Antimicrobial Resistance Hidden within Multiserovar Salmonella Populations

Abstract

Salmonella enterica can exist in food animals as multiserovar populations, and different serovars can harbor diverse antimicrobial resistance (AMR) profiles. Conventional Salmonella isolation assesses AMR only in the most abundant members of a multiserovar population, which typically reflects their relative abundance in the initial sample. Therefore, AMR in underlying serovars is an undetected reservoir that can readily be expanded upon antimicrobial use. CRISPR-SeroSeq profiling demonstrated that 60% of cattle fecal samples harbored multiple serovars, including low levels of Salmonella serovar Reading in 11% of samples, which were not found by culture-based Salmonella isolation. An in vitro challenge revealed that Salmonella serovar Reading was tetracycline resistant, while more abundant serovars were susceptible. This study highlights the importance of AMR surveillance in multiserovar populations.

Keywords: CRISPR-SeroSeq; Salmonella; antimicrobial resistance; cattle; serovar Reading; tetracycline.

FIG 1

Salmonella populations in feedlot cattle feces are diverse and consist of multiple serovars. CRISPR-SeroSeq identified multiple serovars in 60% and 45% of Salmonella enriched fecal samples from day 0 (55 total samples) and day 20 (11 total samples), respectively. Day 0 samples that contain serovar Reading (yellow) are noted with an asterisk above the bar. Each color represents a different serovar as indicated, and the colors used are the same colors used by Ohta et al. (16). At the bottom of the figure, the gray numbers show the numbers of different serovars per sample. The cattle identification numbers are shown as black numbers; each unique number corresponds to a single steer. All cattle in this study were treated with in-feed CTC.

FIG 2

Salmonella serovar Reading that is present at low levels in untreated cattle is resistant to tetracycline. (A) CRISPR-SeroSeq identified six fecal samples collected before CCFA and CTC treatment that contained low levels of Salmonella serovar Reading. The relative Salmonella serovar frequency is represented by the thickness of the line in the Sankey plots, and each serovar is represented by a different color as labeled on the right and matches the color used by Ohta et al. (16). Each node on the left side of the Sankey plots represents an enriched fecal sample from a single steer; the cattle identification number is shown in bold type, and each number is unique to a single steer. The percentage values represent the percentages of Salmonella serovar Reading found in the samples. (B) Fecal samples enriched from cattle at day 0 were recovered from frozen glycerol stocks by culturing for 5 h in LB broth (white bars) and then cultured in the presence (black bars) or absence (diagonal bars) of 16 μg/ml tetracycline for 48 h. TaqMan-based qPCR assays targeting CRISPR sequences unique to each serovar were used to assess relative changes in serovar abundance. The numbers above the black bars represent the fold differences in DNA between treated and untreated samples at 48 h, calculated as the log₂ of the difference in C_T values. This experiment is representative of three independent experiments performed on separate days with different aliquots of the frozen culture. Sample 2313 was not included in these experiments as we could not reproducibly detect Salmonella serovar Reading at the 5-h time point. We suspect this is because its relative frequency was very low (0.2%), and the necessitated repeated freezing and thawing of the glycerol cultures damaged the integrity of that sample. For sample 2392, Salmonella serovar Mbandaka was undetectable after 40 cycles when cultured in tetracycline.