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Year : 2015  |  Volume : 142  |  Issue : 5  |  Page : 614-617

TEM mediated extended spectrum cephalosporin resistance in clinical & environmental isolates of Gram negative bacilli: A report from northeast India

Microbiology Laboratory, Department of Biotechnology & Bioinformatics, North Eastern Hill University, Shillong 793 022, Meghalaya, India

Date of Web Publication9-Dec-2015

Correspondence Address:
S R Joshi
Microbiology Laboratory, Department of Biotechnology & Bioinformatics, North Eastern Hill University, Shillong 793 022, Meghalaya
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0971-5916.171294

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How to cite this article:
Upadhyay S, Joshi S R. TEM mediated extended spectrum cephalosporin resistance in clinical & environmental isolates of Gram negative bacilli: A report from northeast India. Indian J Med Res 2015;142:614-7

How to cite this URL:
Upadhyay S, Joshi S R. TEM mediated extended spectrum cephalosporin resistance in clinical & environmental isolates of Gram negative bacilli: A report from northeast India. Indian J Med Res [serial online] 2015 [cited 2020 Sep 24];142:614-7. Available from:


The increase in the number of extended spectrum beta lactamase (ESBL) producing Gram-negative bacilli (GNB) has always been a threat to healthcare. ESBL producing bacteria are commonly isolated from hospitalized patients but their dissemination into the environment especially water bodies has been a cause of concern [1] . s0 ince ESBL genes are usually found encoded on mobile vectors such as plasmids (blaCTX-M , blaSHV ) and transposons (blaTEM ), the transfer of resistance between bacteria is easily facilitated [2],[3] .

The present study was aimed to investigate the presence of ESBL producing bacteria belonging to Enterobacteriaceae in water bodies (rivers, ponds, lakes) and clinical setting of a high altitude city of north eastern India, Shillong, the capital city of Meghalaya.

Water samples were collected in sterile vials from 31 water bodies including rivers, ponds, and tap water supply from Shillong, Meghalaya [Table 1]. For further analysis, serial dilutions were made starting with 1 ml of water samples diluted in 9 ml of saline solution (0.9% NaCl). A volume of 100 µl from each well homogenized dilution was inoculated onto the MacConkey agar plates (Hi-Media, Mumbai, India). Plates were incubated at 37°C for 24 h under aerobic conditions. All lactose fermenting and non-fermenting colonies with different colouration and morphology were picked from the selective plates, subcultured and stored in glycerol stock (2%) at -80°C.
Table 1. Location, number of water samples collected and resistance profile of isolates

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To further compare the ESBL producers obtained from water samples with the clinical isolates, 10 consecutive, non-duplicate, cephalosporin resistant clinical isolates were taken from the Microbiology department of Nazareth Hospital and The Children`s Hospital, Shillong. The study was carried out in the Microbiology laboratory of North Eastern Hill University (NEHU), Shillong, from August to December 2013.

All isolates were selected on the basis of their initial screening for presence of ESBL by combined disc diffusion method according to CLSI (Clinical and Laboratory Standards Institute) recommendation [4] . For amplification and characterization of blaESBL genes, a set of six primers was used namely: blaTEM , blaCTX-M , blaSHV , blaOXA-2 , blaOXA-10 and blaGES as described elsewhere [5] . Reaction mixture was prepared using Promega PCR master mix (Promega, USA) and reaction were run under the following conditions: initial denaturation 94°C for 5 min, 33 cycles of 94 °C for 35 sec, 51°C for 1 min, 72°C for 1 min and final extension at 72°C for 7 min. The amplicons were sequenced and compared by performing BLAST analysis ( Negative control used in the PCR reaction was Escherichia coli ATCC 25922, and three previously confirmed isolates from our laboratory of E. coli producing blaTEM, blaCTX-M , blaSHV were taken as positive control.

Screening for the presence of AmpC (class of C beta lactamases) and metallo beta-lactamases (MBL) was performed by cefoxitin disc test [6] and imipenem EDTA combined disc test [7] , respectively. Multiplex PCR was performed for detection of MBL and AmpC gene types. PCR conditions and primers were as described previously [8],[9] . For detection of class 1 and class 2 integron, integrase genes PCR were performed [10] . To find the genetic association of blaESBL gene with integrons, PCR was performed using forward primer of the conserved region (5'CS) of integron gene and reverse primer of the characterized ESBL gene as well as 3'CS region of integron and forward primer of ESBL gene [8] . Integrons containing functional genes known as gene cassettes, were amplified by 59 base elements PCR and amplicons were sequenced [11] .

Antimicrobial susceptibility testing was performed by Kirby Bauer disc diffusion method (CLSI, 2011) [4] on Muller-Hinton agar plates using antibiotics viz. cefopodoxime (10 μg), amikacin (30 μg), gentamicin (10 μg), ciprofloxacin (30 μg), trimithoprim/sulphamethoxazole (1.25 / 23.75 μg), tigecycline

(15 μg), cefepime (30 μg), imipenem (10 μg), meropenem (10 μg) and aztreonam (30 μg) [4] (Hi-Media, Mumbai, India).

A total of four bacterial isolates belonging to Enterobacteriaceae family and non-fermenting rods were confirmed in this study as ESBL producers [Table 2], two of which i.e. E. coli (R59) and Pseudomonas aeruginosa (R60) were from the environmental source (river water) and the remaining two were from clinical samples. Both the environmental isolates showed susceptibility towards imipenem, kanamycin, gentamicin, ciprofloxacin, tigecycline and polymixin B. Among the two ESBL producing isolates obtained from clinical samples, one KP7 (Klebsiella pneumoniae) was from the sputum of a one year old child admitted in intensive care unit, showed susceptibility to imipenem, ciprofloxacin, co-trimoxazole and polymyxin B while other isolate EC12 (E. coli) from the stool sample of a 16 yr old female was susceptible only towards polymyxin B.
Table 2. Details of blaTEM harbouring isolates

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Multiplex PCR assay yielded the product with expected size for blaTEM for two of the environmental isolates (R59 & R60) from river flowing outside the hospital area and also from the clinical isolates. However, sequencing could not reveal the exact variant of TEM type ESBL. Chromosomal AmpC was found in all four isolates while none of these were carrying any MBL gene. Among TEM negative environmental isolates, AmpC production was suspected in 12 isolates, but none of these were harbouring any plasmid AmpC gene as targeted by multiplex PCR. Presence of TEM and class 1 integron was demonstrated in all the four isolates while TEM was found to be cassette mediated only in case of clinical isolates.

The two isolates obtained from water bodies showed susceptibility towards all the tested antibiotics except cephalosporins. However, the clinical isolate EC12 conferred resistance against quinolones and aminoglycosides, whereas KP7 was resistant to all the antibiotics except polymixin B.

The prevalence of ESBL in north-eastern part of India found to be varied from 67-74 per cent [12] . These resistance determinants detected in the characterized isolates may easily disseminate to the community during irrigation using river water [1],[13],[14],[15] . Presence of these resistant genes within bacteria in river traces indicates their origin from anthropogenic sources, such as hospital, and municipal effluents [16] . It is quite alarming that this resistant determinant (TEM) is maintained within the organism in lotic water system where antibiotic pressure is minimum or absent.

In our country, most of the studies are focused on the beta lactamase gene obtained from isolates from hospital environment. However, this study underscores the persistence of the blaESBL along with gene capture mechanism in isolates from clinical and environmental sources. Maintenance of these resistant determinants in environmental reservoir system and their transmission to human host was evidenced by a recent study which showed that the healthy travellers acquired carbapenemases producing enterobacteriaceae upon their visit to India without any contact with local healthcare centres [17] . Thus, it may be suggested that appropriate measures are required to reduce the burden of antibiotic resistance in the environment as well as proper treatment of municipal and hospital waste water and improvement of water quality.

The present investigation was a pilot study, preliminary in nature and further research is necessary to determine the transferability of the resistant determinant or mechanism of gene transfer. Further, the presence of TEM mediated extended spectrum cephalosporin resistance would further restrict the therapeutic alternatives and infection control management strategies.

   Acknowledgment Top

The authors acknowledge the Departments of Microbiology, Nazareth Hospital and The Children`s Hospital, Shillong, for providing the clinical isolates. This work was sponsored by the University Grants Commission (UGC) under the Dr D.S. Kothari Postdoctoral Fellowship Scheme (No.F.4-2/2006(BSR)/13-927/2013(BSR) to the first author (SU).

   References Top

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Schink AK, Kadlec K, Schwarz S. Analysis of bla CTX-M carrying plasmids from Escherichia coli isolates collected in the BfT-GermVet study. Appl Environ Microbiol 2011; 77 : 7142-6.  Back to cited text no. 2
Bailey JK, Pinyon JL, Anantham S, Hall RM. Distribution of the bla TEM gene and bla TEM -containing transposons in commensal Escherichia coli. J Antimicrob Chemother 2011; 66 : 745-51.  Back to cited text no. 3
Clinical and Laboratory Standards Institute (CLSI). Performance standards for antimicrobial susceptibility testing; 21 st Informational Supplement: M100-S21. Wayne, PA, USA: CLSI; 2011.  Back to cited text no. 4
Lee S, Park YJ, Kim M, Lee HK, Han K, Kang CS, et al. Prevalence of Ambler class A and D â-lactamases among clinical isolates of Pseudomonas aeruginosa in Korea. J Antimicrob Chemother 2005; 56 : 122-7.  Back to cited text no. 5
Coudron PE, Moland ES, Thomson KS. Occurrence and detection of AmpC beta-lactamases among Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis isolates at a veterans medical center. J Clin Microbiol 2000; 38 : 1791-6.  Back to cited text no. 6
Yong D, Lee K, Yum JH, Shin HB, Rossolini GM, Chong Y. Imipenem-EDTA disk method for differentiation of metallo-b-lactamase-producing clinical isolates of Pseudomonas spp. and Acinetobacter spp. J Clin Microbiol 2002; 40 : 3798-801.  Back to cited text no. 7
Yong D, Toleman MA, Giske CG, Cho HS, Sundmon K, Lee K, et al. Characterization of a new metallo-beta-lactamase gene, blaNDM-1 , and a novel erythromycin esterase gene carried on a unique genetic structure in Klebsiella pneumoniae sequence type 14 from India. Antimicrob Agents Chemother 2009; 53 : 5046-54.  Back to cited text no. 8
Perez-Perez FJ, Hanson ND. Detection of plasmid-mediated AmpC â-lactamase genes in clinical isolates by using multiplex PCR. J Clin Microbiol 2002; 40 : 2153-62.  Back to cited text no. 9
Koeleman JG, Stoof J, Van Der Bijl MW, Vandenbroucke-Grauls CM, Savelkoul PH. Identification of epidemic strains of Acinetobacter baumannii by Integrase gene PCR. J Clin Microbiol 2001; 39 : 8-13.  Back to cited text no. 10
Stokes HW, Holmes AJ, Nield BS, Holley MP, Nevalainen KM, Mabbutt BC, et al. Gene cassette PCR: sequence-independent recovery of entire genes from environmental DNA. Appl Environ Microbiol 2001; 67 : 5240-6.  Back to cited text no. 11
Bora A, Hazarika NK, Shukla SK, Prasad KN, Sarma JB, Ahmed G. Prevalence of blaTEM , blaSHV and blaCTX-M genes in clinical isolates of Escherichia coli and Klebsiella pneumoniae from Northeast India. Indian J Pathol Microbiol 2014; 57 : 249-54.  Back to cited text no. 12
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Ruppe E, Armand-Léfevre L, Estellat C, El-Mniai A, Boussadia Y, Consigny P, et al. Acquisition of carbapenemase- producing Enterobacteriaceae by healthy travellers to India, France, February 2012 to March 2013. Euro Surveill 2014; 19 : pii: 20768.  Back to cited text no. 17


  [Table 1], [Table 2]

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