Molecular Methods for Detection of Antimicrobial Resistance

The field of antibiotic drug discovery and the monitoring of new antibiotic resistance elements have yet to fully exploit the power of the genome revolution. Despite the fact that the first genomes sequenced of free living organisms were those of bacteria, there have been few specialized bioinformatic tools developed to mine the growing amount of genomic data associated with pathogens. In particular, there are few tools to study the genetics and genomics of antibiotic resistance and how it impacts bacterial populations, ecology, and the clinic. We have initiated development of such tools in the form of the Comprehensive Antibiotic Research Database (CARD; http://arpcard.mcmaster.ca). The CARD integrates disparate molecular and sequence data, provides a unique organizing principle in the form of the Antibiotic Resistance Ontology (ARO), and can quickly identify putative antibiotic resistance genes in new unannotated genome sequences. This unique platform provides an informatic tool that bridges antibiotic resistance concerns in health care, agriculture, and the environment.

To develop a rapid and reliable tool to detect by multiplex PCR assays the most frequently widespread beta-lactamase genes encoding the OXA-1-like broad-spectrum beta-lactamases, extended-spectrum beta-lactamases (ESBLs), plasmid-mediated AmpC beta-lactamases and class A, B and D carbapenemases. Following the design of a specific group of primers and optimization using control strains, a set of six multiplex PCRs and one simplex PCR was created. An evaluation of the set was performed using a collection of 31 Enterobacteriaceae strains isolated from clinical specimens showing a resistance phenotype towards broad-spectrum cephalosporins and/or cephamycins and/or carbapenems. Direct sequencing from PCR products was subsequently carried out to identify beta-lactamase genes. Under optimized conditions, all positive controls confirmed the specificity of group-specific PCR primers. Except for the detection of carbapenemase genes, multiplex and simplex PCR assays were carried out using the same PCR conditions, allowing assays to be performed in a single run. Out of 31 isolates selected, 22 strains produced an ESBL, mostly CTX-M-15 but also CTX-M-1 and CTX-M-9, SHV-12, SHV-5, SHV-2, TEM-21, TEM-52 and a VEB-type ESBL, 6 strains produced a plasmid-mediated AmpC beta-lactamase (five DHA-1 and one CMY-2) and 3 strains produced both an ESBL (two SHV-12, one CTX-M-15) and a plasmid-mediated AmpC beta-lactamase (DHA-1). We report here the development of a useful method composed of a set of six multiplex PCRs and one simplex PCR for the rapid screening of the most frequently encountered beta-lactamases. This method allowed direct sequencing from the PCR products.

OXA-48-type carbapenem-hydrolysing class D β-lactamases are increasingly reported in enterobacterial species. To date, six OXA-48-like variants have been identified, with OXA-48 being the most widespread. They differ by a few amino acid substitutions or deletions (one to five amino acids). The enzymes hydrolyse penicillins at a high level and carbapenems at a low level, sparing broad-spectrum cephalosporins, and are not susceptible to β-lactamase inhibitors. When combining permeability defects, OXA-48-like producers may exhibit a high level of resistance to carbapenems. OXA-163 is an exception, hydrolysing broad-spectrum cephalosporins but carbapenems at a very low level, and being susceptible to β-lactamase inhibitors. The bla(OXA-48)-type genes are always plasmid-borne and have been identified in association with insertion sequences involved in their acquisition and expression. The current spread of the bla(OXA-48) gene is mostly linked to the dissemination of a single IncL/M-type self-transferable plasmid of 62 kb that does not carry any additional resistance gene. OXA-48-type carbapenemases have been identified mainly from North African countries, the Middle East, Turkey and India, those areas constituting the most important reservoirs; however, occurrence of OXA-48 producers in European countries is now well documented, with some reported hospital outbreaks. Since many OXA-48-like producers do not exhibit resistance to broad-spectrum cephalosporins, or only decreased susceptibility to carbapenems, their recognition and detection can be challenging. Adequate screening and detection methods are therefore required to prevent and control their dissemination.