Class 1 integrons were detected in fecal and water samples from farm and non- ... Colorado State University, Fort Collins, CO 80523-1171, USA. ABSTRACT. Antibiotic ... Microbial colonies were analyzed for Class 1 integrons with. RTPCR ...
Screening for antibiotic resistance genes and Class 1 integrons in commensal bacteria in agricultural and other environments and their potential transfer to pathogenic bacteria Hua Yang, Oleksandr A. Byelashov, Ifigenia Geornaras, Lawrence Goodridge, Kendra K. Nightingale, Keith E. Belk, Gary C. Smith, and John N. Sofos Center for Meat Safety & Quality and Food Safety Cluster, Department of Animal Sciences, Colorado State University, Fort Collins, CO 80523-1171, USA SAMPLE COLLECTION SITES
ABSTRACT
Figure 1. Total bacterial counts (TSA/DS) and numbers of antibiotic-resistant populations (TSA/DS/Tc and TSA/DS/Cef) in soil, feces, water, and other (feed, bedding, manure, and compost) samples
Antibiotic resistance genes can spread between commensal bacteria and foodborne pathogens via Class 1 integrons. We evaluated the prevalence of tetracycline- and ceftiofur-resistant bacteria which carry these mobile elements in farm and non-farm (urban and natural) environments. Samples from Colorado farms (tap, animal drinking and waste water, soil, feed [unused], bedding [unused], compost, manure and feces) and non-farm locations (lakes, rivers, soil, and animal feces in parks within city limits, and the Rocky Mountain National Park) were analyzed for total microbial counts and tetracycline- and ceftiofur-resistant populations on agar plates and using real-time PCR (RTPCR). Microbial colonies were analyzed for Class 1 integrons with RTPCR, followed by agarose gel electrophoresis and sequencing. The genus of Class 1 integron-positive isolates was identified and MIC for various antibiotics tested. Seventy-two water, 120 fecal, 96 soil, and 42 feed, bedding, compost, and manure samples were analyzed. Levels of total bacteria in soil, feces, feed, bedding, compost, manure and waste water samples were approximately 7 to 9 logs. Overall, in all samples, numbers of tetracycline-resistant bacteria were similar to those of ceftiofurresistant bacteria. The percentages of Class 1 integron-positive isolates were 1.80 for farm and 0.66 for non-farm environments. These isolates included Arthrobacter, Alcaligenes, Brevundimonas, Corynebacterium, and Microbacterium spp. Currently, the rate of conjugative gene transfer between isolates and foodborne pathogens is being determined. These results should contribute to a better understanding of the levels and distribution of antibiotic-resistant bacteria in the environment.
OBJECTIVES (1) To assess the distribution of antibiotic resistance genes and Class 1 integrons in animal fecal samples and agricultural environment samples; non-farm environment samples were tested as well, for comparison. The obtained information should provide a better understanding of the potential reservoirs of antibiotic resistance genes present in farm and other environments. (2) For commensal isolates that are Class 1 integron-positive, conjugation experiments are being conducted in the sample in which the Class 1 integron-positive isolates were originally present to estimate their potential transfer to pathogenic bacteria under various conditions.
EXPERIMENTAL DESIGN Samples (from farm and non-farm environments) RTPCR for Tet and Cef genes
MIC for various antibiotics Bacterial identification
Plating on TSADS or TSADS + Tet or Cef
Tet or Cefresistant isolates RTPCR for Class 1 integrons
Class 1 integron-positive isolates
DNA sequencing for antibiotic-resistance gene cassettes
Conjugative transfer of Class 1 integrons to pathogens
ACKNOWLEDGEMENTS
10
Farm Non-farm
log CFU/g
8 6 4 2 0 TSA/DS
Non-farm environments
• A total of 330 samples were collected over a 7-month period (from June to January) from two cattle operations and non-farm environments including urban and natural (Rocky Mountain National Park) areas in Colorado. • Total microbial populations in soil, feces, feed (unused), bedding (unused), compost, manure, and waste water samples ranged between 7 and 9 log CFU/g or /ml. Numbers of total bacteria in tap water (farm) were comparable to those in rivers and lakes (non-farm) (Fig. 1). • In all samples, numbers of tetracycline- (≥16 µg/ml) and ceftiofur- (≥8 µg/ml) resistant bacteria, including naturally resistant populations, were lower than total bacterial counts in respective samples (Fig. 1). • In soil samples from farm and urban environments, plating and real-time PCR (RTPCR) methods showed similar results for total bacteria (data not shown). • However, the levels of tetracycline-resistant bacteria estimated by RTPCR (
