Repeated isolation of an antibiotic-dependent and temperature-sensitive mutant of Pseudomonas aeruginosa from a cystic fibrosis patient

Abstract

Background: Bacteria adapt to survive and grow in different environments. Genetic mutations that promote bacterial survival under harsh conditions can also restrict growth. The causes and consequences of these adaptations have important implications for diagnosis, pathogenesis, and therapy.

Objectives: We describe the isolation and characterization of an antibiotic-dependent, temperature-sensitive Pseudomonas aeruginosa mutant chronically infecting the respiratory tract of a cystic fibrosis (CF) patient, underscoring the clinical challenges bacterial adaptations can present.

Methods: Respiratory samples collected from a CF patient during routine care were cultured for standard pathogens. P. aeruginosa isolates recovered from samples were analysed for in vitro growth characteristics, antibiotic susceptibility, clonality, and membrane phospholipid and lipid A composition. Genetic mutations were identified by whole genome sequencing.

Results: P. aeruginosa isolates collected over 5 years from respiratory samples of a CF patient frequently harboured a mutation in phosphatidylserine decarboxylase (psd), encoding an enzyme responsible for phospholipid synthesis. This mutant could only grow at 37°C when in the presence of supplemented magnesium, glycerol, or, surprisingly, the antibiotic sulfamethoxazole, which the source patient had repeatedly received. Of concern, this mutant was not detectable on standard selective medium at 37°C. This growth defect correlated with alterations in membrane phospholipid and lipid A content.

Conclusions: A P. aeruginosa mutant chronically infecting a CF patient exhibited dependence on sulphonamides and would likely evade detection using standard clinical laboratory methods. The diagnostic and therapeutic challenges presented by this mutant highlight the complex interplay between bacterial adaptation, antibiotics, and laboratory practices, during chronic bacterial infections.

Figure 1.

Antibiotic susceptibility testing of P. aeruginosa isolate 0122-320 by disc diffusion (a) and Etest (b) on MHA incubated at the temperatures indicated above each plate. Bacterial growth was only observed around the trimethoprim/sulfamethoxazole (SXT) disc and Etest when plates were incubated at 37°C. Antibiotics are labelled above their respective discs on the 30°C MHA plate and are in the same position on MHA incubated at the other indicated temperatures. Zone diameters (c) were compared for 0122-320 when grown at different temperatures, and with those for control strain ATCC 27853. Shown are disc diffusion diameters for piperacillin/tazobactam (TZP), ceftazidime (CAZ), cefepime (FEP), aztreonam (ATM), imipenem (IPM), meropenem (MEM), tobramycin (NN), ciprofloxacin (CIP), and trimethoprim/sulfamethoxazole (SXT).

Figure 2.

Antibiotic-dependent growth of isolate 0122-320 on MHA after 24 h of incubation at 37°C. Growth with trimethoprim (TMP) or sulfamethoxazole (SMX) (a) was tested separately at amounts 2-fold below, equal to, and 2-fold above the amounts present on commercially available BD trimethoprim/sulfamethoxazole (SXT) discs (TMP 1.25 μg/SMX 23.75 μg). The response of 0122-320 to higher amounts of SMX was tested on a separate plate (b). The antibiotic added to each disc is indicated on the left of the plate and the amount is labelled above each disc (a) or above and below each disc (b).

Figure 3.

Relative abundance of phosphatidylserine (PS) in the membranes of isolate 0122-320, revertant 0122-320R, and laboratory strain PAO1 grown under the indicated conditions and temperatures. The graph shows the abundance of two prominent peaks identified as PS by fragmentation analysis, m/z 734.5 and 760.5, relative to the total phospholipid composition in the membrane. Isolates were grown in MHA with and without 15 mM MgCl₂ (Mg++), 2% v/v glycerol (GLY), or sulfamethoxazole (SMX), as indicated. Subinhibitory concentrations of sulfamethoxazole were used for the revertant 0122-320R (2 μg) and PAO1 (40 μg).

Figure 4.

Lipid A profile of isolate 0122-320 (a and b) and revertant 0122-320R (c and d) after growth on MHA at 37°C supplemented with sulfamethoxazole (SMX) (a and c) or MgCl₂ (b and d). Peak intensities are expressed in arbitrary units (a.u.). Subinhibitory concentrations of sulfamethoxazole were used to supplement the media for revertant 0122-320R.