Resistance development of cystic fibrosis respiratory pathogens when exposed to fosfomycin and tobramycin alone and in combination under aerobic and anaerobic conditions

Abstract

Although antibiotics from different classes are frequently prescribed in combination to prevent the development of resistance amongst Cystic Fibrosis (CF) respiratory pathogens, there is a lack of data as to the efficacy of this approach. We have previously shown that a 4:1 (w/w) combination of fosfomycin and tobramycin (F:T) has excellent activity against CF pathogens with increased activity under physiologically relevant anaerobic conditions. Therefore, the aim of this study was to determine whether F:T could delay or prevent the onset of resistance compared to either fosfomycin or tobramycin alone under aerobic and anaerobic conditions. The frequency of spontaneous mutants arising following exposure to fosfomycin, tobramycin and F:T was determined for clinical Pseudomonas aeruginosa and MRSA isolates under aerobic and anaerobic conditions. The effect of sub-inhibitory concentrations of fosfomycin, tobramycin and F:T on the induction of resistance was also investigated, with the stability of resistance and fitness cost associated with resistance assessed if it developed. P. aeruginosa and MRSA isolates had a lower frequency of spontaneous mutants to F:T compared to fosfomycin and tobramycin under both aerobic and anaerobic conditions. There was a maximum two-fold increase in F:T MICs when P. aeruginosa and MRSA isolates were passaged in sub-inhibitory F:T for 12 days. In contrast, sequential resistance to fosfomycin and tobramycin developed quickly (n = 3 days for both) after passage in sub-inhibitory concentrations. Once developed, both fosfomycin and tobramycin resistance was stable and not associated with a biological fitness cost to either P. aeruginosa or MRSA isolates. The results of this study suggest that F:T may prevent the development of resistance compared to fosfomycin or tobramycin alone under aerobic and physiologically relevant anaerobic conditions. F:T may be a potential treatment option in CF patients chronically colonised by MRSA and/or P. aeruginosa.

Figure 1. Fosfomycin, tobramycin and F∶T MICs for P. aeruginosa isolate W050 following serial passage in sub-inhibitory antibiotic concentrations under both aerobic and anaerobic conditions.

Exposure to (A) Fosfomycin aerobic, (B) Fosfomycin anaerobic, (C) Tobramycin aerobic, (D) Tobramycin anaerobic, (E) F∶T aerobic, (F) F∶T anaerobic. Filled circle (•) Fosfomycin MIC; filled square (▪) Tobramycin MIC; filled triangle (▴)F∶T MIC.

Figure 2. Fosfomycin, tobramycin and F∶T MICs for MRSA isolate CFP8 following serial passage in sub-inhibitory antibiotic concentrations under both aerobic and anaerobic conditions.

Exposure to (A) Fosfomycin aerobic, (B) Fosfomycin anaerobic, (C) Tobramycin aerobic, (D) Tobramycin anaerobic, (E) F∶T aerobic, (F) F∶T anaerobic. Filled circle (•) Fosfomycin MIC; filled square (▪) Tobramycin MIC; filled triangle (▴)F∶T MIC.

Figure 3. Fosfomycin, tobramycin and F∶T MICs for P. aeruginosa isolates following serial passage in sub-inhibitory antibiotic concentrations and subsequent removal of antibiotic pressure following 12 passages.

(A) AY4 FOS aerobic, (B) AY4 FOS anaerobic and (C) W050 TOB aerobic. Filled circle (•) Fosfomycin MIC; filled square (▪) Tobramycin MIC; filled triangle (▴)F∶T MIC; broken line, antibiotic pressure removed.

Figure 4. Fosfomycin, tobramycin and F∶T MICs for MRSA isolates following serial passage in sub-inhibitory antibiotic concentrations and subsequent removal of antibiotic pressure following 12 passages.

(A) CFP8 TOB aerobic, (B) 25A TOB anaerobic, (C) CFP8 FOF aerobic. Filled circle (•) Fosfomycin MIC; filled square (▪) Tobramycin MIC; filled triangle (▴),F∶T MIC, broken line, antibiotic pressure removed.

Figure 5. Growth curves showing total viable counts of parent MRSA and P. aeruginosa isolates and resistant mutants which developed following exposure to fosfomycin and tobramycin.

(A) MRSA CFP13, aerobic, (B) MRSA CFP13 anaerobic, (C) P. aeruginosa W050 aerobic and (D) P. aeruginosa W050 anaerobic. Filled circle (•) Parent strain; filled square (▪) Fosfomycin resistant mutant; filled triangle (▴) Tobramycin resistant mutant.