Evaluation of an Expanded Microarray for Detecting Antibiotic Resistance Genes in a Broad Range of Gram-Negative Bacterial Pathogens

Abstract

T he extensive use of antimicrobials has selected resistance in many bacterial species, and this has become a major public health issue worldwide (1). The problem of multidrug resistance is currently most acute in Gram-negative bacteria, where treatment options can be severely limited, and there are few promising new antibiotics with activity against Gram-negative bacteria under advanced development (1). Clonal spread of resistant strains and horizontal transfer of resistance genes both contribute to the rising global prevalence of multiresistant Gram-negative bacteria, with horizontal transfer enabling the acquisition of multidrug resistance by previously susceptible bacteria (2).Most methods currently employed by clinical diagnostic laboratories, e.g., the Vitek (bioMérieux, Marcy l'Etoile, France) and MicroScan (Siemens, Camberley, United Kingdom) (3) systems, provide insight only into the resistance phenotypes, require susceptibility testing following bacterial culture, and can take from approximately 9 to 20 h to obtain results. Molecular methods are faster but present their own challenges. In particular, conventional or real-time PCR, single-gene sequencing, or in situ hybridization usually detects only a few resistance genes simultaneously. Microarrays, however, offer the potential for simultaneous detection of large numbers of resistance genes, allowing prediction of an isolate's repertoire of resistances to multiple antibiotic classes (4-7).We previously developed a microarray that detected 51 resistance genes in Escherichia coli and Salmonella and facilitated epidemiological studies (4, 8). Its coverage included genes that conferred acquired resistance to a range of antimicrobials of clinical importance as well as two integrase genes associated with class 1 and class 2 integrons and not just those genes encoding ␤-lactamases (6). This work sought to extend the microarray to make it relevant to the broader range of Gram-negative bacterial genera commonly encountered in clinical diagnostic or reference laboratories and to include in its coverage newly critical gene groups, including genes encoding carbapenemases. MATERIALS AND METHODS Bacterial strains used and determination of antibiotic susceptibilities.A panel of 132 bacterial isolates from our laboratory collections and clinical isolates representing a diverse range of Enterobacteriaceae and nonfermenter genera, including Pseudomonas and Acinetobacter, was assembled. The antimicrobial susceptibilities (MICs) of these isolates to 19 antibiotics (tobramycin, amikacin, gentamicin, streptomycin, ampicillin, amoxicillin-clavulanic acid [co-amoxiclav], aztreonam, cefotaxime, ceftazidime, cefpirome, cefoxitin, piperacillin, imipenem, meropenem, chloramphenicol, ciprofloxacin, sulfonamide, tetracycline, and trimethoprim) were determined using the British Society for Antimicrobial Chemotherapy (BSAC) agar dilution methodology or disk diffusion (9). Susceptibility was defined using BSAC/European Committee on Antimicrobial Susceptibility Testing (EUCAST) clinic...

Impact of Ciprofloxacin and Clindamycin Administration on Gram-Negative Bacteria Isolated from Healthy Volunteers and Characterization of the Resistance Genes They Harbor

“…Most primers and probes included for this microarray work were described previously (19,20), with the exception of those for bla OXY and cfxA, which were validated during this study. New primers and probes for the genes bla CTX-M group 1, bla CTX-M group 2, qepA, qnrB, qnrC, and qnrS were also included and validated in this study.…”

“…For extraction of DNA, a 10-l loopful of bacteria was lysed, as described previously (20). The DNA was labeled in a linear multiplex reaction using primers described previously (19,20) and some newly designed primers (see below). For the anaerobic samples, the labeled DNA was pooled because previous work (19) with anaerobic bacteria had shown that carriage of resistance genes represented on the microarray was low; each pool comprised material from one volunteer at a single time point (one to six labeling reactions per pool).…”

“…Aerobic bacteria were grown at 37°C on blood agar plates, and anaerobic bacteria were grown on fastidious anaerobe agar as described previously (19). For extraction of DNA, a 10-l loopful of bacteria was lysed, as described previously (20). The DNA was labeled in a linear multiplex reaction using primers described previously (19,20) and some newly designed primers (see below).…”

Detection of the florfenicol resistance gene floR in Chryseobacterium isolates from rainbow trout. Exception to the general rule?

“…Isolates were screened for the presence of 75 antimicrobial resistance (AMR) genes using a 132 previously described DNA microarray (Card et al 2013). Approximately 1 μg of genomic DNA was 133 linearly amplified using antisense primers and simultaneously labelled with biotin.…”

DNA Microarray for Genotyping Antibiotic Resistance Determinants in Acinetobacter baumannii Clinical Isolates

“…Several microarrays for resistance detection in different species, genera, or other groups of bacteria have been developed (3)(4)(5)(6)(7)(8). One clinically relevant bacterial species for which no DNA microarray is yet available is Acinetobacter baumannii.…”