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

Abstract

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.

Antimicrobial Resistance Determinants in Acinetobacter baumannii Isolates Taken from Military Treatment Facilities

“…As all four ARDM identifications met only the minimal requirements for a positive determination, these findings may indicate the potential nonspecific nature of these probes or the presence of probe sequence motifs that are conserved among major facilitator superfamily (MFS) efflux pumps resulting in false-positive identifications. Interestingly, presumptive false-positive tet (30) results have been previously observed with other A. baumannii isolates (26). Determinants conferring resistance to phenicol compounds.…”

“…While some isolates hybridized well to all probes representing the aadA1 and aadA2 family, others barely met the minimal threshold for positive determination; these variations in hybridization efficiency may reflect the presence of alleles which differ in DNA sequence composition by as much as 10% (aadA1, aadA1b, aadA2). These differences could potentially be exploited to identify the specific allele present (versus detecting a member of this gene family) if a wider variety of probes for these related alleles is included in the ARDM content (26).…”

“…Importantly, the content of ARDM v.2 has been refined from previously described ARDM v.1 (26) to minimize unnecessary redundancies, to include probes for Acinetobacter-specific genes, and to broaden the represented variety of genes conferring resistance to quaternary amines, streptothricin antibiotics, and 15 other classes of antimicrobial compounds. Due to the breadth of ARDM v.2 and its associated unbiased sample preparation, the results provide information on determinants not included in previous studies of MTF-derived strains as well as those not typically included in routine investigations of Acinetobacter.…”

“…from four sequenced strains (28), a gene was deemed present if at least half of its representative probes had signals above the 95% probe threshold (mean signal from lowest 2,128 probes ϩ 3 standard deviations [SD]) or if Ͼ70% of its probes had signals above either of two less stringent thresholds (mean signal from lowest 2,016 probes ϩ 3 SD or mean signal from lowest 2,128 probes ϩ 2 SD) (26).…”

Antimicrobial Resistance Prediction in PATRIC and RAST

“…The traditional culture-based detection of AMR phenotypes has drawbacks because many organisms are fastidious growers or may be unculturable89. Molecular approaches, including the high-throughput detection of biomarkers, has improved dramatically over the years101112 but is often limited by the number of markers that can be detected and is constrained by our current knowledge of AMR resistance mechanisms, which are evolving rapidly1131415.…”