Temperature-Dependent Gentamicin Resistance of Francisella tularensis is Mediated by Uptake Modulation

Abstract

Gentamicin (Gm) is an aminoglycoside commonly used to treat bacterial infections such as tularemia – the disease caused by Francisella tularensis. In addition to being pathogenic, F. tularensis is found in environmental niches such as soil where this bacterium likely encounters Gm producers (Micromonospora sp.). Here we show that F. tularensis exhibits increased resistance to Gm at ambient temperature (26°C) compared to mammalian body temperature (37°C). To evaluate whether F. tularensis was less permeable to Gm at 26°C, a fluorescent marker [Texas Red (Tr)] was conjugated with Gm, yielding Tr-Gm. Bacteria incubated at 26°C showed reduced fluorescence compared to those at 37°C when exposed to Tr-Gm suggesting that uptake of Gm was reduced at 26°C. Unconjugated Gm competitively inhibited uptake of Tr-Gm, demonstrating that this fluorescent compound was taken up similarly to unconjugated Gm. Lysates of F. tularensis bacteria incubated with Gm at 37°C inhibited the growth of Escherichia coli significantly more than lysates from bacteria incubated at 26°C, further indicating reduced uptake at this lower temperature. Other facultative pathogens (Listeria monocytogenes and Klebsiella pneumoniae) exhibited increased resistance to Gm at 26°C suggesting that the results generated using F. tularensis may be generalizable to diverse bacteria. Regulation of the uptake of antibiotics provides a mechanism by which facultative pathogens survive alongside antibiotic-producing microbes in nature.

Stressor interaction networks suggest antibiotic resistance co-opted from stress responses to temperature

“…Clinically, the impact of temperature on the effects of antibiotics is also of interest because it suggests some antibiotics could have increased or reduced effectiveness in patients with fever or hypothermia. Previous work has shown there is increased resistance to gentamicin (GEN) in Francisella tularensis, Listeria monocytogenes, and Klebsiella pneumoniae at 26°C when compared to 37°C [32]. This increased resistance seems to be mediated by reduced drug uptake.…”

“…It is natural to ask which response mechanisms evolved first, whether these original responses were co-opted to respond to other stressors, and how much overlap exists among how stressors affect bacteria. It has previously been shown that heat-shock proteins are induced by some antibiotics [31], and that resistance to antibiotics can be temperature-dependent [32]. Furthermore, selection of heatresistant Escherichia coli results in the evolution of resistance to rifampicin, despite the drug being absent during the selection process [33].…”

TpiA is a Key Metabolic Enzyme That Affects Virulence and Resistance to Aminoglycoside Antibiotics through CrcZ in Pseudomonas aeruginosa

“…Tobramycin-Texas Red uptake assay. Tobramycin was conjugated to Texas Red as previously described (19,51). Briefly, 1 g Texas Red sulfonyl chloride was dissolved in 50 l anhydrous N,Ndimethyl formamide on ice.…”

“…The bacterial cells were lysed in PBS by sonication. The Texas Red was excited at 595 nm, and the emission at 615 nm (19,51) was measured with a luminometer (Varioskan Flash; Thermo Scientific). Total bacterial protein concentrations were quantified using a BCA analysis for calibration.…”

Alarm Odor Compounds of the Brown Marmorated Stink Bug Exhibit Antibacterial Activity

“…Disk diffusion assays measuring the antimicrobial effect of stink bug aldehydes were conducted in a similar manner as previously described [ 13 ]. Briefly, broth cultures were diluted to an optical density (A600) of 1.0.…”