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.
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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)