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 ~  Abstract
 ~ Introduction
 ~ Methodology
 ~ Results
 ~ Discussion
 ~ Conclusion
 ~  References
 ~  Article Tables

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  Table of Contents  
ORIGINAL ARTICLE
Year : 2016  |  Volume : 34  |  Issue : 1  |  Page : 33-37
 

First description of SHV-148 mediated extended-spectrum cephalosporin resistance among clinical isolates of Escherichia coli from India


1 Department of Microbiology, Assam University Silchar, Silchar, India
2 Department of Life Science and Bioinformatics, Assam University Silchar, Silchar, India
3 Department of Microbiology, Silchar Medical College and Hospital, Silchar, India

Date of Submission27-Oct-2014
Date of Acceptance05-Oct-2015
Date of Web Publication15-Jan-2016

Correspondence Address:
Amitabha Bhattacharjee
Department of Microbiology, Assam University Silchar, Silchar
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0255-0857.174110

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 ~ Abstract 

Purpose: The present study was aimed to investigate the genetic context, association with IS26 and horizontal transmission of SHV-148 among Escherichia coli in Tertiary Referral Hospital of India. Methodology: Phenotypic characterisation of extended-spectrum beta-lactamases (ESBLs) was carried out as per CLSI criteria. Molecular characterisation of blaSHVand integron was carried out by polymerase chain reaction (PCR) assay and confirmed by sequencing. Linkage of IS26 with blaSHV-148was achieved by PCR. Purified products were cloned on pGEM-T vector and sequenced. Strain typing was performed by pulsed field gel electrophoresis with Xba I digestion. Transferability experiment and antimicrobial susceptibility was performed. Results: A total of 33 isolates showed the presence of SHV-148 variant by sequencing and all were Class 1 integron borne. PCR and sequencing results suggested that all blaSHV-148 showed linkage with IS26 and were present in the upstream portion of the gene cassette and were also horizontally transferable through F type of Inc group. Susceptibility results suggest that tigecycline was most effective. Conclusion: The present study reports for the first time of SHV-148 mediated extended spectrum cephalosporin resistance from India. Association of their resistance gene with IS26 and Class 1 integron and carriage within IncF plasmid signifies the potential mobilising unit for the horizontal transfer.


Keywords: Antimicrobial resistance, extended-spectrum beta-lactamases, horizontal gene transfer, IncF, integron, IS26, SHV-148


How to cite this article:
Maurya AP, Talukdar AD, Chanda DD, Chakravarty A, Bhattacharjee A. First description of SHV-148 mediated extended-spectrum cephalosporin resistance among clinical isolates of Escherichia coli from India. Indian J Med Microbiol 2016;34:33-7

How to cite this URL:
Maurya AP, Talukdar AD, Chanda DD, Chakravarty A, Bhattacharjee A. First description of SHV-148 mediated extended-spectrum cephalosporin resistance among clinical isolates of Escherichia coli from India. Indian J Med Microbiol [serial online] 2016 [cited 2019 Sep 15];34:33-7. Available from: http://www.ijmm.org/text.asp?2016/34/1/33/174110



 ~ Introduction Top


Infection with multidrug-resistant  Escherichia More Details coli is a challenging public health issue which creates increasing difficulty in the diagnosis and therapeutic options.[1],[2],[3] Evolution of resistance to β-lactam antibiotics in gram negative pathogens, particularly in E. coli commonly results in the production of extended-spectrum beta-lactamases (ESBLs) which confer resistance to expanded spectrum cephalosporins and monobactam.[2],[4] Plasmid encoded blaSHV represent a significant subgroup of Class A β-lactamases in which most of them are found in members of enterobacteriaceae family.[5] This gene is extensively disseminated along with integron gene cassette and mobilised through plasmid.[6] In addition, detection of this bla gene is understood to be highly significant as they are often related with treatment failure.[7]

The present study was aimed to investigate the genetic context and association with IS26 as well as horizontal transmission dynamics of SHV-148 among E. coli in Tertiary Referral Hospital of India.


 ~ Methodology Top


Bacterial strain and phenotypic testing

A total number of 121 consecutive, nonduplicate isolates of E. coli were collected for a period of 1 year (December 2011–November 2012) from Silchar Medical College and Hospital, India [Table 1]. All the isolates were phenotypically tested for ESBL production by initial screening followed by confirmation by combined disc diffusion method as per CLSI guidelines.[8]
Table 1: Clinical details and molecular background of SHV-148 harbouring Escherichia coli

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Molecular characterisation and sequencing

Polymerase chain reaction (PCR) was carried out for molecular characterisation and detection of blaESBL genes. A set of three primers were used: blaTEM, blaCTX-Mand blaSHV primers [9] under the following reaction conditions: Initial denaturation 95°C for 2 min, 34 cycles of 95°C for 20 s, 54°C for 1 min, 72°C for 1 min and final extension at 72°C for 7 min. Amplified PCR products were purified by Gene Jet PCR purification Kit (Thermo scientific, Lithuania). 25 µl of purified PCR products were used for sequencing along with ESBL gene primers. A previously confirmed isolate of E. coli harbour SHV obtained from the Department of Microbiology, IMS, BHU, Varanasi, was used as positive control for molecular characterisation.[10] The amplified product was sequenced, and sequencing result was compared by NCBI BLAST search.

Detection of genetic environment of extended-spectrum beta-lactamase genes

Integrase gene PCR was carried out for characterisation of Class 1 and Class 2 integron in all isolates using primers as described previously.[11] The location of SHV genes within integron was determined by amplification of whole cassette 5CS and 3CS primers.[11] All the purified PCR products were cloned on the pGEM-T vector (Promega, Madison, USA) and sequenced. The nucleotide sequences were analysed by using the software available on the National Centre of Biotechnology Information website (http://www.ncbi.nlm.nih.gov). Further linkage of IS26 with SHV gene was determined by PCR with following reaction conditions: Initial denaturation 95°C for 2 min, 35 cycles of 95°C for 20 s, 50°C for 45 s, 72°C for 2 min and final extension at 72°C for 7 min. The primer which was used in the study is described earlier.[12]

Plasmid preparation, transferability, incompatibility group typing and stability assay

Bacterial isolates were grown overnight in Luria Bertani (LB) broth (Hi-Media, Mumbai, India) containing 0.5 µg/ml of cefotaxime and plasmids were extracted using the QIAGEN ® Plasmid Mini Kit (Germany). The transformation was carried out using E. coli JM107 as recipient and selected on LB agar plates containing cefotaxime 0.5 µg/ml. Conjugation experiments were performed between the clinical isolates and streptomycin resistant E. coli strain B, as the recipient (Genei, Banglore). Incompatibility groups were determined by PCR-based replicon typing, targeting 18 different replicon types among all the transformants and transconjugants carrying blaSHV gene.[13] Plasmid fitness analysis of all blaSHV-148 producers as well as their transformants and transconjugants were analysed by serial passages method for 110 consecutive days at 1:1000 dilutions in LB broth without antibiotic pressure as described previously.[14] PCR assay was carried out for the presence of blaSHV in the isolates after each passage.

Strain typing

Genotyping of all the blaSHV harbouring E. coli isolates were performed by digestion of agarose plugs with Xba I and analysed by pulsed field gel electrophoresis (CHEF DRIII system, BIO-RAD, USA).

Susceptibility testing

Antimicrobial susceptibility of blaSHV harbouring isolates, transformants and transconjugants were determined by Kirby Bauer disc diffusion method.[15] Following antibiotics were used: Amikacin (30 μg), ciprofloxacin (30 μg), gentamicin (10 μg), tigecycline (15 μg), trimithoprim/sulphamethoxazole (1.25/23.75 μg), cefoxitin (30 μg), cefopodoxime (10 μg), ceftriaxone (30 μg), cefepime (30 μg), imipenem (10 μg), meropenem (10 μg), and aztreonam (30 μg) (Hi-Media, Mumbai, India). MIC's of donor strains and transformants as well as transconjugants with cefotaxime, ceftazidime, ceftriazone, cefepime, imipenem, meropenem, ertapenem and aztreonam (Hi-Media, Mumbai, India) were also determined.[8]


 ~ Results Top


All 33 isolates of E. coli were obtained from urine, pus, oral swabs, drain tip, sputum and stool collected from adult patients (20 to 60 years) in surgery, medicine, female burn unit, paediatrics, medicine, gynaecology and orthopaedics wards/clinics [Table 1]. Of these E. coli isolates, 71 strains (58.67%) were found resistant to expanded spectrum cephalosporins and were phenotypically ESBL positive. These 43 strains showed amplification with the target primers of which 33 were confirmed to be SHV-148 variant by sequencing [Table 1]. All these SHV-148 harbouring strains were carrying Class 1 integron, and the SHV-148 gene was located within integron gene cassette. blaSHV-148 was associated with IS26 and was present in the upstream portion of the gene cassette which was further confirmed by sequencing. Transformation and conjugation result could establish the fact that blaSHV-148 was horizontally transferable and was carried within IncF group of plasmids. The gene was quite stable in the wild type which retained the gene till 91 consecutive passages. Tigecycline was found to be most effective against the isolates. Donor strain, transformants as well as transconjugants, showed high MIC50 and MIC90 against all tested cephalosporins but were found below the break point against ertapenem and meropenem [Table 2] and [Table 3]. Typing results revealed that twenty-seven different clones were distributed in different wards and clinics of this hospital setting.
Table 2: MIC50 and MIC90 values of blaSHV-148 with other β-lactamases harbouring isolates, their transformants and transconjugants

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Table 3: MIC values of isolates, transformants and transconjugants harbouring only blaSHV-148

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 ~ Discussion Top


The prevalence rate of ESBL producing E. coli strains in clinical settings differs considerably over time and geographic location although it is a global problem. In our study 58.67% isolates were found to be ESBL positive, however in another report the authors reported a prevalence rate of 78% in India.[16] In our study, it was observed that an SHV-148 was associated with 5'-end of a Class 1 integron which may help in the mobilisation of this resistant determinant in the hospital environment. In the current study, it was also found that IS26 underlining the most important role in the mobilisation and expression of this gene. Transferability results of our study supported that SHV gene was horizontally transferable in this hospital settings between different wards/clinics. The transferability of blaSHV too has been reported earlier.[2] In our study blaSHV-148 was found to be stable till 91 passages and the IncF group plasmid is likely to be the genetic tool for the dissemination of this resistant gene in our hospital. This was also supported by the typing results. This study further advocates that tigecycline could be used as treatment option considering the development of resistance against carbapenem drug.


 ~ Conclusion Top


Thus this study, to the best of our knowledge, is the first report of SHV-148 mediated extended spectrum cephalosporin resistance from India. Presence of insertion sequence IS26 upstream of SHV-148 advocates its significant role in the mobilisation of blaSHV-148 and also emphasises emergence of this resistance genes from this part of the world, which may be tracked to implement proper infection control policy.

Financial support and sponsorship

University Grants Commissions (UGC-MRP) Government of India and Department of Biotechnology (DBT-NER twinning Scheme and Assam University Biotech Hub).

Conflicts of interest

There are no conflicts of interest.

 
 ~ References Top

1.
Alsultan AA, Aboulmagd E, Amin TT. ESBL-producing E. coli and K. pneumoniae in Al-Ahsa, Saudi Arabia: Antibiotic susceptibility and prevalence of blaSHV and blaTEM. J Infect Dev Ctries 2013;7:1016-9.  Back to cited text no. 1
    
2.
Fernandes R, Amador P, Oliveira C, Prudêncio C. Molecular characterization of ESBL-producing Enterobacteriaceae in Northern Portugal. ScientificWorldJournal 2014;2014:782897.  Back to cited text no. 2
    
3.
Leila N, Fereshteh S, Vajihe SN, Shoeib N. PER, CTX-M, TEM and SHV Beta-lactamases in clinical isolates of Klebsiella pneumoniae isolated from Tehran, Iran. Iran J Basic Med Sci 2010;13:111-8.  Back to cited text no. 3
    
4.
Tärnberg M, Nilsson LE, Monstein HJ. Molecular identification of (bla) SHV, (bla) LEN and (bla) OKP beta-lactamase genes in Klebsiella pneumoniae by bi-directional sequencing of universal SP6- and T7-sequence-tagged (bla) SHV-PCR amplicons. Mol Cell Probes 2009;23:195-200.  Back to cited text no. 4
    
5.
Bradford PA. Extended-spectrum beta-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. 5
    
6.
Davies J, Davies D. Origins and evolution of antibiotic resistance. Microbiol Mol Biol Rev 2010;74:417-33.  Back to cited text no. 6
    
7.
Pitout JD, Laupland KB. Extended-spectrum beta-lactamase-producing Enterobacteriaceae: An emerging public-health concern. Lancet Infect Dis 2008;8:159-66.  Back to cited text no. 7
    
8.
Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing; Twenty-First Informational Supplement. M100-S21. Wayne, PA, USA: CLSI; 2011.  Back to cited text no. 8
    
9.
Lee S, Park YJ, Kim M, Lee HK, Han K, Kang CS, et al. Prevalence of Ambler class A and D beta-lactamases among clinical isolates of Pseudomonas aeruginosa in Korea. J Antimicrob Chemother 2005;56:122-7.  Back to cited text no. 9
    
10.
Bhattacharjee A, Sen MR, Prakash P, Anupurba S. Role of beta-lactamase inhibitors in enterobacterial isolates producing extended-spectrum beta-lactamases. J Antimicrob Chemother 2008;61:309-14.  Back to cited text no. 10
    
11.
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. 11
    
12.
Eckert C, Gautier V, Arlet G. DNA sequence analysis of the genetic environment of various blaCTX-M genes. J Antimicrob Chemother 2006;57:14-23.  Back to cited text no. 12
    
13.
Carattoli A, Bertini A, Villa L, Falbo V, Hopkins KL, Threlfall EJ. Identification of plasmids by PCR-based replicon typing. J Microbiol Methods 2005;63:219-28.  Back to cited text no. 13
    
14.
Locke JB, Rahawi S, Lamarre J, Mankin AS, Shaw KJ. Genetic environment and stability of cfr in methicillin-resistant Staphylococcus aureus CM05. Antimicrob Agents Chemother 2012;56:332-40.  Back to cited text no. 14
    
15.
Castanheira M, Farrell SE, Deshpande LM, Mendes RE, Jones RN. Prevalence of ß-lactamase-encoding genes among Enterobacteriaceae bacteremia isolates collected in 26 U.S. hospitals: Report from the SENTRY Antimicrobial Surveillance Program (2010). Antimicrob Agents Chemother 2013;57:3012-20.  Back to cited text no. 15
    
16.
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.  Back to cited text no. 16
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