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

<div><p>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 <i>Acinetobacter baumannii</i>, <i>Escherichia coli</i> and <i>Klebsiella pneumoniae</i> 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.</p></div

ARDM v.1, PCR and phenotypic β-lactamase/ESBL profiles of the clinical and reference strains used in this study.

<p>N/A – not available. R – resistant, I – intermediate, S – sensitive.</p>a<p>The identified family to which the CTX-M allele belongs is indicated in parentheses.</p>b<p>While no official ATM resistance criteria for <i>A. baumannii</i> are available, all isolates of this species designated as resistant (R) in the above table showed no zone of inhibition around ATM disks.</p>c<p>Weakly positive detection by PCR.</p

ARDM content allocations for genes conferring resistance to different classes of antimicrobial compounds.

<p>Two hundred thirty-nine different resistance determinants are represented by probes on each sub-array.</p

Electrochemical signals from MDR reference strain, <i>A. baumannii</i> BAA-1710 (A) and antibiotic sensitive reference strain, <i>A. baumannii</i> 17978 (B).

<p>Each data point represents one probe; data are sorted according to alleles represented by each probe. In each panel, the horizontal dotted line indicates the “probe threshold” used to determine whether probes were considered positive (mean of lowest 95% probes+3 SD). For panel A, peaks correspond to the following alleles: <b><i>a</i></b>: <i>tet(G)</i> [9/9 probes positive]; <b><i>b</i></b>: <i>cat4</i> [5/7 probes positive]; <b><i>c</i></b>: <i>dfrA1</i> [9/9 probes positive]; <b><i>d</i></b>: <i>catA1</i> [6/8 probes positive]; <b><i>e</i></b>: <i>arr-3</i> [8/8 probes positive]; <b><i>f</i></b><i>: aph6/str(B)</i> [8/10 probes positive], <i>aph3″/str(A)</i> [10/10 probes positive], <i>aadA1b</i> [10/10 probes positive], <i>aadB</i> [10/10 probes positive]; <b><i>g</i></b>: <i>qacEΔ1</i> [10/10 probes positive]; <b><i>h</i></b>: <i>aadA2</i> [2/9 probes positive - allele deemed negative], <i>aadA1</i> [7/7 probes positive]; <b><i>i</i></b>: <i>bla</i>_VEB [9/9 probes positive]; <b><i>j</i></b>: <i>bla</i>_OXA-10 [7/7 probes positive]; <b><i>k</i></b>: <i>dfrA10</i> [8/10 probes positive]; <b><i>l</i></b>: <i>bla</i>_PSE-1/CARB [2/10 probes positive – allele deemed negative]; <b><i>m</i></b>: <i>aacC1</i> [9/9 probes positive], <i>aphA1</i> [10/10 probes positive]; <b><i>n</i></b>: <i>ant(2′)-Ia</i> [10/10 probes positive]; <b><i>o</i></b>: <i>sulI</i> [10/10 probes positive]; <b><i>p</i></b>: <i>tet(A)</i> [9/9 probes positive]. For panel B, the <i>sulII</i> allele (peak indicated) was deemed positive with 7/7 probes positive; other alleles had one or two positive probes, but were deemed negative.</p

Clinical isolates from Egyptian hospitals.

a<p>Clinical sites from which the isolates were obtained. CUH – Cairo University Hospital, Cairo, Egypt; AFH – Abbasia Fever Hospital, Cairo, Egypt; ASU – Assiut Fever Hospital, Assiut, Egypt; SAH – Shebeen Al Kom Hospital, Shebeen Al Kom, Egypt; ALX – Alexandria Fever Hospital, Alexandria, Egypt; TBRI – Theodor Bilharz Research Institute, Cairo, Egypt.</p>b<p>This ESBL strain containing <i>bla</i>_CTX-M-15 has been previously described under the designation of E450 (35).</p

ARDM analysis of reference strains.

a<p>ARDM-positive genes that have been validated by sequencing or PCR for strains whose genomes have not been fully sequenced are shown in bold.</p

Genetic MDR profiles from clinical isolates, excluding ESBLs.

a<p>Abbreviations: TET – tetracyclines; CHLOR – chloramphenicol; QAC – quaternary ammonium compounds; SUL – sulfonamides; TRI – trimethoprim; FQ - fluoroquinolones.</p>b<p>Genes responsible for resistance to β-lactams, which are not included in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069507#pone-0069507-t003" target="_blank">Table 3</a>.</p>c<p>Based on results obtained with the reference strains and PCR validation (data not shown) and may reflect false positives and/or truncated genes.</p