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. 2022 Dec 21;10(6):e0184222.
doi: 10.1128/spectrum.01842-22. Epub 2022 Dec 1.

The Effects of Antibiotic Combination Treatments on Pseudomonas aeruginosa Tolerance Evolution and Coexistence with Stenotrophomonas maltophilia

Jack P Law  1 , A Jamie Wood  1   2 , Ville-Petri Friman  1
Affiliations

The Effects of Antibiotic Combination Treatments on Pseudomonas aeruginosa Tolerance Evolution and Coexistence with Stenotrophomonas maltophilia

Jack P Law et al. Microbiol Spectr. .

Abstract

The Pseudomonas aeruginosa bacterium is a common pathogen of cystic fibrosis (CF) patients due to its ability to evolve resistance to antibiotics during treatments. While P. aeruginosa resistance evolution is well-characterized in monocultures, it is less well-understood in polymicrobial CF infections. Here, we investigated how exposure to ciprofloxacin, colistin, or tobramycin antibiotics, administered at sub-minimum inhibitory concentration (MIC) doses, both alone and in combination, shaped the tolerance evolution of P. aeruginosa (PAO1 lab and clinical CF LESB58 strains) in the absence and presence of a commonly co-occurring species, Stenotrophomonas maltophilia. The increases in antibiotic tolerances were primarily driven by the presence of that antibiotic in the treatment. We observed a reciprocal cross-tolerance between ciprofloxacin and tobramycin, and, when combined, the selected antibiotics increased the MICs for all of the antibiotics. Though the presence of S. maltophilia did not affect the tolerance or the MIC evolution, it drove P. aeruginosa into extinction more frequently in the presence of tobramycin due to its relatively greater innate tobramycin tolerance. In contrast, P. aeruginosa dominated and drove S. maltophilia extinct in most other treatments. Together, our findings suggest that besides driving high-level antibiotic tolerance evolution, sub-MIC antibiotic exposure can alter competitive bacterial interactions, leading to target pathogen extinctions in multispecies communities. IMPORTANCE Cystic fibrosis (CF) is a genetic condition that results in thick mucus secretions in the lungs that are susceptible to chronic bacterial infections. The bacterial pathogen Pseudomonas aeruginosa is often associated with morbidity in CF and is difficult to treat due to its high resistance to antibiotics. The resistance evolution of Pseudomonas aeruginosa is poorly understood in polymicrobial infections that are typical of CF. To study this, we exposed P. aeruginosa to sublethal concentrations of ciprofloxacin, colistin, or tobramycin antibiotics in the absence and presence of a commonly co-occurring CF species, Stenotrophomonas maltophilia. We found that low-level antibiotic concentrations selected for high-level antibiotic resistance. While P. aeruginosa dominated in most antibiotic treatments, S. maltophilia drove it into extinction in the presence of tobramycin due to an innately higher tobramycin resistance. Our findings suggest that, besides driving high-level antibiotic tolerance evolution, sublethal antibiotic exposure can magnify competition in bacterial communities, which can lead to target pathogen extinctions in multispecies communities.

Keywords: cystic fibrosis; experimental evolution; interspecies interactions.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

FIG 1
FIG 1
Methods schematic. (A) The combinations of antibiotic treatments and bacterial cultures that were used during the selection experiment. (B) The procedure followed during the selection experiment.
FIG 2
FIG 2
Growth of each evolved P. aeruginosa replicate population in the treatment concentrations of antibiotic relative to their growth without antibiotic. Growth was measured in separate growth assays at the end of the selection experiment. The panel columns show the two P. aeruginosa strains, and the panel rows show the growth in the presence of different antibiotics. Each point represents the mean growth in antibiotic for three technical replicates of one replicate population, minus the growth without antibiotic of the same replicate population (ΔepOD600Abx, as defined in Materials and Methods). The boxes show the means of all replicates (center line; ΔepOD600Abx¯, as defined in Materials and Methods), the and upper and lower limits represent the ±SEM. The horizontal dashed line represents the ΔepOD600Abx of the ancestor. The solid dark gray line represents the growth equal to that without antibiotic (i.e., the relative change in OD600 = 0). The shapes show monocultures (circle; “Mono”) and cocultures (triangle; “Co”). The colors show antibiotic treatments, with lighter and darker shades representing the absence and presence of the S. maltophilia competitor, respectively. An asterisk indicates a statistically significant difference (P < 0.05) between the antibiotic treatment and the “No Antibiotic” control treatment via a post hoc pairwise comparison. A cross-tolerance between ciprofloxacin and tobramycin can be seen in panels A, B, E, and F, comparing the “No Antibiotic” treatment in columns 1 and 2 to the CIP and TOB treatments in columns 3, 4, 7, and 8.
FIG 3
FIG 3
Growth of each evolved P. aeruginosa replicate population without antibiotic relative to the respective ancestor. Growth was measured in separate growth assays at the end of the selection experiment. The panel columns show the two P. aeruginosa strains. Each point represents the mean growth without antibiotic for three technical replicates of one replicate population, minus growth of the ancestor under the same conditions (ΔepOD600E, as defined in Materials and Methods). The boxes show the means of all replicates (center line; ΔepOD600E¯, as defined in Materials and Methods), and the upper and lower limits represent the ±SEM. The horizontal dashed line represents the growth equal to that of the ancestor (i.e., the relative change in OD600 = 0). The shapes show monocultures (circle; “Mono”) and cocultures (triangle; “Co”). The colors show antibiotic treatments, with lighter and darker shades representing the absence and presence of the S. maltophilia competitor, respectively. An asterisk indicates a statistically significant difference (P < 0.05) between the antibiotic treatment and the “No Antibiotic” control treatment via a post hoc pairwise comparison. The cost of tobramycin tolerance can be seen in both panels by comparing the “No Antibiotic” treatment in columns 1 and 2 to the TOB treatments in columns 7 and 8.
FIG 4
FIG 4
The MIC of the three individual antibiotics for each evolved replicate population of the P. aeruginosa strains. The panel columns show the P. aeruginosa strain, and the panel rows show the MIC of each antibiotic. The dashed line represents the MIC of the respective ancestors. The gray line shows the MIC50 of each treatment across replicate populations, as defined in Materials and Methods. The dashed blue line shows the treatment concentration. The size of each point represents the number of replicates with the specified MIC. The color of the points represents the treatment. The white center dot represents a monoculture, and black represents a coculture. The number of extinctions in each treatment and coculture is shown beneath the x axis. The MIC was measured in triplicate for each replicate. An asterisk represents a significant difference (P < 0.05) between the antibiotic treatment and the “No Antibiotic” control treatment via a post hoc pairwise independence test. A cross-tolerance between ciprofloxacin and tobramycin can be seen in panels A, B, E, and F, comparing the “No Antibiotic” treatment in columns 1 and 2 to the CIP and TOB treatments in columns 3, 4, 7, and 8.
FIG 5
FIG 5
Optical density of bacterial populations and composition of cocultures at the final time point of the selection experiment. (A–D) Boxplots of the optical density of the bacterial population (OD600) of each replicate population from eight treatments (see the legend for the boxplot fill colors). The panel columns show the P. aeruginosa strain, and the panel rows show monocultures or cocultures. The points represent individual replicates (N = 6). The shapes show the species present at the final time point: P. aeruginosa, circles; S. maltophilia, diamonds; both, red squares. (E and F) The presence of surviving species in each coculture replicate (N = 6). The colors represent the surviving species as follows: P. aeruginosa, orange; S. maltophilia, blue; both, red.
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