Abstract
With the alarming proliferation of antibiotic resistance, it is important to understand the de novo development of bacterial adaptation to antibiotics in formerly susceptible lineages, in the absence of external genetic input from existing resistance pools. A strain of ceftiofur susceptible Salmonella enterica serovar Enteritidis ABB07-SB3071 (MIC = 1.0 μg/ml) was successively exposed to sub-MIC of ceftiofur to allow its adaptation for tolerance to a concentration of 2.0 μg/ml of this antibiotic. Genomic and proteomic comparative analyses of the parental strain and induced tolerant derived lineages were performed to characterize underlying mechanisms of de novo adaptation (tolerance). Expression and localization of specific drug-, heme-, sugar-, amino acid-, and sulfate-transporters were altered, as was the localization of the cell membrane stabilizing protein OsmY in the tolerant strains adapted to 2.0 μg/ml compared to the parental isolate lines. This redistribution of existing transporters acts to minimize the concentrations of ceftiofur in the periplasm, by decreasing facilitated import and increasing active efflux and cytosolic sequestration as determined by high performance liquid chromatography quantification of residual total and extracellular ceftiofur after growth. Genetic, subcellular localization, and abundance changes of specific regulators of transcription, translation, and post-translational dynamics in the derived ceftiofur tolerant lineages decrease metabolic strain on cell walls and enhance periplasmic envelop stability against stress. This produces slower growing, more tolerant populations, which deplete free ceftiofur concentrations significantly more than susceptible parental populations (P < 0.05), as measured by recoverable levels of ceftiofur from cultures of equivalent cellular density incubated with equal ceftiofur concentrations. Genetic and abundance changes to specific carbon and nitrogen metabolism enzymes, not traditionally associated with beta-lactam metabolism, establish an enzymatic framework with the potential to detoxify/degrade ceftiofur, while mutations and changes in subcellular localization in specific cell surface factors enhance the stability of the Gram-negative cell envelop despite the compromising effect of ceftiofur. The observed changes highlight generalizable mechanisms of de novo tolerance without horizontal gene transfer, and thus can inform policies to combat antibiotic tolerance and minimize induction of de novo tolerance.
Highlights
Salmonella spp. infections are among the top three most prevalent sources of food-borne illness in Canada causing over 87,000 illness per year, and are an ongoing global health concern (Varga et al, 2015)
Based on Sensititre broth microdilution automated system results (Clinical and Laboratory Standards Institute [CLSI], 2018), MICs of ceftiofur and ceftriaxone, a closely ceftiofur-related antibiotic used in human medicine against the adapted lineages were 8.0 and 0.5 μg/ml compared to the parental strain (1.0 and 0. 25 μg/ml)
Compared to the parental strain, the 2.0 μg/ml ceftiofur-adapted lineages showed elevated MICs for several other antimicrobial agents including amoxicillin/clavulanic acid (2.0 vs. 8.0 μg/ml), ampicillin (1.0 vs. 16 μg/ml), chloramphenicol (8.0 vs. 16 μg/ml), ciprofloxacin (0.015 vs. 0.06 μg/ml), and nalidixic acid (2.0 vs. 8.0 μg/ml) (Table 1). These results clearly indicate that exposure of susceptible Enteritidis isolates to sub-MICs can lead to crossresistance to multi-antimicrobials
Summary
Salmonella spp. infections are among the top three most prevalent sources of food-borne illness in Canada causing over 87,000 illness per year, and are an ongoing global health concern (Varga et al, 2015). With the proliferation of antibiotic resistance in both sectors the need to understand how this pathogen changes and adapts to evade control strategies is a pressing need. As cephalosporins are among the front line antibiotics for the treatment of salmonellosis in humans the increasing prevalence of extended-spectrum cephalosporin resistant Salmonella in North America and Europe is concerning (Liakopoulos et al, 2016). Antimicrobial resistance acquisition processes are innate and ancient but may be exacerbated through the widespread use of antibiotics, especially in the absence of clear understandings of how tolerance develops. Resistance describes the inherited ability to grow at relatively high concentrations of a substance (Brauner et al, 2016), whereas a tolerant organism is heritably able to grow at higher levels of a substance than an ancestor, but may or may not be a high enough level to qualify as resistance
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