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  Table of Contents    
ORIGINAL ARTICLE  
Year : 2014  |  Volume : 57  |  Issue : 2  |  Page : 249-254
Prevalence of blaTEM , blaSHV and blaCTX-M genes in clinical isolates of Escherichia coli and Klebsiella pneumoniae from Northeast India


1 Department of Biotechnology, Gauhati University, Guwahati, Assam, India
2 Department of Microbiology, Gauhati Medical College and Hospital, Guwahati, Assam, India
3 Department of Microbiology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India
4 Department of Microbiology, Northumbria Healthcare NHS Foundation Trust, North Shields, NE29 8NH, United Kingdom

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Date of Web Publication19-Jun-2014
 

   Abstract 

Aim: This study was carried out to determine the presence of blaTEM , blaSHV and blaCTX-M genes in extended-spectrum β-lactamase (ESBL) producing Escherichia coli (E. coli) and Klebsiella pneumoniae (K. pneumoniae) at a tertiary care referral hospital in Northeast India. Materials and Methods: A total of 270 E. coli and 219 K. pneumoniae isolates were recovered during the period between August 2009 and July 2010. Kirby-Bauer disk diffusion method was performed to determine the antibiotic resistance pattern. Screening and phenotypic confirmatory test for ESBL production were performed using standard disc diffusion methods. Each of the initial ESBL screening test isolate was investigated for the presence of blaTEM , blaSHV and blaCTX-M genes via polymerase chain reaction (PCR) using gene-specific primers. Results: Phenotypic confirmatory test able to detect ESBL production in 73.58% of E. coli and 67.24% of K. pneumoniae. However, PCR amplification showed the presence of one or more ESBL genes in each of the initial ESBL screening positive isolate. Among three ESBL genotypes, the most prevalent genotype was found to be blaCTX-M in E. coli (88.67%) and blaTEM in K. pneumoniae (77.58%) ESBL producing isolates. Majority of ESBL producing isolates possess more than one ESBL genes. Conclusion: This study constituted a primer report on high prevalence of blaTEM and blaCTX-M genes in ESBL producing isolates of E. coli and K. pneumoniae and denotes the need of more extensive studies on these antibiotic genes to determine the magnitude of the problem of antibiotic resistance exiting in this locality.

Keywords: bla CTX-M, blaSHV , blaTEM , Escherichia coli, extended-spectrum β-lactamase, Klebsiella pneumoniae

How to cite this article:
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

How to cite this URL:
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 [serial online] 2014 [cited 2023 Mar 20];57:249-54. Available from: https://www.ijpmonline.org/text.asp?2014/57/2/249/134698



   Introduction Top


In the wide-ranging and complex world of β-lactamases, extended-spectrum β-lactamases (ESBLs) have played a leading role in the clinical field in recent decades. Their importance resides in the fact that they significantly expand the spectrum of previous β-lactamases to include hydrolysis of all penicillins, cephalosporins (with the exception of cephamycins) and aztreonam. [1] Furthermore, most of the genes encoding ESBLs are generally found on plasmids that conferred resistance to multiple antibiotic classes and are readily transferable between and within bacterial pathogens. [2] Majority of ESBLs are derived from the amino acid substitutions of their parent enzymes TEM and SHV β-lactamases, but there are several other types of acquired ESBLs found in Enterobacteriaceae. [3] Among these ESBLs, TEM, SHV and CTX-M types of are of being major clinical concern and predominated in Gram-negative bacteria. Production of TEM, SHV and CTX-M types of ESBLs are mediated by blaTEM, blaSHV and blaCTX-M genes, respectively. [4]

ESBL producing  Escherichia More Details coli (E. coli) and Klebsiella pneumoniae (K. pneumoniae) are being increasingly identified in many parts of the world and are prevalent in several countries in the Asia-Pacific region. [5] However, there are still scanty reports on the prevalence of ESBL producing E. coli and K. pneumoniae in Northeast Indian hospitals, primarily on molecular identification of clinically important β-lactamases. Considering the accelerated prevalence of ESBL producing E. coli and K. pneumoniae from different parts of our country, the present study was designed to ascertain the present scenario of ESBL production in clinical isolates of E. coli and K. pneumoniae as well as to reveal the prevalence of three major types of ESBL genotypes in both bacterial species at a tertiary care teaching hospital in Northeast India.


   Materials and methods Top


Bacterial isolates

A total of 489 consecutive, non-duplicate isolates of E. coli (n = 270) and K. pneumoniae (n = 219) were recovered from various clinical specimens at a tertiary teaching hospital in Northeast India. Distribution of the sources of these isolates was: Urine (n = 325), sputum (n = 83), pus (n = 38) and blood (n = 43). Samples were obtained from both outpatients and inpatients between August 2009 and July 2010. Standard microbiological techniques were used for isolation and identification of the isolates. [6] Prior to their testing, all the isolates were stored in 15% glycerol-supplemented Luria-Bertani medium at –80°C. The study was carried out with consent from the institutional ethics committee.

Antibiotic susceptibility testing

Kirby-Bauer disk diffusion method was performed on Mueller-Hinton agar (MHA) plates to determine the susceptibilities of different β-lactam and non-β-lactam antibiotics and results were interpreted as per Clinical and Laboratory Standards Institute (CLSI) guidelines. [7] The following antibiotics were tested: Ampicillin (10 μg), cephalothin (30 μg), cefotaxime (30 μg), ceftazidime (30 μg), cefpodoxime (10 μg), ceftriaxone (30 μg), aztreonam (30 μg), cefoxitin (30 μg), amikacin (30 μg), gentamicin (10 μg), co-trimoxazole (25 μg), ciprofloxacin (5 μg), nalidixic acid (30 μg), imipenem, (10 μg), meropenem (10 μg), ertapenem (10 μg) and piperacillin/tazobactam (100/10 μg). Minimum inhibitory concentration (MIC) of imipenem, meropenem and ertapenem were determined for the carbapenem resistant isolates by using Etest strips (bioMerieux, France) following the manufacturer's protocol. All the antibiotic discs and media were procured from HiMedia Laboratories Pvt, Ltd., Mumbai, India. E. coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 strains were used for quality control.

Screening for ESBL production

By disc diffusion susceptibility testing, any isolate with a zone diameter of ≤17 mm for cefpodoxime, ≤27 mm for cefotaxime, ≤22 mm for ceftazidime, ≤25 mm for ceftriaxone or ≤27 mm for aztreonam was considered to be screening positive isolate for ESBL production. [7]

Phenotypic confirmatory test for ESBL production

The phenotypic confirmatory test for ESBL production was performed as per CLSI guidelines. [7] For this purpose, following four antibiotic discs were used: Cefotaxime (30 μg), ceftazidime (30 μg), cefotaxime-clavulanic acid (30/10 μg) and ceftazidime-clavulanic acid (30/10 μg) (HiMedia Laboratories Pvt, Ltd., Mumbai, India). Discs were placed 25 mm apart on a MHA plate inoculated with 0.5 McFarland suspension of the test isolate. Plates were incubated at 35°C for 18 h at ambient atmosphere. After incubation the zone diameters around each of the disc were measured. A difference of ≥5 mm between the zone diameters of either of the cephalosporin discs and their respective cephalosporin/clavulanic acid disc was considered as positive phenotypic confirmatory test for ESBL production. E. coli ATCC 25922 was used as negative control and K. pneumoniae ATCC 700603 was used as ESBL positive control.

Detection of ESBL genotypes by polymerase chain reaction (PCR)

Each of the isolate positive in the initial screening test for ESBL production was examined for the presence of blaTEM , blaSHV and blaCTX-M genes by PCR amplification with the primers and conditions described previously [Table 1]. [8],[9],[10] The template deoxyribonucleic acid (DNA) was prepared from freshly cultured bacterial isolates by suspending 2-3 colonies in 500 μl of molecular grade water and then vortexed to get a uniform suspension. Bacterial cells were lysed by heating at 95°C for 10 min. Cellular debris was removed by centrifugation at 12,000 rpm for 2 min and the supernatant was directly used for DNA template for PCR. The concentration of each reagent in a PCR mixture was 100 ng of DNA template, 1X PCR buffer(Bangalore Genei, Bangalore, India), 0.5 μM of each primer, 250 μM of each dNTP (Fermentas) and 1 U of Taq polymerase (Bangalore Genei, Bangalore, India), in a final volume of 25 μl prepared by addition of molecular grade water. The PCR amplification for individual bla gene was carried out in a Peltier Thermal Cycler (PTC-100, MJ Research, USA). A previously confirmed K. pneumoniae isolate possessing blaTEM , blaSHV and blaCTX-M genes was used as a positive control and a negative control (nuclease-free water) was included in each run.
Table 1: List of primers used for PCR amplification

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   Results Top


Antibiotic resistance pattern of E. coli and K. pneumoniae isolates to different β-lactam and non-β-lactam antibiotics was found to vary widely. The percentage resistance rates of E. coli and K. pneumoniae isolates against 17 selected antimicrobial agents recovered from different clinical sources is shown in the [Figure 1], [Figure 2], [Figure 3], [Figure 4]. Majority of the E. coli and K. pneumoniae isolates were found to be multi-drug resistant (MDR) i.e., resistant to three or more antibiotics used in the study. A total of 86.3% of E. coli and 87.6% of K. pneumoniae isolates exhibited the MDR phenotypes. The distribution of MDR E. coli and K. pneumoniae isolates in various clinical samples are shown in the [Figure 5].
Figure 1: Antibiotic resistance pattern of urinary isolates of  Escherichia coli Scientific Name Search siella pneumonia

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Figure 2: Antibiotic resistance pattern of sputum isolates of Escherichia coli and Klebsiella pneumonia

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Figure 3: Antibiotic resistance pattern of pus isolates of Escherichia coli and Klebsiella pneumonia

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Figure 4: Antibiotic resistance pattern of blood isolates of Escherichia coli and Klebsiella pneumonia

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Figure 5: Distribution of multi-drug resistant Escherichia coli and Klebsiella pneumoniae Scientific Name Search  isolates in various clinical samples

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Interestingly, a total of 14 E. coli (5.18%) and 19 K. pneumoniae isolates (8.67%) isolates were found to be resistant to all 3 carbapenems used in the study. The distribution of carbapenem resistant E. coli and K. pneumoniae isolates in various clinical samples are shown in the [Figure 1], [Figure 2], [Figure 3], [Figure 4]. Among the E. coli carbapenem resistant isolates, the range for MIC values of imipenem, meropenem and ertapenem were 2-8 μg/ml, 3-16 μg/ml, 8->32 μg/ml and 2-6 μg/ml, respectively. Among the K. pneumoniae carbapenem resistant isolates, the range for MIC values of imipenem, meropenem and ertapenem were 2->32 μg/ml, 2->32 μg/ml and 6->32 μg/ml, respectively.

Out of 270 E. coli isolates, a total of 212 isolates (78.52%) showed positive results in initial screening test of ESBL production. Out of 219 K. pneumoniae isolates, a total of 174 isolates (79.45%) showed positive results in initial screening test of ESBL production. Distribution of ESBL screening positive E. coli and K. pneumoniae isolates in various clinical samples are shown in the [Table 2]. Among the isolates screened for ESBL production, 73.58% (156/212) E. coli and 67.24% (117/174) of K. pneumoniae showed positive results in phenotypic confirmatory test of ESBL production.
Table 2: Distribution of ESBL screening positive E. coli and K. pneumoniae isolates in various clinical samples

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In PCR detection of ESBL genotypes, all the ESBL screening positive isolates of both E. coli and K. pneumoniae were found to possess one or more ESBL genes tested in this study ([Figure 6], [Figure 7], [Figure 8]). Overall, 78.52% (212/270) of E. coli and 79.45% (174/219) of K. pneumoniae isolates were positive for one or more ESBL genes. Distribution of ESBL genotypes in E. coli and K. pneumoniae isolates from various clinical samples are shown in the [Table 3] and [Table 4], respectively. Overall incidence of ESBL genotypes in E. coli and K. pneumoniae isolates illustrates in [Figure 9].
Figure 6: Agarose gel showing polymerase chain reaction amplified product of blaTEM genes, lane M = 1 kb deoxyribonucleic acid (DNA ) ladder (Biochem Life Sciences, India), lane 1 and 3-5 = blaTEM positive amplicons (1080 bp), lane 2 = negative control (no template DNA added)

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Figure 7: Agarose gel showing polymerase chain reaction amplified product of blaSHV genes, lane M = 1 kb deoxyribonucleic acid (DNA ) ladder (Biochem Life Sciences, India), lane 1 and 3-5 = blaSHV positive amplicons (929 bp), lane 2 = negative control (no template DNA added)

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Figure 8: Agarose gel showing polymerase chain reaction amplified product of blaCTX-M genes, lane M = 1 kb deoxyribonucleic acid (DNA ) ladder (Biochem Life Sciences, India), lane 1 and 3-5 = blaCTX-M positive amplicons (544 bp), lane 2 = negative control (no template DNA added)

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Figure 9: Overall incidence of extended-spectrum ƒÀ-lactamase genotypes in screening positive Escherichia coli and Klebsiella pneumoniae isolates

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Table 3: Distribution of ESBL genotypes in E. coli isolates from various clinical samples

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Table 4: Distribution of ESBL genotypes in K. pneumoniae isolates from various clinical samples

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   Discussion Top


In India, majority of tertiary care hospitals are now facing the problem of treating infections with MDR E. coli and K. pneumoniae. [11] In this study, we also observed the high prevalence of MDR E. coli and K. pneumoniae isolates in various clinical samples. The overall prevalence of MDR phenotypes in E. coli K. pneumoniae isolates were 86.3% and 87.6%, respectively. Among the MDR E. coli and K. pneumoniae isolates, majority of them were ESBL producers. A very high rate of MDR phenotype was observed for blood isolates of both the organism, which was 94.00% for E. coli and 92.30% for K. pneumoniae. Previous reports from other parts of India also indicated the high prevalence of MDR E. coli and K. pneumoniae. [12],[13] Furthermore, the prevalence of MDR Enterobacteriaceae appears to be increasing throughout the world. [14]

Carbapenems are often the last line of effective treatment available for infections caused by MDR Enterobacteriaceae. [15] In the present study, imipenem, meropenem and ertapenem were found to be the most active antimicrobial agents, including the ESBLs producing isolates. However, the incidence of carbapenem resistant in E. coli (5.18%) and K. pneumoniae (8.67%) isolates reveals the most serious threat to the clinician. Among the carbapenem resistant isolates, higher level of resistance was observed against ertapenem than other carbapenems. Although carbapenem resistant isolates of both the organisms were negative in CLSI phenotypic confirmatory test for ESBL production, each of the isolate was found to possess one or more ESBL genes in genotypic detection. Increasing resistance to carbapenems and the event of superbug in Indian hospitals and community has now spurred the government and regulatory agencies to impose strict statutory guidelines for rational use of antibiotics.

By following the CLSI screening criteria for ESBL production, overall 78.52% of E. coli and 79.45% of K. pneumoniae isolates were screened for detection of ESBL production. Presence of one or more ESBL genes in all the screening positive isolates indicated the high prevalence of ESBL producing E. coli (78.52%) and K. pneumoniae (79.45%) in this geographical region. However, true prevalence of ESBL producing E. coli and of K. pneumoniae isolates in the community cannot be deduced as the samples were collected from only one tertiary centre.

In contrast to genotypic detection, the CLSI phenotypic confirmatory test for ESBL production found to be positive for only 57.78% of E. coli and 53.42% K. pneumoniae isolates in this study. There are a number of instances by which the phenotypic confirmatory tests for ESBL detection may be falsely positive or negative. Multiple factors contribute to this, including production of multiple different β-lactamase types by a single bacterial isolate and the production of ESBLs by organisms that constitutively produce the AmpC β-lactamases, varying substrate affinities and the inoculum effect. [16] PCR with oligonucleotide primers that are specific for a ESBL gene is the easiest and most reliable methods used to detect the presence of a ESBL. However, following PCR amplification of the blaTEM and blaSHV genes with oligonucleotide primers, sequencing is needed to discriminate different variants of TEM or SHV ESBLs. The PCR amplification of CTX-M-specific products without sequencing usually provides sufficient evidence that a blaCTX-M gene is responsible for ESBL production in an isolate. [17]

CTX-M-type ESBLs have emerged as the most common type of ESBL globally and their incidence easily surpassing those of SHV and TEM ESBLs in most locales. [18] Among three ESBL genotypes included in this study, the most prevalent genotype was found to be blaCTX-M in E. coli (88.67%) and blaTEM in K. pneumoniae (77.58%) ESBL producing isolates. The less prevalent ESBL genotype was blaSHV and the prevalence rate of blaSHV in ESBL producing K. pneumoniae isolates (50.57%) was higher than E. coli isolates (13.20%). All the ESBL producing isolates of both the organisms were found to be positive for one or more ESBL genotypes. None of the E. coli ESBL producing isolates was found to possess blaSHV alone, but this ESBL genotype was present in combination with blaTEM or/and blaCTX-M . In contrast, 8.04% of ESBL producing K. pneumoniae isolates were found to possess blaSHV alone, in addition to combination with blaTEM or/and blaCTX-M . It was observed that blaCTX-M alone was more prevalent in E. coli (45.28%, 96/212), while blaTEM and blaCTX-M together (27.58%, 48/174) were more prevalent in K. pneumoniae isolates. A previous study from India reported blaTEM and blaCTX-M together predominated in E. coli (39.2%), while blaTEM , blaSHV and blaCTX-M together predominated in Klebsiella spp (42.6%). [19] In the present study, the combination of blaTEM and blaCTX-M was found to possess in 33.01% of E. coli, while blaTEM , blaSHV and blaCTX-M together was found to possess in 20.11% of K. pneumoniae isolates. Previous studies from Indian hospitals also documented the presence of these ESBL genotypes in combination among the majority of third generation cephalosporin resistant E. coli and K. pneumoniae isolates. [20],[21] The occurrence of TEM, SHV and CTX-M along with impermeability can cause resistance to carbapenems; this is worrisome especially in India where the ESBL prevalence is very high. [19]


   Conclusion Top


Knowledge of the antibiotic resistance patterns and resistance genes of bacterial pathogens in a geographical area is of paramount importance for surveillance and control of antibiotic resistance as well as for guiding appropriate and judicious antibiotic usage. Though, the molecular methods are not possible to carry out routinely in the laboratories of developing countries, some extra-efforts such as PCR should be carried out for the correct identification of the genes involved in antibiotic resistance. Finally, more studies are needed from other hospitals of Northeast India to determine the magnitude of the problem of antibiotic resistance exiting in emerging bacterial pathogens like E. coli and K. pneumoniae.


   Acknowledgments Top


The first author (Arijit Bora) acknowledges University Grants Commission (UGC), India for providing UGC-RFSMS fellowship to carry out his research and Department of Science and Technology, Government of India for providing the visiting fellowship for N.E. Research student to carry out the molecular analysis part of this study in Department of Microbiology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, India.

 
   References Top

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Correspondence Address:
Giasuddin Ahmed
Department of Biotechnology, Gauhati University, Guwahati - 781 014, Assam
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0377-4929.134698

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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]

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15 Characterisation of extended spectrum ß-lactamase among Escherichia coli and Klebsiella pneumoniae associated with skin and urogenital tract infections in dog
SUSMI T PAULSON, SURYA SANKAR, BINU K MANI, V R AMBILY, THANKSY S AKKARA, ANU BOSEWELL, M MINI
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16 High Prevalence of Multidrug-Resistant Klebsiella pneumoniae Harboring Several Virulence and ß-Lactamase Encoding Genes in a Brazilian Intensive Care Unit
Roumayne L. Ferreira,Brenda C. M. da Silva,Graziela S. Rezende,Rafael Nakamura-Silva,André Pitondo-Silva,Emeline Boni Campanini,Márcia C. A. Brito,Eulália M. L. da Silva,Caio César de Melo Freire,Anderson F. da Cunha,Maria-Cristina da Silva Pranchevicius
Frontiers in Microbiology. 2019; 9
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17 Multidrug-Resistant Escherichia albertii: Co-occurrence of ß-Lactamase and MCR-1 Encoding Genes
Qun Li,Hong Wang,Yanmei Xu,Xiangning Bai,Jianping Wang,Zhengdong Zhang,Xiang Liu,Yimao Miao,Ling Zhang,Xinqiong Li,Nianli Zou,Guodong Yan,Xi Chen,Jie Zhang,Shanshan Fu,Ruyue Fan,Jianguo Xu,Juan Li,Yanwen Xiong
Frontiers in Microbiology. 2018; 9
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18 T4-like Escherichia coli phages from the environment carry bla CTX-M
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19 Molecular characterization of extended spectrum ß -lactamases enterobacteriaceae causing lower urinary tract infection among pediatric population
Nahla O. Eltai,Asmaa A. Al Thani,Khalid Al-Ansari,Anand S. Deshmukh,Eman Wehedy,Sara H. Al-Hadidi,Hadi M. Yassine
Antimicrobial Resistance & Infection Control. 2018; 7(1)
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20 Molecular detection of ß-lactamase and integron genes in clinical strains of Klebsiella pneumoniae by multiplex polymerase chain reaction
Mansour Sedighi,Masoumeh Halajzadeh,Rashid Ramazanzadeh,Noor Amirmozafari,Mohsen Heidary,Serve Pirouzi
Revista da Sociedade Brasileira de Medicina Tropical. 2017; 50(3): 321
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21 Discriminatory Power of Three Typing Techniques in Determining Relatedness of Nosocomial Klebsiella pneumoniae Isolates from a Tertiary Hospital in India
Swathi Purighalla, Sarita Esakimuthu, Mallika Reddy, George K. Varghese, Vijay S. Richard, Vasan K. Sambandamurthy
Indian Journal of Medical Microbiology. 2017; 35(3): 361
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22 New Delhi metallo-ß-lactamase and extended spectrum ß-lactamases co-producing isolates are high in community-acquired urinary infections in Assam as detected by a novel multiplex polymerase chain reaction assay
VV Borah, KK Saikia, P Chandra, NK Hazarika, R Chakravarty
Indian Journal of Medical Microbiology. 2016; 34(2): 173
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23 Prevalence of CTX-M resistance marker and integrons amongEscherichia coliandKlebsiella pneumoniaeisolates of clinical origin
S. Shaikh,S.M.D. Rizvi,R. Anis,S. Shakil
Letters in Applied Microbiology. 2016; 62(5): 419
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24 T4-like coliphage FKAZ14 virulent to pathogenic and extended spectrum ß-lactamase-producing Escherichia coli of poultry origin
Kaikabo Adamu Ahmad,Abdulkarim Sabo Mohanmmed,Faridah Abas,Sieo Chin Chin
Virologica Sinica. 2015; 30(1): 73
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25 Multidrug-resistantEscherichia coliin Asia: epidemiology and management
Hanna E Sidjabat,David L Paterson
Expert Review of Anti-infective Therapy. 2015; 13(5): 575
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