The complex interplay of iron, biofilm formation, and mucoidy affecting antimicrobial resistance ofPseudomonas aeruginosa

Abstract

Pseudomonas aeruginosa is a Gram-negative opportunistic bacterial pathogen that is refractory to a variety of current antimicrobial therapeutic regimens. Complicating treatment of such infections is the ability of P. aeruginosa to form biofilms, as well as several innate and acquired resistance mechanisms. Previous studies suggest iron plays a role in resistance to antimicrobial therapy, including the efficacy of an FDA-approved iron chelator, deferasirox (DSX), or Gallium, an iron analog, in potentiating antibiotic-dependent killing of P. aeruginosa biofilms. Here we show that iron-replete conditions enhance resistance of P. aeruginosa nonbiofilm growth against tobramycin and tigecycline. Interestingly, the mechanism of iron-enhanced resistance to each of these antibiotics is distinct. Whereas pyoverdine-mediated iron uptake is important for optimal resistance to tigecycline, it does not enhance tobramycin resistance. In contrast, heme supplementation results in increased tobramycin resistance, while having no significant effect on tigecycline resistance. Thus, non-siderophore bound iron plays an important role in resistance to tobramycin, while pyoverdine increases the ability of P. aeruginosa to resist tigecycline treatment. Lastly, we show that iron increases the minimal concentration of tobramycin, but not tigecycline, required to eradicate P. aeruginosa biofilms. Moreover, iron depletion blocks the previous observed induction of biofilm formation by sub-inhibitory concentrations of tobramycin, suggesting iron and tobramycin signal through overlapping regulatory pathways to affect biofilm formation. These data further support the role of iron in P. aeruginosa antibiotic resistance, providing yet another compelling case for targeting iron acquisition for future antimicrobial drug development.

The prrF -Encoded Small Regulatory RNAs Are Required for Iron Homeostasis and Virulence of Pseudomonas aeruginosa

“…We show that the ⌬prrF1,2 mutant is defective for iron and heme homeostasis. Additionally, we show that ⌬prrF1,2 mutant biofilms are not enhanced by iron in the presence of aminoglycoside antibiotics, a known trait of P. aeruginosa biofilm formation (39,40). Further, we demonstrate that iron and heme regulation of PrrF and PrrH is conserved in several P. aeruginosa clinical isolates.…”

“…For example, aminoglycoside antibiotics have been shown to increase biofilm formation when added to P. aeruginosa cultures at subinhibitory concentrations (39). Moreover, we recently showed that induction of biofilm formation by exposure to tobramycin, an aminoglycoside antibiotic that is often used to treat airway infections in cystic fibrosis patients, is dependent upon iron (40). We therefore determined if altered iron homeostasis in the ⌬prrF1,2 mutant would affect the capacity of aminoglycosides to enhance biofilm formation.…”

“…For growth curves, qPCR, mass spectrometry, and Northern blotting, strains were diluted from L broth overnights into M9 minimal medium purchased from Teknova containing 2% glucose and grown for 4 h, and then they were subcultured into fresh M9 for an additional 8 h at 37°C (41). For biofilm analysis, strains were grown in Chelex-treated, dialyzed Trypticase soy broth (DTSB) prepared as previously described (40). Ferric chloride (FeCl 3 ) was added to a final concentration of 100 M where indicated below.…”

“…Biofilm growth and measurement. The MBEC (minimal biofilm eradication concentration) assay physiology and genetics biofilm device (formerly known as the Calgary biofilm device; Innovotech) was used to assay biofilm formation and the MBEC of tobramycin against PAO1 and the ⌬prrF1,2 mutant as previously described (40). Briefly, 96-well MBEC plates containing DTSB, supplemented with 100 M FeCl 3 , were inoculated in duplicate with approximately 1 ϫ 10 7 CFU ml Ϫ1 of bacteria.…”

Iron Depletion Enhances Production of Antimicrobials by Pseudomonas aeruginosa

“…Escherichia coli strains were routinely grown in L broth or on L agar plates, and P. aeruginosa strains were maintained in brain heart infusion (BHI) broth or on BHI agar plates. For iron-depleted medium, Chelex-treated, dialyzed Trypticase soy broth (DTSB) was prepared as previously described (44). Ferric chloride (FeCl 3 ) was added to DTSB at a final concentration of 100 M for iron-replete conditions.…”