Abstract Bacterial population exposed to stressful antibiotic conditions consists of various subpopulations such as tolerant, persister, and resistant cells. The aim of this study was to evaluate the phenotypic heterogeneity of Salmonella Typhimurium preadapted to sublethal concentrations of antibiotics. Salmonella Typhimurium cells were treated with 1/2 × MIC of antibiotics for the first 48 h and successively 1 × MIC for the second 24 h at 37°C, including untreated control (CON), no antibiotic and 1 × MIC ciprofloxacin (NON-CIP), 1/2 × MIC ciprofloxacin and 1 × MIC ciprofloxacin (CIP-CIP), 1/2 × MIC tetracycline and 1 × MIC ciprofloxacin (TET-CIP), no antibiotic and 1 × MIC tetracycline (NON-TET), 1/2 × MIC ciprofloxacin and 1 × MIC tetracycline (CIP-TET), and 1/2 × MIC tetracycline and 1 × MIC tetracycline (TET-TET). All treatments were evaluated by antibiotic susceptibility, ATP level, relative fitness, cross-resistance, and persistence. S. Typhimurium cells were more susceptible to non-adapted NON-CIP and NON-TET (&amp;gt;3-log reduction) than pre-adapted CIP-CIP, TET-CIP, CIP-TET, and TET-TET. CON exhibited the highest ATP level, corresponding to the viable cell number. The relative fitness levels were more than 0.95 for all treatments, except for NON-CIP (0.78). The resistance to ciprofloxacin and tetracycline was increased at all treatments with the exception of NON-TET. The persister cells were noticeably induced at CIP-TET treatment, showing more than 5 log CFU mL−1. The results suggest that the antibiotic preadaptation led to heterogeneous populations including persisters that can develop to resistance. This study provides new insight in the bacterial persistence associated with their potential risk and paves the way to design antibiotic therapy targeting dormant bacteria. 

Assessment of phenotypic heterogeneity in Salmonella Typhimurium preadapted to ciprofloxacin and tetracycline.

Bacterial survival upon sharp fluctuations of environmental parameters and exposure to lethal doses of stressors (antibiotics, disinfectants, heat shock, and others) is ensured by the use of different strategies of resistance, an important place among which is occupied by the forms with reduced or stopped metabolism, antibiotic tolerant (AT) persister (P) cells, anabiotic dormant forms (DFs), and viable but non-culturable (VBNC) cells. Elucidating the role of these forms of bacterial resistance to an impact of chemical and biological toxicants and physical stressors is of great fundamental and practical interest. The aim of this research was to study the dynamics of the resistance forms in bacteria developing in biofilms and, for comparison in liquid media, upon exposure to lethal doses of antibiotics and heat shock (80 °C, 15 min). In the trials, the experimental model of the development of monospecies and binary forms of bacterial biofilms including contaminants of meat products (eight strains of genera Pseudomonas, Escherichia, Salmonella, Staphylococcus, and Kocuria) on the fiberglass filters was used. It was established that survival of populations in the presence of lethal doses of antibiotics and upon heating was ensured by persister cells forming in bacterial populations and, at the late stages of the development of biofilm or planktonic cultures (28 days of incubation), by anabiotic DFs. With that, the number of thermoresistant (TR) DFs (103–104 CFU/mL) in dying biofilms (28 days) developing in the standard conditions (composition and volume of a medium, pH, growth temperature) weakly depended on the bacterial taxonomic status. This study demonstrates the heterogeneity of DF populations of biofilm bacterial cultures in terms of the depth of dormancy, as a result of which the number of thermoresistant DFs after heating can exceed their total number before heating (due to the effect of DF revival, resuscitation). When studying the dynamics of TR cells and P cells in bacterial biofilm and planktonic cultures, it was found that their number (CFU/mL) in populations decreased up to the absence of TR cells and P cells on the 21st day of growth and was restored on the 28th day of growth. The revealed phenomenon can be explained only by cardinal changes in the ultrastructural organization of cells, namely, cytoplasm vitrification due to a sharp decrease in an amount of free water in a cell, which, according to the results obtained, occurs in the period between the 21st and 28th days of incubation. A high degree of correlation between the number of AT cells and TR cells (0.5–0.92) confirms the hypothesis that regards P cells as precursors of DFs.

/pdf/forms-of-bacterial-survival-in-model-biofilms-3u9cbwkf.pdf

Forms of Bacterial Survival in Model Biofilms

The foodborne pathogenic bacterium Campylobacter jejuni utilizes chemotaxis to assist in the colonization of host niches. A key to revealing the relationship among chemotaxis and pathogenicity is the discovery of signaling molecules perceived by the chemoreceptors. The C. jejuni chemoreceptor Tlp11 is encoded by the highly infective C. jejuni strains. In the present study, we report that the dCache-type ligand-binding domain (LBD) of C. jejuni ATCC 33560 Tlp11 binds directly to novel ligands methyl pyruvate, toluene, and quinoline using the same pocket. Methyl pyruvate elicits a strong chemoattractant response, while toluene and quinoline function as the antagonists without triggering chemotaxis. The sensory LBD was used to control heterologous proteins by constructing chimeras, indicating that the signal induced by methyl pyruvate is transmitted across the membrane. In addition, bioinformatics and experiments revealed that the dCache domains with methyl pyruvate-binding sites and ability are widely distributed in the order Campylobacterales. This is the first report to identify the class of dCache chemoreceptors that bind to attractant methyl pyruvate and antagonists toluene and quinoline. Our research provides a foundation for understanding the chemotaxis and virulence of C. jejuni and lays a basis for the control of this foodborne pathogen.

Identification of a dCache-type chemoreceptor in Campylobacter jejuni that specifically mediates chemotaxis towards methyl pyruvate

This chapter provides an overview of current and emerging approaches to decontaminate raw meat. The introduction of Hazard Analysis Critical Control Points has been the key motivator for introducing interventions with meat processing and stimulated R&D to develop pathogen controls based on physical, chemical, or biological approaches. To this end, a broad range of decontamination methods has been evaluated on the laboratory scale with a number now being applied commercially. The advantages and disadvantages of different interventions applied alone or in combination will be described. Recent advances in meta-analysis to provide an assessment on different combinations of interventions within meat processing will be provided. The underlying reasons why laboratory-based studies typically overestimate the efficacy of interventions when applied on a commercial scale will be discussed. Finally, knowledge gaps and future research directions will be described. 

Interventions and current challenges in enhancing the microbiological safety of raw meat

Persister cells (PCs), a subpopulation occurring within normal cells, exhibit a transient tolerance to antibiotics because of their dormant state. PCs are categorized into two types: type I PCs, which emerge during the stationary phase, and type II PCs, which emerge during the logarithmic phase. Using the conventional colony-forming method, we previously demonstrated that type I PCs of 

Characterization of Escherichia coli Persisters from Biofilm Culture: Multiple Dormancy Levels and Multigenerational Memory in Formation

Persisters are a form of dormancy in bacteria that provide temporary resistance to antibiotics. The following reports on the formation of Escherichia coli O157:H7 E318 type II persisters from a pro...

The progress of type II persisters of Escherichia coli O157:H7 to a non-culturable state during prolonged exposure to antibiotic stress with revival being aided through acid-shock treatment and provision of methyl pyruvate

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The progress of type II persisters of Escherichia coli O157:H7 to a non-culturable state during prolonged exposure to antibiotic stress with revival being aided through acid-shock treatment and provision of methyl pyruvate