Caracterização da formação de células persisters em salmonella enterica

Abstract

Salmonella enterica is an important zoonotic pathogen associated with foodborne diseases due to the consumption of contaminated foods, especially those derived from animal origin. Different non-typhoid S. enterica serovars are considered pathogens adapted to infect and survive inside phagocytic cells, triggering gastroenteritis in animals, including humans, but usually self-limiting. The use of antimicrobial therapy is only necessary in cases of severe salmonellosis, being fluoroquinolones and third-generation cephalosporins the drugs of choice. However, prevalence of multiresistant isolates of S. enterica in samples from different origins has been increasingly reported, which could lead to failures in the antimicrobial treatment against infections. On the other hand, therapeutic failure and recalcitrant infections may also be associated with persister cells. Therefore, this study aimed to evaluate the persister cell levels in S. enterica isolates exposed to ciprofloxacin and ceftazidime, as well as the influence of previous exposure to animal feed additives on tolerance to ciprofloxacin. Additionally, it identified differentially expressed transcripts in persister cells from different S. enterica serovars exposed to ciprofloxacin and ceftazidime in planktonic culture. For this, 10 S. enterica isolates were evaluated and characterized as weak producers of biofilm on polystyrene surface and susceptible to ciprofloxacin, ceftazidime, and colistin. All isolates were able to form distinct fractions of persister cells after exposure to 100X the Minimum Inhibitory Concentration (MIC) value of ciprofloxacin or ceftazidime in planktonic culture and biofilm. The levels of persisters in biofilms were higher than those found in planktonic culture for both drugs, and it was possible to observe heterogeneity in these levels among S. enterica isolates against the same challenge. In addition, small colony variants (SCV) were found among surviving cells after exposure to ciprofloxacin in all S. enterica isolates. Nevertheless, the SCV phenotype showed to be unstable, since reversion to the normal colony phenotype (NCP) was observed when sub-cultures derived from these colonies were performed in the absence of the stressor, even after repeated cycles of exposure to ciprofloxacin. Likewise, it was possible to verify that similar persister levels were found in a S. enterica isolate after successive cycles of exposure to the same drug, with no selection of a highly persistent phenotype, demonstrating the non-inheritable condition of the persisters. We also found heterogeneity in persister levels following exposure to drugs with different mechanisms of action, indicating that persistence is not a multitolerant phenotype. These findings are in agreement with the heterogeneous patterns of gene expression found on exposure to ciprofloxacin and ceftazidime. Cells from SCVs and NCPs, obtained from planktonic culture and biofilm exposed to ciprofloxacin were evaluated by scanning electron microscopy, and it was observed that the two phenotypes presented similar shape and size, regardless of the culture condition analyzed. However, division septum and filamentous cells were found in all morphotypes and culture conditions analyzed. Planktonic cultures of a subgroup of six S. enterica isolates were also exposed to concentrations above the MIC of colistin, and one S. Agona isolate was unable to form persisters against this drug. The other isolates not only presented surviving cells after colistin treatment, but, interestingly, after 48 h of exposure, a resumption of growth was observed in the presence of the drug. The possible selection of resistant mutants and stable hetero-resistant cells was discarded in this surviving population that was able to grow in the presence of colistin. Furthermore, a previous exposure to sub-inhibitory concentrations of organic acids, colistin, and even ciprofloxacin did not influence persister cell levels after exposure to lethal concentrations of ciprofloxacin. Thus, these results suggest that the antimicrobials tested, which were or are still employed as feed additives added to animal feed or drinking water, did not induce antimicrobial tolerance nor select highly persistent mutants. Overall, the findings of this work suggest that, in addition to the possible presence of several adaptive strategies for survival against antimicrobial stressors among S. enterica isolates, a single isolate may originate physiologically distinct populations of persisters, where cells growing under distinct stress conditions may adopt different and perhaps complementary survival strategies.