TY - JOUR
T1 - Development of a polymerase chain reaction assay for the detection of antibiotic resistance genes in community DNA
AU - Patterson, Sheila K.
AU - Singer, Randall S.
N1 - Copyright:
Copyright 2017 Elsevier B.V., All rights reserved.
PY - 2006/3
Y1 - 2006/3
N2 - 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.
AB - 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.
KW - Antibiotic resistance
KW - Community DNA
KW - DNA extraction
UR - http://www.scopus.com/inward/record.url?scp=33645363273&partnerID=8YFLogxK
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U2 - 10.1177/104063870601800206
DO - 10.1177/104063870601800206
M3 - Article
C2 - 16617698
AN - SCOPUS:33645363273
SN - 1040-6387
VL - 18
SP - 172
EP - 181
JO - Journal of Veterinary Diagnostic Investigation
JF - Journal of Veterinary Diagnostic Investigation
IS - 2
ER -