Salicylate reduces the antimicrobial activity of ciprofloxacin against extracellular Salmonella enterica serovar Typhimurium, but not against Salmonella in macrophages.
OBJECTIVE
Salicylate, a potent inducer of the MarA activator in Salmonella enterica, is the principal metabolite of aspirin, which is often consumed for medicinal and cosmetic uses. Our research was aimed at testing if salicylate activates the mar regulon in macrophage-associated Salmonella (intracellular bacteria), and investigating its effects on bacterial susceptibility to ciprofloxacin extracellularly and intracellularly.


METHODS
J774 macrophages were infected with S. enterica serovar Typhimurium (wild-type and marA null mutant), treated with ciprofloxacin with and without pre-exposure to salicylate, and the surviving bacteria were counted. Similar experiments were conducted with bacteria in broth (extracellular bacteria). Phe-Arg-beta-naphthylamide (PAbetaN) was added to investigate the role of efflux pumps in resistance. The transcriptional regulation of marRAB, acrAB and micF in extracellular and intracellular Salmonella Typhimurium with and without salicylate and ciprofloxacin was investigated using green fluorescent protein as a marker protein and quantitative real time PCR.


RESULTS
Pre-exposure of Salmonella to salicylate increased the resistance of extracellular but not intracellular bacteria to ciprofloxacin, although salicylate stimulated the expression of mar genes in intracellular and extracellular bacteria. Using marA mutants and the inhibitor PAbetaN, we showed that the improved resistance in extracellular bacteria is derived from the induction of acrAB by salicylate, which is mediated by MarA.


CONCLUSIONS
In intracellular bacteria, the expression of acrAB is already higher when compared with extracellular cells; therefore, salicylate does not result in significant acrAB induction intracellularly and subsequent resistance enhancement. Results show that conclusions raised from extracellular studies cannot be applied to intracellular bacteria, although the systems have similar functions.
Hartog
Efrat
E
Faculty of Biotechnology and Food Engineering, Technion, Haifa 32000, Israel.
Menashe
Ofir
O
Kler
Edna
E
Yaron
Sima
S
D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D000893:Anti-Inflammatory Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000096:biosynthesis; D002460:Cell Line; D002939:Ciprofloxacin / Q000494:pharmacology*; D015169:Colony Count, Microbial; D004336:Drug Antagonism; D015870:Gene Expression; D020869:Gene Expression Profiling; D008264:Macrophages / Q000382:microbiology*; D026901:Membrane Transport Proteins / Q000096:biosynthesis; D051379:Mice; D050296:Microbial Viability / Q000187:drug effects; D012459:Salicylates / Q000494:pharmacology*; D012486:Salmonella typhimurium / Q000187:drug effects*
D000900:Anti-Bacterial Agents; D000893:Anti-Inflammatory Agents; D001426:Bacterial Proteins; D026901:Membrane Transport Proteins; D012459:Salicylates; D002939:Ciprofloxacin