Prophage Gifsy-1 Induction in Salmonella enterica Serovar Typhimurium Reduces Persister Cell Formation after Ciprofloxacin Exposure.

Persister cells are drug-tolerant bacteria capable of surviving antibiotic treatment despite the absence of heritable resistance mechanisms. It is generally thought that persister cells survive antibiotic exposure through the implementation of stress responses and/or energy-sparing strategies. Exposure to DNA gyrase-targeting antibiotics could be particularly detrimental for bacteria that carry prophages integrated in their genomes. Gyrase inhibitors are known to induce prophages to switch from their dormant lysogenic state into the lytic cycle, causing the lysis of their bacterial host. However, the influence of resident prophages on the formation of persister cells has only been recently appreciated. Here, we evaluated the effect of endogenous prophage carriage on the generation of bacterial persistence during Salmonella enterica serovar Typhimurium exposure to both gyrase-targeting antibiotics and other classes of bactericidal antibiotics. Results from the analysis of strain variants harboring different prophage combinations revealed that prophages play a major role in limiting the formation of persister cells during exposure to DNA-damaging antibiotics. In particular, we present evidence that prophage Gifsy-1 (and its encoded lysis proteins) are major factors limiting persister cell formation upon ciprofloxacin exposure. Resident prophages also appear to have a significant impact on the initial drug susceptibility, resulting in an alteration of the characteristic biphasic killing curve of persister cells into a triphasic curve. In contrast, a prophage-free derivative of S. Typhimurium showed no difference in the killing kinetics for ß-lactam or aminoglycoside antibiotics. Our study demonstrates that induction of prophages increased the susceptibility toward DNA gyrase inhibitors in S. Typhimurium, suggesting that prophages have the potential for enhancing antibiotic efficacy. IMPORTANCE Bacterial infections resulting from antibiotic treatment failure can often be traced to nonresistant persister cells. Moreover, intermittent or single treatment of persister cells with ß-lactam antibiotics or fluoroquinolones can lead to the formation of drug-resistant bacteria and to the emergence of multiresistant strains. It is therefore important to have a better understanding of the mechanisms that impact persister formation. Our results indicate that prophage-associated bacterial killing significantly reduces persister cell formation in lysogenic cells exposed to DNA-gyrase-targeting drugs. This suggests that therapies based on gyrase inhibitors should be favored over alternative strategies when dealing with lysogenic pathogens.

Salmonella Central Carbon Metabolism Enhances Bactericidal Killing by Fluoroquinolone Antibiotics.

The efficacy of killing by bactericidal antibiotics has been reported to depend in large part on the ATP levels, with low levels of ATP leading to increased persistence after antibiotic challenge. Here, we show that an atp operon deletion strain of Salmonella enterica serovar Typhimurium lacking the ATP synthase was at least 10-fold more sensitive to killing by the fluoroquinolone antibiotic ciprofloxacin and yet showed either increased survival or no significant difference compared with the wild-type strain when challenged with aminoglycoside or ß-lactam antibiotics, respectively. The increased cell killing and reduced bacterial survival (persistence) after fluoroquinolone challenge were found to involve metabolic compensation for the loss of the ATP synthase through central carbon metabolism reactions and increased NAD(P)H levels. We conclude that the intracellular ATP levels per se do not correlate with bactericidal antibiotic persistence to fluoroquinolone killing; rather, the central carbon metabolic pathways active at the time of challenge and the intracellular target of the antibiotic determine the efficacy of treatment.

The role of ATP pools in persister cell formation in (fluoro)quinolone-susceptible and -resistant strains of Salmonella enterica ser. Typhimurium.

In this study, we investigated the reported dependence on the ATP pools for persister cell formation in fluoroquinolone-resistant variants of the facultative intracellular pathogen Salmonella enterica serovar Typhimurium. We compared the generation of persister cell populations after ciprofloxacin challenge of wildtype and a nalidixic acid-resistant variant of S. Typhimurium with reduced ciprofloxacin-susceptibility, as well as strains containing a deletion of the atp operon or harbouring the cloned atp genes. A gyrA mutation (D87Y) was found to contribute to increased stationary phase formation of persister cells in S. Typhimurium. However, in contrast to expectations from prior studies, while treatment with the ATP synthase poison arsenate showed the expected increase in persister cells surviving ciprofloxacin treatment, a more direct approach using a strain of Salmonella deleted for the atp operon showed severe reductions in persister cell formation. Persister cell formation was recovered after introduction of the cloned atp operon which restored the reduced ATP levels. These results suggest either an alternative explanation for previous studies, or that persister cell formation in Salmonella is differently regulated.