Development of a Polymerase Chain Reaction Assay for the Detection of Antibiotic Resistance Genes in Community DNA

Abstract

Abstract. Many methods are used to detect antibiotic resistance genes in samples. The objective of the study reported here was to compare polymerase chain reaction (PCR) analysis of community DNA with fecal culturing for detecting antibiotic resistance genes in cattle samples. In the laboratory-based portion of this study, known concentrations of an Escherichia coli strain with 3 antibiotic resistance genes (cmy-2, flo, and cat) were added to feces from dairy cattle. These genes were used to assess the effect of various primer pairs, chromosomally versus plasmid-encoded genes, and gene copy number on the sensitivity of PCR amplification. Gene-specific PCR amplification was performed on the community DNA extracted from the feces. Feces were cultured for the inoculated strain. In the field-based portion of the study, 80 cattle fecal samples of unknown gene status were compared by use of similar methods. Culture and PCR amplification from community DNA extractions produced variable results, and this variability was most noticeable at dilutions that approached the detection limit of the assay. Typically, PCR amplification had a higher sensitivity than did culture for detecting the gene of interest. However, the sensitivity of culture was improved by plating on selective media containing antibiotics. The community DNA approach enables assessment of bacterial communities in complex samples such as feces, a task that can be prohibitive by budget or time constraints associated with culture methods. Through a strategic combination of culture and community DNA approaches, the relationship between specific selection pressures and the persistence and dissemination of specific resistance genes can be elucidated.

Effects of chlortetracycline and copper supplementation on the prevalence, distribution, and quantity of antimicrobial resistance genes in the fecal metagenome of weaned pigs

“…These isolate-based methods likely underestimate the true magnitude and also the underlying dynamics of AMR since the majority of bacteria are noncultivatable on existing culture media (Kim et al, 2011). Quantitative methods, based on the determination of gene copies from total community DNA, can give more accurate information on the impact of antimicrobial use on AMR element load in any given bacterial ecology (Patterson and Singer, 2006), such as the pig gut. Therefore, we used a culture-independent total community DNA approach to investigate the impact of in-feed therapeutic doses of CTC and elevated levels of copper supplementation on the presence, and quantity, of antimicrobial and copper resistance genes among the gut bacteria of pigs.…”

“…Antimicrobial resistance has been largely studied using culture-based methods; these typically involve bacterial isolation followed by sensitivity testing, or sometimes the testing of bacterial DNA isolated from bacterial culture for the presence of AMR genes (Patterson and Singer, 2006;Agga et al, 2014). These isolate-based methods likely underestimate the true magnitude and also the underlying dynamics of AMR since the majority of bacteria are noncultivatable on existing culture media (Kim et al, 2011).…”

Effects of Therapeutic Ceftiofur Administration to Dairy Cattle on Escherichia coli Dynamics in the Intestinal Tract

“…DNA was extracted from each fecal sample by using the QIAamp DNA stool mini kit (Qiagen Inc., Valencia, CA) and a modified protocol as previously described (29). Briefly, the following modifications were made to the manufacturer's recommended protocol: 0.1 g of sample was used instead of 0.2 g; all centrifugation steps were increased by 30 s; samples were incubated at 95°C instead of 70°C during the lysis step (no.…”

“…PCR templates were then stored at 4°C until used in PCR amplification. For detecting the plasmid-borne bla CMY-2 gene in the extracted fecal community DNA, we used a previously published protocol for which specificity had already been established (29). Briefly, a nested PCR design was used to ensure the most sensitive results, and this increased sensitivity was evaluated previously (29).…”

“…For detecting the plasmid-borne bla CMY-2 gene in the extracted fecal community DNA, we used a previously published protocol for which specificity had already been established (29). Briefly, a nested PCR design was used to ensure the most sensitive results, and this increased sensitivity was evaluated previously (29). Samples were amplified by one set of primers, and the product of that reaction was diluted and used as the template for a second PCR amplification.…”