There is significant concern that agricultural use of antimicrobials leads to spill over of antimicrobial resistance (AMR) into the general population. Animal manures contain a large and diverse reservoir of AMR genes. While poultry litter is a rich source of nitrogen, it contains an abundance of AMR genes and genetic elements associated with its dissemination. Reduce this reservoir and its potential to transmit AMR to pathogens is diminished. Methods. The ability of litter microbiota to transmit AMR was assessed using a rifampicin (Rp)-resistant Salmonella Typhimurium LT2 strain, as the recipient, and an Escherichia coli pR100, plasmid donor strain. Autoclaved poultry litter served as a negative control in plasmid transfer studies. Abundance of phenotypic AMR was assessed for litter microbiota to the antibiotics: ampicillin (Ap; 25 ï g/ml), chloramphenicol (Cm; 25 ï g/ml), streptomycin (Sm; 100 ï g/ml), and tetracycline (Tc; 25 ï g/ml). qPCR was used to estimate gene load of streptomycin-resistance and sulfonamide-resistance genes aadA and sul, respectively in the poultry litter community. AMR gene load was determined relative to total population; using 16S rRNA qPCR to estimate the total bacterial load. Different growth conditions (temperature, media) and cell densities (filter vs liquid matings) were assessed to determine conditions optimal for AMR transfer from the litter community to the recipient Salmonella strain. Results. Poultry litter contained 108 CFU/g, with Gram-negative enterics representing a minor population (< 103 CFU/g). There was high abundance of resistance to Sm (106 CFU/g) and tetracycline (107 CFU/g); and a sizeable antimicrobial resistance gene (aadA- 2.63 E+6; sul- 7.01 E+10) load in poultry litter. Autoclaving reduced viable bacterial counts by >6 log10 and the antimicrobial resistance gene load was undetectable and by 4 log10 for aadA and sul, respectively. While plasmid transfer was observed between the Salmonella recipient with R100 plasmid, no AMR Salmonella were observed in matings with live or dead, poultry litter microbiota under conditions optimal for F-plasmid transfer. Conclusions. While poultry litter microbiota contains a significant AMR gene load, potential to transmit resistance is low, under conditions commonly used to assess plasmid conjugation. Autoclaving poultry litter significantly reduces the litter resistome. Several processes (ex. composting) are currently being examined to identify one that ameliorates poultry litter. / Master of Science / There is significant concern that agricultural use of antimicrobials leads to spill over of antimicrobial resistance (AMR) into the general population. Animal manures contain a large and diverse reservoir of AMR genes. While poultry litter is a rich source of nitrogen, it contains an abundance of AMR genes and genetic elements associated with its dissemination. Reduce this reservoir and its potential to transmit AMR to pathogens is diminished. Methods. The ability of litter microbiota to transmit AMR was assessed using a rifampicin (Rp)-resistant Salmonella Typhimurium LT2 strain, as the recipient, and an Escherichia coli pR100, plasmid donor strain. Autoclaved poultry litter served as a negative control in plasmid transfer studies. Abundance of phenotypic AMR was assessed for litter microbiota to the antibiotics: ampicillin (Ap; 25 ï g/ml), chloramphenicol (Cm; 25 ï g/ml), streptomycin (Sm; 100 ï g/ml), and tetracycline (Tc; 25 ï g/ml). qPCR was used to estimate gene load of streptomycin-resistance and sulfonamide-resistance genes aadA and sul, respectively in the poultry litter community. AMR gene load was determined relative to total population; using 16S rRNA qPCR to estimate the total bacterial load. Different growth conditions (temperature, media) and cell densities (filter vs liquid matings) were assessed to determine conditions optimal for AMR transfer from the litter community to the recipient Salmonella strain. Results. Poultry litter contained 108 CFU/g, with Gram-negative enterics representing a minor population (< 103 CFU/g). There was high abundance of resistance to Sm (106 CFU/g) and tetracycline (107 CFU/g); and a sizeable antimicrobial resistance gene (aadA- 2.63 E+6; sul- 7.01 E+10) load in poultry litter. Autoclaving reduced viable bacterial counts by >6 log10 and the antimicrobial resistance gene load was undetectable and by 4 log10 for aadA and sul, respectively. While plasmid transfer was observed between the Salmonella recipient with R100 plasmid, no AMR Salmonella were observed in matings with live or dead, poultry litter microbiota under conditions optimal for F-plasmid transfer. Conclusions. While poultry litter microbiota contains a significant AMR gene load, potential to transmit resistance is low, under conditions commonly used to assess plasmid conjugation. Autoclaving poultry litter significantly reduces the litter resistome. Several processes (ex. composting) are currently being examined to identify one that ameliorates poultry litter.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/110877 |
| Date | 17 May 2021 |
| Creators | Oxendine, Aaron Casey |
| Contributors | Animal and Poultry Sciences, Maurer, John J., Gerrard, David E., Johnson, Sally E. |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | ETD, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
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