The effect of bacterial warfare on resistance in Salmonella. Towards a more optimal selection of Live Microbial Formulations.

The rapid rise of antibiotic resistance is compromising our ability to effectively control bacterial infections. There is a pressing need for alternative treatments to substitute or complement the use of antibiotics. Probiotics are increasingly attracting attention in this regard, particularly due to their multifaceted modes of action to combat pathogens. Despite their promising potential and long history of use, the exact ways in which they interact with pathogens are not well characterized. Therefore, understanding these social interactions will be pivotal to optimize probiotic treatment in the future. In light of the rising use of probiotics as an alternative treatment to combat antibiotic-resistant pathogens, it will become crucial to assess their efficacy against pathogens that previously developed resistance to clinically employed antibiotics. We hypothesized that antibiotic resistance mechanisms can affect probiotic efficacy, and therefore sought to examine the exact interplay between probiotics and antibiotic-resistant pathogens. In this work, we investigate by use of a series of in vitro assays whether some of the interactions could improve probiotic efficacy, and in this way potentially provide us with an advantage in the fight against antibiotic resistance.

In the first part of this research, we examine whether combinatorial treatment with probiotics and antibiotics can serve as a promising risk-spreading strategy in case the pathogen carries a public resistance mechanism. We investigate whether the public resistance mechanism can provide protection to a probiotic in cotreatment with an antibiotic, potentiating the probiotic to inhibit the pathogen when the antibiotic alone is not able to. Hereto, we perform competition experiments between β-lactamase-producing Salmonella Typhimurium and susceptible Escherichia coli Nissle under cefotaxime treatment. We vary factors that were predicted to influence exploitation of the susceptible probiotic, like antibiotic dose, timing of treatment and inoculum density/frequency. We present evidence that protection of the susceptible probiotic occurs under many of the tested conditions. Protection was also observed for another strain that inhibits Salmonella, Bacillus subtilis PS216. This strain populates a distinct niche from Salmonella, indicating the genericity of our findings, and underlining the strongly shared character of the resistance mechanism. Additionally, we find that protection of, and inhibition by the probiotic are correlated, indicating that protection of the probiotic potentiates its inhibitory actions. In current clinical practice, combinatorial treatment options are limited due to killing of the probiotic by the antibiotic. Our work highlights the future potential of applying combinatorial treatment as a risk-spreading strategy to combat pathogens that potentially carry a public resistance mechanism.

In the second part of this research, we study whether resistance against clinical antibiotics can increase (= collateral sensitivity) or decrease (= cross-resistance) susceptibility against probiotics. To study this, a high-throughput flow cytometry-based assay is first optimized to allow the growth and rapid enumeration of a large number of cocultures containing Salmonella and probiotics. To achieve this, a suitable growth medium is selected, and a search is conducted for a genomically integrated fluorescent protein bright enough to allow distinction between Salmonella and probiotics. As the Biofab:DsRed2 construct displays this property, it is used in all subsequent experiments to label Salmonella. Next, the optimized flow cytometry assay is employed to compete a wide range of antibiotic-resistant Salmonellae with eight probiotic strains in a search for combinations that display alterations in the fitness of Salmonella. The antibiotic-resistant strains either originate from experimental evolution with antibiotics or are plasmid-encoded. Within our dataset, no strong indications for cross-resistance are found, whereas collateral sensitivity events are abundant. The carriage of certain resistance mechanisms substantially lowers Salmonella’s fitness in competition with certain probiotics, and in some cases leads to faster outcompetition when competed for multiple days. Moreover, aberrant growth in monospecies conditions is found not to be correlated to a reduced fitness in competition with probiotics. These findings suggest that specific resistance mechanisms directly influence probiotic susceptibility. Whole genome sequencing reveals an overall low mutation load for most strains and allows the formulation of preliminary hypotheses on underlying mechanisms that could guide further work.

Our research endeavors highlight the potential of probiotic employment to combat antibiotic-resistant pathogens, as two distinct scenarios were explored in which the potential benefits were demonstrated of utilizing probiotics as an alternative or complementary treatment to antibiotics. However, a deeper understanding of underlying mechanisms will be imperative to improve the rational selection of probiotic strains to enhance their efficacy against antibiotic-resistant pathogens. Additionally, the transition to in vivo experiments will be of paramount importance to assess the clinical relevance of our findings and to translate them to tangible probiotic treatment regimens in the future.