Microbiology Section DOI : 10.7860/JCDR/2017/28540.10591
Year : 2017 | Month : Sep | Volume : 11 | Issue : 9 Page : DL01 - DL02

Universal Presence of blaNDM-1 Gene in Carbapenem-Resistant Gram-Negative Bacilli in an Indian Hospital in 2015

Biswaroop Chatterjee1, Naresh Khanduri2, Barnali Kakati3, Aarti Kotwal4

1 Associate Professor, Department of Microbiology, Himalayan Institute of Medical Sciences, Dehradun, Uttarakhand, India.
2 PhD Scholar, Department of Microbiology, Himalayan Institute of Medical Sciences, Dehradun, Uttarakhand, India.
3 Associate Professor, Department of Microbiology, Himalayan Institute of Medical Sciences, Dehradun, Uttarakhand, India.
4 Associate Professor, Department of Microbiology, Himalayan Institute of Medical Sciences, Dehradun, Uttarakhand, India.


NAME, ADDRESS, E-MAIL ID OF THE CORRESPONDING AUTHOR: Dr. Biswaroop Chatterjee, Associate Professor, Department of Microbiology, Himalayan Institute of Medical Sciences, Jolly Grant, Dehradun-248016, Uttarakhand, India.
E-mail: doctorbiswaroopchatterjee@gmail.com
Abstract

Dear Editor,

Carbapenem-resistant Gram-Negative Bacilli (GNB) which is simultaneously resistant to most other antimicrobials is now found in many hospitals worldwide [1]. Resistant strains are associated with high mortality; therefore, it is important to investigate resistance mechanisms to guide efforts to combat them.

The study started with all GNB strains (n=1544) isolated from routine clinical specimens received at the diagnostic laboratory of our tertiary-care hospital during the period January 2015 to June 2015. Out of these strains, 194 were found to be carbapenem-resistant by the Kirby-Bauer disc diffusion method, interpreted according to Clinical Laboratory Standard Institute (CLSI) guidelines [2], were included in the study. Subsequently, six isolates of Elizabethkingia meningoseptica and four of Stenotrophomonas maltophilia were excluded because of intrinsic carbapenem-resistance. Carbapenem Minimum Inhibitory Concentrations (MIC) of the remaining strains (n=184) were determined using E-test strips (bioMérieux, India) on Muller Hinton II Agar (Becton Dickinson, USA) [3]; MIC values were interpreted according to CLSI guidelines [2]. Mean carbapenem MICs of resistant isolates were above 25 μg/mL for all organisms/carbapenem combinations studied. Isolates were identified with standard biochemical methods [4], supplemented with Vitek 2 GNID panels if needed. Resistant strains were comprised of 104 isolates of Acinetobacter calcoaceticus-baumannii Complex (ACBC), 49 of family Enterobacteriaceae, and 31 of Pseudomonas aeruginosa. Among Carbapenem-Resistant Enterobacteriaceae (CRE), Klebsiellapneumoniae (17) and Escherichia coli (15) were the most common species, followed by Enterobacter cloacae (7), Enterobacter aerogenes (5), Citrobacter freundii (4), and Citrobacter koseri (1).

PCR was performed for blaNDM-1, blaVIM, blaKPC and blaOXA-48 carbapenemase genes with positive and negative controls in each run [5-7]. Phenotypic tests for carbapenemases were also used; these included Modified Hodge Test (MHT), Carba NP Test (CNPT), Blue Carba test (BCT), and Carba Acineto NP test (CANPT). MHT and CNPT were performed according to CLSI protocols [2], while BCT and CANPT were performed according to protocols in publications reporting these tests for the first time [8,9]. No phenotypic assay for carbapenemase detection had sensitivity above 90% in our hands when compared with PCR.

All GNB isolates with acquired carbapenem resistance carried the blaNDM-1 gene [Table/Fig-1]. In addition, the blaVIM gene was detected in 24 isolates, which included P. aeruginosa (n=20), Acinetobacter calcoaceticus-baumannii complex (n=3) and Enterobacter cloacae (n=1). The blaOXA-48 was detected only in K. pneumoniae (n=8). No isolate carried the blaKPC gene.

Carbapenemase genes in carbapenem-resistant Gram-negative bacilli. (Total strains 184)

AcinetobacterP. aeruginosaEnterobacteriaceae
Total strains 184
Number of strains carrying the gene concernedNDM-11043149
VIM032001
OXA-480008
KPC000

Studies on New Delhi Metallo-beta-lactamase 1 (NDM-1) in Southern Asia, starting with the seminal article by Kumarasamy KK et al. in 2010, are too numerous to quote [10]. The prevalence of blaNDM-1 gene in India has increased steadily since then, and a PubMed search revealed an article from 2012 reporting its presence in all (n=17) carbapenem-resistant isolates of K. pneumoniae in Guwahati, Assam, India [11]. Another study from 2014 reported the presence of blaNDM-1 gene in all [12] carbapenem-resistant isolates in Sharjah, UAE, where many patients travel frequently to Southern Asia [13]. However, ours is the first to report the universal presence of the blaNDM-1 gene in such a large number (n=184) of carbapenem-resistant isolates. Ours is also the first to report the high incidence (12.56%) of carbapenem-resistance in clinical isolates of GNB, and the presence of blaNDM-1, from our mountainous state in Northern India.

The universal presence of blaNDM-1 in our carbapenem-resistant isolates, along with similar or identical findings in other places in Asia, is worrisome because Ambler Class B metallo-beta-lactamases are not inhibited by the newly developed beta-lactamase inhibitors, avibactam and relebactam, which target serine carbapenemases of Ambler Class A and C only. This emphasizes the need to develop inhibitors of Ambler Class B carbapenemases. Fortunately, cyclobutanone and bisthiazolidine derivatives have displayed promising activity against metallo-beta-lactamases, and it is hoped that structural modifications will improve their activity to clinically significant levels in the near future [12,14].

To conclude, it is important to monitor the nature of carbapenemases to provide impetus to the development of newer inhibitors, and also guide their subsequent use, especially on an empiric basis. Since it is neither feasible nor economical to do this on all carbapenem-resistant isolates, nationally coordinated surveys must be done periodically with significant numbers of geographically representative isolates to maintain an up-to-date picture of resistance mechanisms in different parts of the country.

References

[1]Walsh TR, Emerging carbapenemases: A global perspective Int J Antimicrob Agents 2010 36(13):S8-S13.  [Google Scholar]

[2]Clinical and Laboratory Standard Institute. Performance standard for Antimicrobial Susceptibility Testing: Twenty-Fifth Informational Supplement M100-S25 Wayne, PA:CLSI: 2015  [Google Scholar]

[3]Steward CD, Mohammed JM, Swenson JM, Stocker SA, Williams PP, Gaynes RP, Antimicrobial Susceptibility Testing of Carbapenems: Multicenter Validity Testing and Accuracy Levels of Five Antimicrobial Test Methods for Detecting Resistance in Enterobacteriaceae and Pseudomonas aeruginosa Isolates J Clin Microbiol 2003 41(1):351-58.  [Google Scholar]

[4]Lennette EH, Balows A, Hausler WJ, Shadomy HJ, Manual of Clinical Microbiology 1985 4th EdnWashington, D.CAmerican Society for Microbiology  [Google Scholar]

[5]Monteiro J, Santos AF, Asensi MD, Peirano G, Gales AC, First report of KPC-2 producing Klebsiella pneumoniae strains in Brazil Antimicrob Agents Chemother 2009 53(1):333-34.  [Google Scholar]

[6]Monteiro J, Widen RH, Pignatari ACC, Kubasek C, Silbert S, Rapid detection of carbapenemase genes by multiplex real-time PCR J Antimicrob Chemother 2012 67(4):906-09.  [Google Scholar]

[7]Mendes RE, Kiyota KA, Monteiro J, Castanheira M, Andrade SS, Gales AC, Rapid detection and identification of metallo-β-lactamase-encoding genes by multiplex real-time PCR assay and melt curve analysis J Clin Microbiol 2007 45(2):544-47.  [Google Scholar]

[8]Pires J, Novais A, Peixe L, Blue-Carba, an Easy Biochemical Test for Detection of Diverse Carbapenemase Producers Directly from Bacterial Cultures J Clin Microbiol 2013 51(12):4281-83.  [Google Scholar]

[9]Dortet L, Poirel L, Errera C, Nordmann P, CarbAcineto NP test for rapid detection of carbapenemase-producing Acinetobacter spp J Clin Microbiol 2014 52(7):2359-64.  [Google Scholar]

[10]Kumarasamy KK, Toleman MA, Walsh TR, Bagaria J, Butt F, Balakrishnan R, Emergence of a new antibiotic resistance mechanism in India, Pakistan, and the UK: a molecular, biological, and epidemiological study Lancet Infect Dis 2010 10(9):597-602.  [Google Scholar]

[11]Bora A, Ahmed G, Detection of NDM-1 in clinical isolates of Klebsiella pneumoniae from North-East India J Clin Diagn Res 2012 6(5):794-800.  [Google Scholar]

[12]Drawza SM, Papp-Wallace KM, Bonomo RA, New β-Lactamase Inhibitors: a Therapeutic Renaissance in an MDR World Antimicrob Agents Chemother 2014 58(4):1835-46.  [Google Scholar]

[13]Dash N, Panigrahi D, Zarouni MA, Darwish D, Ghazawi A, Sonnevend A, High incidence of New Delhi metallo-beta-lactamase-producing Klebsiella pneumoniae isolates in Sharjah, United Arab Emirates Microb Drug Resist 2014 20(1):52-56.  [Google Scholar]

[14]Gonzalez MM, Kosmopoulou M, Mojica MF, Castillo V, Hinchliffe P, Pettinati I, Bisthiazolidinones: a substrate-mimicking scaffold as an inhibitor of the NDM-1 carbapenemase ACS Infect Dis 2015 1(11):544-54.  [Google Scholar]