapenem was determined by the E-test method (Biomerieux, Craponne, France). Interpretative criteria used were as per the E-test manufacturer's guidelines and CLSI 2010. [9] E. coli ATCC 25922, S. aureus ATCC 29213, P. aeruginosa ATCC 27853, S. aureus ATCC 25923 and E. faecalis ATCC 29212 were used as quality control.

Screening for and confirmation of extended-spectrum β-lactamases

All the E. coli, Klebsiella spp. and Enterobacter spp. isolates were screened for ESBL production by the double-disc method, MICs were determined and the results were interpreted as per the CLSI 2010 guidelines. [9] K. pneumoniae ATCC 700603 (positive control) and E. coli ATCC 25922 (negative control) were used for quality control of the ESBL tests. [9]

Extended-spectrum β-lactamase gene identification

Uniplex polymerase chain reaction (PCR) for the detection of TEM, SHV and CTX-M genes was carried out among 121 study isolates (determined to be ESBL producers by phenotypic methods) selected based on clinical significance of infection. DNA template preparation was performed as follows: fresh culture of the test organism and the control strains was suspended in 500 μl of saline and vortexed to get a uniform suspension. The bacterial cells were lysed by heating at 100°C for 10 min, cellular debris removed by centrifugation at 8000 rpm for 5 min and supernatant used as template. The master mix for the PCR was prepared as follows: 2.5 μl of PCR buffer, 2.5 l of 25 mM MgCl 2 , 2.5 μl of 2 mM DNTPs, 0.3 μl of Taq polymerase (Fermentas, Glen Brunie, MD, USA), 10.2 μl of MilliQ H 2 O and 1 μl of each of the forward and the reverse primers. Finally, after dispensing 20 μl of the master mix in the individual amplification tubes, 5 μl of the extracted DNA was added in the corresponding tubes to make up the total volume to 25 μl. The PCR primers and cycling conditions used are indicated in [Table 1]. The PCR products were analysed by electrophoresis with 1.5% agarose gels in 0.5X TBE buffer. The gels were stained with 5 μg/ml ethidium bromide and the PCR products were visualized with UV light. E. coli A-2-23 was used as the control strain for the PCR (Kind courtesy Dr Nina Langeland, Haukeland University Hospital, Norway).
Table 1: Primer pairs used for amplifi cation of the TEM, SHV and CTX-M ESBL types

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 ~ Results Top


Antimicrobial susceptibility

The proportion of ESBLs among the 300 study isolates was 78% (n = 234). [Table 2] shows the distribution of the MIC 50 and MIC 90 values of the study antimicrobials. The study isolates showed excellent susceptibility to imipenem (100%), meropenem (100%), ertapenem (98.7%), amikacin (89.7%) and piperacillin/tazobactam (85.3%). Reduced susceptibility to ertapenem among four Klebsiella spp. was encountered. The MIC 50 and MIC 90 of E. coli and Klebsiella spp. to ertapenem were 0.064 μg/ml and 0.38 μg/ml. ESBL isolates demonstrated a higher degree of multidrug resistance as compared with non-ESBLs.
Table 2: MIC50 and MIC90 distribution among E. coli (n = 167), Klebsiella spp. (n = 122) and Enterobacter spp. (n = 11)

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Molecular characterization

From [Table 3], it can be observed that ί-lactamases, 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%). The four Klebsiella strains non-susceptible to ertapenem were positive for TEM, SHV and CTX-M ESBL types. These strains were recovered from the Intensive Care Unit of a single institution around the same time period and had similar susceptibility profiles, thereby suggesting a single clone. In general, higher resistance was observed among isolates carrying all three (TEM, SHV, CTX-M) ESBL types [Figure 1].
Figure 1: Antimicrobial resistance pattern versus extended-spectrum ƒÀ-lactamase types among 121 study isolates (E. coli – 56, Klebsiella spp. – 61, Enterobacter spp. – 4)

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Table 3: Distribution of TEM, SHV and CTX-M ESBL types among 121 study isolates

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 ~ Discussion Top


Overall, the ESBL rate in the India SENTRY surveillance was 84%. Of the 163 Gram negative pathogens tested as part of the SARI (2004-2006) study, the prevalence of ESBL was 88%. [2] Studies from Lucknow, Delhi and Nagpur have reported the percentage of ESBL-positive isolates to be elevated. Notably, all of the ESBL-producing isolates were consistently susceptible only to carbapenems. [10],[11],[12],[13] 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, Bangalore, Chennai, Tamil Nadu and Kolkata, indicating that ESBL-positive strains are seen all over the country and are not restricted to any single city or region. [14] The high rate of ESBL prevalence in India and its widespread dissemination is a cause for worry.

Carbapenems are regarded as reserve agents for treating multidrug-resistant Gram negative bacterial infections. Carbapenem resistance among clinically significant Enterobacteriaceae in India remains low, with high rates of clinical success reported with its use in patients. Resistance to carbapenems is mediated through metallo beta lactamases, combinations of reduced accumulation and periplasmic space hydrolysis by β-lactamases.[7] In a recent study from a tertiary care hospital in India, 7% of the ESBL-producing clinical isolates were found to be resistant to ertapenem. [6] One potential benefit of ertapenem over many cephalosporins and extended-spectrum penicillins is its stability in the presence of ESBL and AmpC enzymes. For clinicians, this may be an important advantage to keep in mind in patients who fail beta-lactam therapy. The study results suggest that ertapenem can be used as an alternative to imipenem and meropenem. Amikacin and piperacillin/tazobactam may also be used in situations where susceptibility reports are available for ESBL-producing clinical isolates while levofloxacin and ceftriaxone are unsuitable as empiric choices to treat Gram negative infections in our setting.

The CTX-M group of ESBLs are diverse, with 30 alleles divided into five distinct phylogenetic groups. [15] 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. [2],[3] In a study conducted at a tertiary care centre in India, it was found that the majority of the ESBL-positive clinical isolates of Klebsiella spp. carried both TEM and SHV genes followed by TEM alone. [11] In our present study, TEM and CTX-M were predominantly found in E. coli (39.2%), while among Klebsiella spp., TEM, SHV and CTX-M occurred together (42.6%). The occurrence of TEM, SHV and CTX-M along with impermeability can cause resistance to carbapenems; [7],[16] this is worrisome especially in India where the ESBL prevalence is very high. The four Klebsiella strains non-susceptible to ertapenem were positive for all three ESBL types and were nosocomial in origin. This underscores the importance of rational antibiotic therapy, preventing spread of such strains in a hospital environment and understanding the clinical implications of ESBL types.

High rates of antimicrobial resistance among organisms causing clinically significant infections suggest monitoring mechanisms of antimicrobial resistance as well as comprehensively evaluating the recently introduced antimicrobial agents for their in vitro activity.


 ~ Acknowledgements Top


The authors are grateful to the Christian Medical College Institutional Review Board for approving the conduct of this study vide min no. 5926 dated 20 th June 2006 and Cipla India for funding the SARI study.

 
 ~ References Top

1.Paterson DL, Bonomo RA. Extended-spectrum â-Lactamases: A clinical update. Clin Microbiol Rev 2005;18:657-86.  Back to cited text no. 1
    
2.Mathai D, Manoharan A, Vasanthan G. Epidemiology and Implications of ESBL (2009). Crit Care Update 2009;14:152-62.  Back to cited text no. 2
    
3.Hawkey PM. Prevalence and clonality of extended-spectrum beta-lactamases in Asia. Clin Microbiol Infect 2008;14:159-65.  Back to cited text no. 3
    
4.Zhanel GG, Wiebe R, Dilay L, Thomson K, Rubinstein E, Hoban DJ, et al. Comparative review of the carbapenems. Drugs 2007;67:1027-52.  Back to cited text no. 4
    
5.Zhanel GG, Johanson C, Embil JM, Noreddin A, Gin A, Vercaigne L, et al. Ertapenem: Review of a new carbapenem. Expert Rev Anti Infect Ther 2005;3:23-39.  Back to cited text no. 5
    
6.Behera B, Mathur P, Das A, Kapil A. Ertapenem susceptibility of extended spectrum â-lactamase-producing Enterobacteriaceae at a tertiary care centre in India. Singapore Med J 2009;50:628.  Back to cited text no. 6
    
7.Livermore DM, Sefton AM, Scott GM. Properties and potential of ertapenem. J Antimicrob Chemother 2003;52:331-44.  Back to cited text no. 7
    
8.Jain A, Mondal R. TEM and SHV genes in extended spectrum beta-lactamase producing Klebsiella species and their antimicrobial resistance pattern. Indian J Med Res 2008;128:759-64.  Back to cited text no. 8
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9.CLSI. Performance Standards for Antimicrobial Susceptibility Testing; Twentieth Informational Supplement. CLSI Document M100-S20, Wayne, PA: Clinical and Laboratory Standards Institute; 2010 .   Back to cited text no. 9
    
10.Jain A, Mondal R. Prevalence and antimicrobial resistance pattern of extended spectrum â-lactamase producing Klebsiella spp isolated from cases of neonatal septicaemia. Indian J Med Res 2007;125:89-94.  Back to cited text no. 10
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11.Lal P, Kapil A, Das BK, Sood S. Occurrence of TEM and SHV gene in extended spectrum beta-lactamases (ESBLs) producing Klebsiella sp. isolated from a tertiary care hospital. Indian J Med Res 2007;125:173-8.  Back to cited text no. 11
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12.Hansotia JB, Agarwal V, Pathak AA, Saoji AM. Extended spectrum beta-lactamase mediated resistance to third generation cephalosporins in Klebsiella pneumoniae in Nagpur, central India. Indian J Med Res 1997;105:158-61.  Back to cited text no. 12
    
13.Manchanda V, Singh NP, Goyal R, Kumar A, Thukral SS. Phenotypic characteristics of clinical isolates of Klebsiella pneumoniae and evaluation of available techniques for detection of extended spectrum beta-lactamases. Indian J Med Res 2005;122:330-7.  Back to cited text no. 13
    
14.Hawser SP, Bouchillon SK, Hoban DJ, Robert E. Badal RE, Hsueh PR, et al. Emergence of High Levels of Extended-Spectrum-Lactamase-Producing Gram-Negative Bacilli 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. 14
    
15.Jemima SA, Verghese S. Multiplex PCR for blaCTX-M and blaSHV in the extended spectrum beta lactamase (ESBL) producing Gram-negative isolates. Indian J Med Res 2008;128:313-7.  Back to cited text no. 15
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16.Shah PM, Isaacs RD. Ertapenem, the first of a new group of carbapenems. J Antimicrob Chemother 2003;52:538-42.  Back to cited text no. 16
    


    Figures

  [Figure 1]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]

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