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Title: Poultry litter environment selects for the development of antibiotic resistance (AR) in Salmonella Heidelberg via conjugative IncX plasmids

Author
item OLADEINDE, ADELUMOLA - Orise Fellow
item Cook, Kimberly - Kim
item Plumblee Lawrence, Jodie
item Cox Jr, Nelson

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 5/16/2017
Publication Date: N/A
Citation: N/A

Interpretive Summary: The fitness of S. Heidelberg in poultry litter (PL) was determined following growth in nutrient rich media (BPW) or in poultry litter extracts (PLE) made from nutrient present in PL. S. Heidelberg BHI inoculum (from nutrient rich media) decreased rapidly in PL (even within 24 hr) while the concentration of S. Heidelberg PLE inoculum decreased by only two orders of magnitude in 9 days. To investigate the role of the inoculum source on S. Heidelberg, whole genome sequencing was performed on isolates recovered from BHI, BHI/PL, PLE and PLE/PL. All recovered isolates harbored a conjugative IncX (37kb) plasmid with several virulence and resistant determinants. For PLE/PL and Nal resistant isolates, a 23-bp insertion was detected in the promoter/operator region of the stbD/E toxin-antitoxin system. In addition, PLE/PL isolates carried a 23S rRNA mutation and Nal resistant isolates carried gyrA mutation. These findings suggest that acquired and mutational resistance contribute to the fitness of S. Heidelberg.

Technical Abstract: The fitness of S. Heidelberg in poultry litter (PL) was determined following growth preconditioning in either Brain Heart Infusion (BHI) broth or poultry litter extracts (PLE, a centrifuged and filter sterilized PL slurry). Isolates were monitored by direct culture count for up to 9 days. The concentration of S. Heidelberg BHI inoculum decreased by 5-orders of magnitude in PL within 24 h and was below limit of detection after 5 days, however, the concentration of S. Heidelberg PLE inoculum decreased by only two orders of magnitude in 9 days. To investigate the role of the inoculum source on the development of Nalidixic acid (Nal) resistance, we inoculated Brilliant Green Sulfur (BGS) agar containing 200 ppm of Nal with S. Heidelberg BHI or S. Heidelberg PLE or S. Heidelberg BHI passed through PL or S. Heidelberg PLE passed through PL and incubated at 37oC for 72 h. One colony was observed after 48 h for S. Heidelberg BHI. After being sub-cultured onto blood agar, multiple colony picks were required before stable Nal resistance was observed again. For PLE inoculum, a colony of S. Heidelberg appeared on Nal plates after 72 h and retained Nal resistance through subsequent passes on selective and non-selective media. Whole genome sequencing was performed on S. Heidelberg isolates recovered from BHI, BHI/PL, PLE and PLE/PL before and after passage on Nal plates. Plasmid and resistance gene identification was completed using SPAdes genome assembler and the CARD database. Recovered BHI isolates harbored a mutation in gyrA and one conjugative IncX plasmid. Isolates from PLE/PL before passage on Nal plates carried either 1 (22-36.8 kb) or 3 (12 kb) plasmids, while all isolates after passage on Nal plates carried 2 (12 kb and 25 kb). In addition to these plasmids containing sequences identical to the larger IncX plasmid, each also carried unique accessory genes including virulence and resistance determinants. These findings suggest that acquired and mutational resistance contribute to the fitness of S. Heidelberg.