|Year : 2016 | Volume
| Issue : 3 | Page : 359-361
Multidrug resistance mediated by co-carriage of extended-spectrum beta-lactamases, AmpC and New Delhi metallo-beta-lactamase-1 genes among carbapenem-resistant Enterobacteriaceae at five Indian medical centres
A Manoharan1, GS Barla2, R Peter2, M Sugumar2, D Mathai3
1 Pushpagiri Research Center, Pushpagiri Institute of Medical Sciences and Research Center, Thiruvalla, Kerala, India
2 Department of Medicine, Benjamin M Pulimood Laboratories for Infection, Immunity and Inflammation, Christian Medical College, Vellore, Tamil Nadu, India
3 Department of Medicine, Apollo Institute of Medical Sciences, Education and Research, Hyderabad, Telangana, India
|Date of Submission||24-Mar-2015|
|Date of Acceptance||28-Jun-2016|
|Date of Web Publication||12-Aug-2016|
Pushpagiri Research Center, Pushpagiri Institute of Medical Sciences and Research Center, Thiruvalla, Kerala
Source of Support: None, Conflict of Interest: None
In this study, we evaluated the coexistence of extended-spectrum beta-lactamases (ESBL), AmpC and New Delhi metallo-beta-lactamase-1 (NDM-1) genes among carbapenem-resistant Enterobacteriaceae (CRE) recovered prospectively from patients at multiple sites. The study included 285 CRE strains from 2782 Gram-negative Bacilli collected from multiple centres during 2007-2010, of which 87 were characterised. Standard and reference laboratory methods were used for resistance determination. Detection of blaNDM-1 , blaAmpC , blaTEM , blaSHV and blaCTX-M was done by polymerase chain reaction. High levels of antimicrobial resistance observed among study isolates. Co-carriage of ESBLs, AmpC and NDM-1 was 26.3%. Nosocomial origin among the co-carriage isolates was 64.3%, with 9.2% associated mortality.
Keywords: AmpC, carbapenem-resistant Enterobacteriaceae, extended-spectrum beta-lactamases, New Delhi metallo-beta-lactamase
|How to cite this article:|
Manoharan A, Barla G S, Peter R, Sugumar M, Mathai D. Multidrug resistance mediated by co-carriage of extended-spectrum beta-lactamases, AmpC and New Delhi metallo-beta-lactamase-1 genes among carbapenem-resistant Enterobacteriaceae at five Indian medical centres. Indian J Med Microbiol 2016;34:359-61
|How to cite this URL:|
Manoharan A, Barla G S, Peter R, Sugumar M, Mathai D. Multidrug resistance mediated by co-carriage of extended-spectrum beta-lactamases, AmpC and New Delhi metallo-beta-lactamase-1 genes among carbapenem-resistant Enterobacteriaceae at five Indian medical centres. Indian J Med Microbiol [serial online] 2016 [cited 2021 Jan 21];34:359-61. Available from: https://www.ijmm.org/text.asp?2016/34/3/359/188350
| ~ Introduction|| |
Antibiotic resistance is a global public health problem. Among Gram-negative organisms, antibiotic resistance is mediated through multiple mechanisms,  often resulting in multidrug resistance (MDR) and pandrug resistance. This poses a problem to clinicians regarding choice of antibiotic therapy.
Carbapenems are a group of beta-lactams, which are active against many clinically significant pathogens and stable against a wide variety of beta-lactamases including extended-spectrum beta-lactamases (ESBLs) and AmpC type enzymes due to their widest spectrum of antimicrobial activity. However, over the past decade, there have been increasing reports on the emergence and dissemination of carbapenem-resistant Enterobacteriaceae (CRE) worldwide. The recent emergence and spread of the New Delhi metallo-beta-lactamase-1 (NDM-1) is a case in point. 
In India, studies have recorded CRE prevalence rates of 12-50%.  However, limited scientific reports are available on the co-carriage status of multiple resistance genes (such as TEM, SHV, CTX-M, AmpC and NDM-1) among CRE. The objective of this study was to determine the presence CRE at multiple Indian medical centres and characterise the mediators of antimicrobial resistance (AMR) among these pathogens.
| ~ Materials and Methods|| |
Setting and study design
A total of 285 CRE among 2782 Gram-negative isolates recovered from clinically significant infections at five participating centres over 3 years (2007-2010) were included. The details of the participating sites and study methods are provided elsewhere.  From this collection, 87 isolates (Klebsiella sp. [n = 49]; Escherichia coli [n = 28] and Enterobacter sp. [n = 10] from bloodstream infections [BSIs; n = 37], urinary tract infections [UTI; n = 18], skin and soft tissue [SSTI; n = 23] and respiratory tract infections [RTI; n = 9]) were further characterised.
Study protocols and interpretations used were as per previous publication.  Carbapenemase production was determined by modified Hodge test. Results were further confirmed using combined disc diffusion test with imipenem (10 μg) alone and combination with EDTA (930 μg).  Minimum inhibitory concentration (MIC) was determined using E-strips (BioMerieux, Craponne, France).
Molecular characterisation of extended-spectrum beta-lactamases, AmpC and New Delhi metallo-beta-lactamase
The protocols and primers used were as previously published. ,,
| ~ Results|| |
Among 285 CRE isolates, 95.8% were ESBL producers. Maximum resistance to carbapenems was detected in Klebsiella sp. (56.5%), followed by E. coli (31.9%) and Enterobacter sp. (11.6%). A significant proportion of the isolates was from BSI (41.1%). 1.9% of patients were identified with infection within 48 h after admission, and 65% were hospitalised for more than 48 h. High rates of MDR were found among nosocomial infections (64.2%), followed by hospital-acquired infections (20.7%) and community-acquired infections (15.1%). Among these patients, 31.9% had a history of surgical interventions.
A high nosocomial infection rate (64.3%) was observed among the 87 CRE. Mortality was 9.2% among the patients with co-carriage isolates, with majority having a nosocomial origin of infection.
Antimicrobial resistance pattern
Of the 87 CRE, 28 (32.2%) were E. coli, 49 (56.3%) were Klebsiella sp. and 10 (11.4%) were Enterobacter sp. Following antibiotic susceptibility test, highest resistance was detected for piperacillin-tazobactam (97.7%, n = 85) and levofloxacin (97.7%, n = 85) each followed by amikacin (93.1%, n = 81). About 88.5% (n = 77) isolates were tigecycline susceptible.
Tigecycline showed the highest activity against CRE [Table 1]; all E. coli isolates were tigecycline susceptible (100%); 18.4% of Klebsiella isolates were resistant (MIC value ranging from 1 to 32 μg/ml). In general, high AMR was observed among the co-carriage strains with corresponding high MIC values to the study antimicrobials.
|Table 1: Distribution of minimum inhibitory concentration (μg/mL) and resistance among 87 co - carriage isolates |
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Molecular characterisation of multidrug resistance genes
Among the 87 CRE, 99% (n = 86) carried ESBLs genes followed by 63.2% NDM (n = 55) and 46% AmpC (n = 40). On analysis, it was found that in E. coli blaTEM , blaCTX-M , blaAmpC and blaNDM-1 occurred together (42.9%) whereas 12.2% of Klebsiella sp. carried blaNDM-1 along with blaTEM , blaSHV , blaCTX-M and blaAmpC . Overall 26.4% (n = 23) carried all the tested resistance determinant genes.
| ~ Discussion|| |
Multidrug resistance in Enterobacteriaceae is an emerging and evolving public health threat. Increasing reports of the rapid dissemination of CRE across multiple sites in India is a matter of concern. Tigecycline in this study continued to remain effective against 88.5% of CRE isolates, which again was well corroborated with the findings of Kumar et al., where they found 100% activity against all MDR Gram-negatives, with most of the isolates exhibiting an MIC value ranging from 0.25 to 8 μg/ml.  Although tigecycline remained the most active drug in our in-vitro study, emerging tigecycline resistance in Klebsiella pneumonia has been reported worldwide. , Moreover, it was consistent with our findings where 18.4% of the Klebsiella isolates were tigecycline resistant.
BSIs due to MDR Gram-negative bacteria are highly associated with ESBL production. In this study, the majority of the isolates were from BSI (n = 37/87) and were ESBL (99%) producers, which was similar to the findings of SMART study and other Indian multicentric study, where the ESBL rate among E. coli (79%) and Klebsiella pneumonia (72-74%) was high. ,
In this study, the majority of the (94.2%) isolates carried two or more of the beta-lactamase genes. These data are in agreement with the findings of another Indian study, where a majority of the isolates (73.68%) possessed two or more additional bla genes and 26.32% of isolates had only one additional bla gene in addition to blaNDM-1 . 
In this study, we found 26.3% of CRE having multiple co-carriage genes, which pose a significant problem to clinicians in choosing appropriate antibiotics. In conclusion, increasing MDR among Enterobacteriaceae is mediated by multiple enzyme-based resistance mechanisms. Nosocomial origin of a majority of study strains is a cause for worry.
| ~ Conclusion|| |
NDM carbapenemase is significantly associated with carbapenem resistance, and its detection in clinical microbiology laboratories is essential.
We acknowledge the Indian Council of Medical Research, New Delhi, for financial assistance (Reference-5/3/3/5/99-ECD-I [Vol VI]), and Christian Medical College, Vellore Institutional Review Board, for approving conduct of this study (R.C. No. 5342 dated April 20, 2004).
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| ~ References|| |
Drawz SM, Bonomo RA. Three decades of beta-lactamase inhibitors. Clin Microbiol Rev 2010;23:160-201.
Nordmann P, Naas T, Poirel L. Global spread of carbapenemase-producing Enterobacteriaceae
. Emerg Infect Dis 2011;17:1791-8.
Coudron PE. Inhibitor-based methods for detection of plasmid-mediated AmpC beta-lactamases in Klebsiella
spp. Escherichia coli
, and Proteus mirabilis
. J Clin Microbiol 2005;43:4163-7.
Manoharan A, Sugumar M, Kumar A, Jose H, Mathai D, Khilnani GC, et al
. Phenotypic and molecular characterization of AmpC β-lactamases among Escherichia coli
species and Enterobacter
species from five Indian medical centers. Indian J Med Res 2012;135:359-64.
Yong D, Toleman MA, Giske CG, Cho HS, Sundman K, Lee K, et al.
Characterization of a new metallo-beta-lactamase gene, bla (NDM-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.
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.
Nordmann P, Gniadkowski M, Giske CG, Poirel L, Woodford N, Miriagou V; European Network on Carbapenemases. Identification and screening of carbapenemase-producing Enterobacteriaceae
. Clin Microbiol Infect 2012;18:432-8.
Kumar S, Bandyopadhyay M, Mondal S, Pal N, Ghosh T, Bandyopadhyay M, et al.
Tigecycline activity against metallo-ß-lactamase-producing bacteria. Avicenna J Med 2013;3:92-6.
Sood S. Identification and differentiation of carbapenemases in Klebsiella pneumoniae
: A phenotypic test evaluation study from Jaipur, India. J Clin Diagn Res 2014;8:DC01-3.
Neonakis IK, Stylianou K, Daphnis E, Maraki S. First case of resistance to tigecycline by Klebsiella pneumoniae
in a European University Hospital. Indian J Med Microbiol 2011;29:78-9.
Hawser SP, Bouchillon SK, Hoban DJ, Badal RE, Hsueh PR, Paterson DL. Emergence of high levels of extended-spectrum-beta-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.
Reinert RR, Low DE, Rossi F, Zhang X, Wattal C, Dowzicky MJ. Antimicrobial susceptibility among organisms from the Asia/Pacific Rim, Europe and Latin and North America collected as part of TEST and the in vitro
activity of tigecycline. J Antimicrob Chemother 2007;60:1018-29.
Bora A, Ahmed G. Detection of NDM-1 in clinical isolates of Klebsiella pneumoniae
from Northeast India. J Clin Diagn Res 2012;6:794-800.