Molecular Characterization of Multidrug Resistant Hospital Isolates Using the Antimicrobial Resistance Determinant Microarray

PLOS ONE, Dec 2019

Molecular methods that enable the detection of antimicrobial resistance determinants are critical surveillance tools that are necessary to aid in curbing the spread of antibiotic resistance. In this study, we describe the use of the Antimicrobial Resistance Determinant Microarray (ARDM) that targets 239 unique genes that confer resistance to 12 classes of antimicrobial compounds, quaternary amines and streptothricin for the determination of multidrug resistance (MDR) gene profiles. Fourteen reference MDR strains, which either were genome, sequenced or possessed well characterized drug resistance profiles were used to optimize detection algorithms and threshold criteria to ensure the microarray's effectiveness for unbiased characterization of antimicrobial resistance determinants in MDR strains. The subsequent testing of Acinetobacter baumannii, Escherichia coli and Klebsiella pneumoniae hospital isolates revealed the presence of several antibiotic resistance genes [e.g. belonging to TEM, SHV, OXA and CTX-M classes (and OXA and CTX-M subfamilies) of β-lactamases] and their assemblages which were confirmed by PCR and DNA sequence analysis. When combined with results from the reference strains, ∼25% of the ARDM content was confirmed as effective for representing allelic content from both Gram-positive and –negative species. Taken together, the ARDM identified MDR assemblages containing six to 18 unique resistance genes in each strain tested, demonstrating its utility as a powerful tool for molecular epidemiological investigations of antimicrobial resistance in clinically relevant bacterial pathogens.

A PDF file should load here. If you do not see its contents the file may be temporarily unavailable at the journal website or you do not have a PDF plug-in installed and enabled in your browser.

Alternatively, you can download the file locally and open with any standalone PDF reader:

https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0069507&type=printable

Molecular Characterization of Multidrug Resistant Hospital Isolates Using the Antimicrobial Resistance Determinant Microarray

et al. (2013) Molecular Characterization of Multidrug Resistant Hospital Isolates Using the Antimicrobial Resistance Determinant Microarray. PLoS ONE 8(7): e69507. doi:10.1371/journal.pone.0069507 Molecular Characterization of Multidrug Resistant Hospital Isolates Using the Antimicrobial Resistance Determinant Microarray Tomasz A. Leski 0 1 Gary J. Vora 0 1 Brian R. Barrows 0 1 Guillermo Pimentel 0 1 Brent L. House 0 1 Matilda Nicklasson 0 1 Momtaz Wasfy 0 1 Mohamed Abdel-Maksoud 0 1 Chris Rowe Taitt 0 1 Shamala Devi Sekaran, University of Malaya, Malaysia 0 a Current address: Agricultural Research Service, US Department of Agriculture , Beltsville, Maryland , United States of America b Current address: US Naval Medical Research Center-Frederick, Fort Detrick, Maryland, United States of America c Current address: University of Gothenburg , Go teborg , Sweden 1 1 Center for Bio/Molecular Science and Engineering, US Naval Research Laboratory , Washington, D.C. , United States of America, 2 National Research Council Postdoctoral Associate at US Naval Research Laboratory , Washington, D.C. , United States of America, 3 US Naval Medical Research Unit No. 3, Cairo , Egypt Molecular methods that enable the detection of antimicrobial resistance determinants are critical surveillance tools that are necessary to aid in curbing the spread of antibiotic resistance. In this study, we describe the use of the Antimicrobial Resistance Determinant Microarray (ARDM) that targets 239 unique genes that confer resistance to 12 classes of antimicrobial compounds, quaternary amines and streptothricin for the determination of multidrug resistance (MDR) gene profiles. Fourteen reference MDR strains, which either were genome, sequenced or possessed well characterized drug resistance profiles were used to optimize detection algorithms and threshold criteria to ensure the microarray's effectiveness for unbiased characterization of antimicrobial resistance determinants in MDR strains. The subsequent testing of Acinetobacter baumannii, Escherichia coli and Klebsiella pneumoniae hospital isolates revealed the presence of several antibiotic resistance genes [e.g. belonging to TEM, SHV, OXA and CTX-M classes (and OXA and CTX-M subfamilies) of blactamases] and their assemblages which were confirmed by PCR and DNA sequence analysis. When combined with results from the reference strains, ,25% of the ARDM content was confirmed as effective for representing allelic content from both Gram-positive and -negative species. Taken together, the ARDM identified MDR assemblages containing six to 18 unique resistance genes in each strain tested, demonstrating its utility as a powerful tool for molecular epidemiological investigations of antimicrobial resistance in clinically relevant bacterial pathogens. - The evolution, increasing prevalence and dissemination of pathogenic bacteria resistant to multiple antimicrobial agents is currently recognized as one of the most important problems in global public health [1]. The rapid spread of antibiotic resistance genes, facilitated by mobile genetic elements such as plasmids and transposons, has led to the emergence of multidrug resistant (MDR) strains of many clinically important species that now frequently leave clinicians out of therapeutic options [2,3]. Traditional phenotypic methods currently used to determine antimicrobial resistance profiles (e.g., disk diffusion, broth microdilution) remain critical in guiding appropriate treatment options. However, techniques such as these are unable to determine the actual molecular mechanisms of resistance, and are especially lacking in situations where the observed phenotype is a result of the interaction of multiple gene products with overlapping activities [4]. Molecular techniques, such as PCR and DNA sequencing, have recently been employed to mitigate some of these deficiencies by identifying genes and genetic assemblages responsible for antibiotic resistance and MDR, monitoring the spread of resistance determinants, and elucidating the genetic elements responsible for the dissemination of resistance determinants. The use of DNA microarrays is another promising technology for the identification of antimicrobial resistance determinants in any number of species. Due to their inability to determine whether resistance determinants are expressed or gene products are functional, DNA microarrays are not intended to replace standard phenotypic testing. Rather, microarrays provide a powerful platform for molecular epidemiology and broad-based screening. This contention has been supported by a number of recent reports that have successfully applied a variety of microarray platforms for both limited [5,6,7,8] and broad spectrum [9,10,11] detection of antibiotic resistance genes. Furthermore, as they allow for the simultaneous detection of a large number of genes in a single assay, microarrays can be used to track determinants directed against multiple classes of (...truncated)


This is a preview of a remote PDF: https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0069507&type=printable

Tomasz A. Leski, Gary J. Vora, Brian R. Barrows, Guillermo Pimentel, Brent L. House, Matilda Nicklasson, Momtaz Wasfy, Mohamed Abdel-Maksoud, Chris Rowe Taitt. Molecular Characterization of Multidrug Resistant Hospital Isolates Using the Antimicrobial Resistance Determinant Microarray, PLOS ONE, 2013, 7, DOI: 10.1371/journal.pone.0069507