Prolonged anoxic exposure impacts antibiotic sensitivity profiles of Pseudomonas aeruginosa

Abstract

Chronic respiratory tract infections with Pseudomonas aeruginosa frequently occur in patients with cystic fibrosis, chronic obstructive pulmonary disease, and bronchiectasis. A hallmark of these conditions is the accumulation of mucus plugs, creating oxygen-limited niches. Within these microenvironments, P. aeruginosa undergoes cellular modifications that may alter its antibiotic sensitivity. Although the acute effects of anoxia are well studied, the impact of prolonged anoxic exposure on antibiotic sensitivity remains unclear. In this study, we developed anoxic-conditioned P. aeruginosa strains by passaging a laboratory strain for 22 days in an anoxic environment. We performed time-kill assays with both parental and anoxic-conditioned strains in anoxic and aerobic environments, using ceftazidime, ciprofloxacin, colistin, and tobramycin. The anoxic-conditioned strains exhibited increased susceptibility to tobramycin and reduced sensitivity to colistin and ceftazidime. These differences were attributed to altered killing rates (as with tobramycin) or reduced regrowth under anoxic conditions (as with colistin). For ciprofloxacin, a steeper killing rate was observed against the anoxic-conditioned strains, but 24-h outcomes were similar to the parental strain. Overall, our findings demonstrate that long-term anoxia alters antibiotic sensitivity in P. aeruginosa differently than acute anoxia, with important implications for treating chronic infections in oxygen-limited environments.

Keywords: Pseudomonas aeruginosa; anoxia; antibiotic sensitivity; metabolic specialization; oxygen.

Figure 1.

Change in cell density of the parental and anoxic-conditioned P. aeruginosa strains after 24 h of antibiotic exposure in aerobic and anoxic environments. Orange lines and points represent the anoxic-conditioned P. aeruginosa strain, evolved over 22 days in an anoxic environment prior to antibiotic exposure, while navy lines and points represent the parental PAO1 strain. Solid lines and points denote the mean log₁₀ change in cell density, calculated from the three biological replicates, which are represented as translucent points.

Figure 2.

Net antibiotic killing rates in parental and anoxic-conditioned P. aeruginosa under aerobic and anoxic environments. Net killing rates during the initial 2 h of antibiotic exposure for each biological replicate (shown as points) were calculated by determining the slope of the change in log₁₀-transformed cell density. Solid lines represent the mean response rate of the mean parental PAO1 strain as well as the conditioned P. aeruginosa strain, which was evolved in an anoxic environment for 22 days prior to treatment.

Figure 3.

Growth rates of the parental and anoxic-conditioned P. aeruginosa strains in aerobic and anoxic environments. Maximal growth rates were calculated from smoothed spline growth curves of four technical replicates per strain and condition. The growth curve is based on optical density at 600 nm (OD600) measurements taken every 30 min. Data points represent µmax values per biological replicate, and the solid line indicates the mean µmax of the three biological replicates.