Inter-species interactions alter antibiotic efficacy in bacterial communities

Abstract

The efficacy of antibiotic treatments targeting polymicrobial communities is not well predicted by conventional in vitro susceptibility testing based on determining minimum inhibitory concentration (MIC) in monocultures. One reason for this is that inter-species interactions can alter the community members' susceptibility to antibiotics. Here we quantify, and identify mechanisms for, community-modulated changes of efficacy for clinically relevant antibiotics against the pathogen Pseudomonas aeruginosa in model cystic fibrosis (CF) lung communities derived from clinical samples. We demonstrate that multi-drug resistant Stenotrophomonas maltophilia can provide high levels of antibiotic protection to otherwise sensitive P. aeruginosa. Exposure protection to imipenem was provided by chromosomally encoded metallo-β-lactamase that detoxified the environment; protection was dependent upon S. maltophilia cell density and was provided by S. maltophilia strains isolated from CF sputum, increasing the MIC of P. aeruginosa by up to 16-fold. In contrast, the presence of S. maltophilia provided no protection against meropenem, another routinely used carbapenem. Mathematical ordinary differential equation modelling shows that the level of exposure protection provided against different carbapenems can be explained by differences in antibiotic efficacy and inactivation rate. Together, these findings reveal that exploitation of pre-occurring antimicrobial resistance, and inter-specific competition, can have large impacts on pathogen antibiotic susceptibility, highlighting the importance of microbial ecology for designing successful antibiotic treatments for multispecies communities.

Fig. 1. PAO1 exposure protection to imipenem is provided by S. maltophilia.

a Imipenem MIC curves for P. aeruginosa PAO1 (orange line), resistant S. maltophilia K279a (black line) and susceptible S. maltophilia K279a ampR^FS strains (grey line). b Assay to detect the inactivation of imipenem by K279a. The ability of PAO1 to grow in sterile filtered supernatant following 24 h incubation/growth in the presence of imipenem with no inoculum, K279a, K279a ampR^FS or PAO1. Line colours represent the inoculum of the initial round of growth from which the supernatant was sourced. c Measured concentration of imipenem by LCMS in sterile filtered supernatant following 24 h incubation/growth with no inoculum or K279a (n = 1). d Growth of PAO1 while in coculture with either K279a or K279a ampR^FS during imipenem treatment, orange line shows PAO1 monoculture control. The horizontal dashed line shows the initial inoculum density of PAO1 (5 × 10⁵ CFU/ml), points above this line show population growth. a and b bold lines show mean and d the median of six biological replicates that are represented by narrow lines of the same colour. a and b shaded areas show standard deviations (n = 6).

Fig. 2. PAO1 exposure protection is dependent on the initial density of resistant K279a.

The MIC of PAO1 for imipenem plotted against the initial density of resistant K279a or susceptible K279a ampR^FS strains. MIC was defined as the concentration of imipenem required to reduce PAO1 growth to below 5% of that in the absence of antibiotic 24 h post inoculation, calculated from broth microdilution cocultures (PAO1 density measured by relative fluorescence; see Supplementary Fig. S5). Points show six independent biological replicates for each condition (five replicates for K279a ampR^FS at 10⁷ CFU/ml). Stars show a significant difference from the No-Competitor control (*p < 0.05, **p < 0.001), Wilcoxon Rank Sum with Holm–Bonferroni correction was used for multiple comparisons.

Fig. 3. S. maltophilia provides no protection to meropenem.

a Meropenem MIC curves for PAO1, S. maltophilia K279a and S. maltophilia K279a ampR^FS. b Growth of PAO1 while in coculture with either K279a or K279a ampR^FS during meropenem treatment, orange line shows PAO1 monoculture control. c Assay to detect the inactivation of meropenem by K279a. The ability of PAO1 to grow in sterile filtered supernatant following 24 h incubation/growth in the presence of meropenem with no inoculum, K279a, K279a ampR^FS or PAO1. Line colours represent the inoculum of the initial round of growth from which the supernatant was sourced. a and c bold lines show mean and b the median of six biological replicates that are represented by narrow lines of the same colour. a and c the shaded areas show standard deviations (n = 6). b the horizontal dashed line shows the initial inoculum density of PAO1 (5 × 10⁵ CFU/ml).

Fig. 4. Modelling the exposure protection as a combined function of antibiotic effect and inactivation rate.

a The x-axis plots increasing α_min and decreasing k that produce a shallower dose-response curve and reduced antibiotic killing rate respectively (lower antibiotic effect, Supplementary Fig. S10) and the y-axis plots increasing V_max and decreasing K_M that increases the rate of antibiotic inactivation (high antibiotic inactivation). Altering α_min and k parameters fivefold 1 to 5, and −1.5 to −7.5, respectively, and V_max and K_M parameters tenfold 1 × 10⁻⁷ to 1 × 10⁻⁶, and 10 to 100, respectively (Supplementary Fig. S14). Shading represents the level of protection provided to the sensitive species S when in coculture as the times increase in MIC of S in monoculture. Initial density R = 5 × 10⁶, initial density S = 5 × 10⁵, b Combined effect of high effect antibiotics and low inactivation rates for selected values indicated by crosses in panel a. Vertical dashed line shows MIC of sensitive population in monoculture and horizontal dashed line shows initial inoculum size of the sensitive population. The MIC is the point at which the cell density of S is reduced below the inoculum density, i.e., the net growth rate is zero. Antibiotic high effect: α_min = −7.5, k = 5, low effect: α_min = −1.5, k = 1, antibiotic inactivation high rate: V_max = 1 × 10⁻⁶, K_M = 10, low rate: V_max = 1 × 10⁻⁷, K_M = 100, for other parameters see Supplementary Table S2.

Fig. 5. Exposure protection provided by clinical S. maltophilia CF isolates is lineage specific and varies between patients.

a The MIC of PAO1 to imipenem when cocultured with clinical S. maltophilia isolates originating from different CF patients. MIC was defined as the concentration of imipenem required to reduce growth to below 5% of that in the absence of antibiotic 24 h post inoculation, calculated from broth microdilution cocultures, PAO1 density measured by relative fluorescence (see Supplementary Fig. S7 and Methods). Points are coloured by whether P. aeruginosa was co-isolated along with the S. maltophilia isolate from sputum samples, grey no P. aeruginosa present, black P. aeruginosa co-isolated with S. maltophilia. Individual MIC curves for each S. maltophilia isolate are presented in Supplementary Fig. S9. Stars show a significant difference from the No-Competitor control and Wilcoxon Rank Sum with Holm–Bonferroni correction was used for multiple comparisons (*p < 0.05, **p < 0.001). b An unrooted maximum likelihood phylogeny build using PhyML 3.0 on the smeT–smeD intergenic region of 76 publicly available S. maltophilia genomes accessible on NCBI together with nine CF S. maltophilia isolates from this study labelled in bold. The tree labels are coloured by the phylogenetic groups described by Gould et al. (2006), group-representatives K279a (Group A), N531 (Group B) and J675a (Group C) are labelled in bold. Branch line widths represent percentage bootstrap support, branching for the four major clades are well supported. The scale bar represents 0.5 substitutions per site within the smeT–smeD intergenic region.