Within-species variability of antibiotic interactions in Gram-negative bacteria

ABSTRACT Treatments with antibiotic combinations are becoming increasingly important even though the supposed clinical benefits of combinations are, in many cases, unclear. Here, we systematically examined how several clinically used antibiotics interact and affect the antimicrobial efficacy against five especially problematic Gram-negative pathogens. A total of 232 bacterial isolates were tested against different pairwise antibiotic combinations spanning five classes, and the ability of all combinations in inhibiting growth was quantified. Descriptive statistics, principal component analysis (PCA), and Spearman’s rank correlation matrix were used to determine the correlations between the different combinations on interaction outcome. Several important conclusions can be drawn from the 696 examined interactions. Firstly, within a species, the interactions are in general conserved but can be isolate-specific for a given antibiotic combination and can range from antagonistic to synergistic. Secondly, additive and antagonistic interactions are the most common observed across species and antibiotics, with 87.1% of isolate-antibiotic combinations being additive, 11.6% antagonistic, and only 0.3% showing synergy. These findings suggest that to achieve the highest precision and efficacy of combination therapy, not only isolate-specific interaction profiling ought to be routinely performed, in particular to avoid using drug combinations that show antagonistic interaction and an expected associated reduction in efficacy, but also discovering rare and potentially valuable synergistic interactions. IMPORTANCE Antibiotic combinations are often used to treat bacterial infections, which aim to increase treatment efficacy and reduce resistance evolution. Typically, it is assumed that one specific antibiotic combination has the same effect on different isolates of the same species, i.e., the interaction is conserved. Here, we tested this idea by examining how several clinically used antibiotics interact and affect the antimicrobial efficacy against several bacterial pathogens. Our results show that, even though within a species the interactions are often conserved, there are also isolate-specific differences for a given antibiotic combination that can range from antagonistic to synergistic. These findings suggest that isolate-specific interaction profiling ought to be performed in clinical microbiology routine to avoid using antagonistic drug combinations that might reduce treatment efficacy.


Fig S2. Evaluation of modified CombiANT
® method for use with colistin together with other antibiotics for all Gram-negative species in this study.The quality control strains for each species were tested.Antibiotics tested against the species are indicated followed by the reservoir they occupied on the CombiANT ® plate.Top row of graphs (a, b, c, d, e) indicates the FICi obtained for three replicates against the pairwise combination of antibiotics.Bottom row of graphs (f, g, h, i, j) indicates the mean and standard error of the mean of the FICi obtained for the top row of graphs.The y axis for the graphs on the left column denotes FICi values for each replicate.The y axis for the graphs on the right column denotes the mean FICi and standard error of the mean calculated based on the FICi on the left column.The red dotted lines represent the clinical level for antagonism (FICi=4), the green dotted lines represent the clinical levels for synergy (FICi=0.5), the black solid line represent the absolute additivity (FICi=1).

Fig S12. Distribution of P. aeruginosa antibiotic interaction FIC indices (FICi) (n=28).
The x axis denotes the FIC indices (FICi), the y axis denotes the percentage of isolates observed.The red dotted lines represent the clinical level for antagonism (FICi=4), the green dotted lines represent the clinical levels for synergy (FICi=0.5), the black dotted lines represent the absolute additivity (FICi=1).

Fig S13. P. aeruginosa antibiotic interaction FIC indices (FICi) (n=53).
The x axis for graphs in both columns denotes the isolate no.The y axis for the graphs on the left column denotes FICi values for each replicate.The y axis for the graphs on the right column denotes the mean FICi and standard error of the mean calculated based on the FICi on the left column.All mean FICi values calculated can be found in supplementary table 3. The red dotted lines represent the clinical level for antagonism (FICi=4), the green dotted lines represent the clinical levels for synergy (FIC=0.5), the black solid line represent the absolute additivity (FICi=1).

Fig S1 .
Fig S1.Evaluation of modified CombiANT ® method for use with colistin.a) colistin impregnated filter paper placed in 3 different reservoirs on the CombiANT ® plates.b) mean and standard error of the mean of the obtained FIC indices in a.

Fig S3 .
Fig S3.Schematic overview of this study.232 Gram-negative clinical isolates were tested against seven different antibiotics resulting in 12 unique pair-wise combinations and 696 interaction profiles quantified.

Fig
Fig S5.A. baumannii antibiotic interaction FIC indices (FICi) (n=48).The x axis for graphs in both columns denotes the isolate number.The y axis for the graphs on the left column denotes FICi values for each replicate.The y axis for the graphs on the right column denotes the mean FICi and standard error of the mean calculated based on the FICi on the left column.All mean FICi values calculated can be found in supplementary table 1.The red dotted lines represent the clinical level for antagonism (FICi=4), the green dotted lines represent the clinical levels for synergy (FICi=0.5), the black solid line represent the absolute additivity (FICi=1).

Fig S6 .
Fig S6.Distribution of E. cloacae antibiotic interaction FIC indices (FICi) (n=50).The x axis denotes the FIC indices (FICi), the y axis denotes the percentage of isolates observed.The red dotted lines represent the clinical level for antagonism (FICi=4), the green dotted lines represent the clinical levels for synergy (FICi=0.5), the black dotted lines represent the absolute additivity (FICi=1).

Fig
Fig S7.E. cloacae antibiotic interaction FIC indices (FICi) (n=50).The x axis for graphs in both columns denotes the isolate number.The y axis for the graphs on the left column denotes FICi values for each replicate.The y axis for the graphs on the right column denotes the mean FICi and standard error of the mean calculated based on the FICi on the left column.All mean FICi values calculated can be found in supplementary table 1.The red dotted lines represent the clinical level for antagonism (FICi=4), the green dotted lines represent the clinical levels for synergy (FICi=0.5), the black solid line represent the absolute additivity (FICi=1).

Fig S8 .
Fig S8.Distribution of E. coli antibiotic interaction FIC indices (FICi) (n=52).The x axis denotes the FIC indices (FICi), the y axis denotes the percentage of isolates observed.The red dotted lines represent the clinical level for antagonism (FICi=4), the green dotted lines represent the clinical levels for synergy (FICi=0.5), the black dotted lines represent the absolute additivity (FICi=1).

Fig
Fig S9.E. coli antibiotic interaction FIC indices (FICi) (n=52).The x axis for graphs in both columns denotes the isolate number.The y axis for the graphs on the left column denotes FICi values for each replicate.The y axis for the graphs on the right column denotes the mean FICi and standard error of the mean calculated based on the FICi on the left column.All mean FICi values calculated can be found in supplementary table1.The red dotted lines represent the clinical level for antagonism (FICi=4), the green dotted lines represent the clinical levels for synergy (FICi=0.5), the black solid line represent the absolute additivity (FICi=1).

Fig S10 .
Fig S10.Distribution of K. pneumoniae antibiotic interaction FIC indices (FICi) (n=53).The x axis denotes the FIC indices (FICi), the y axis denotes the percentage of isolates observed.The red dotted lines represent the clinical level for antagonism (FICi=4), the green dotted lines represent the clinical levels for synergy (FICi=0.5), the black dotted lines represent the absolute additivity (FICi=1).

Fig
Fig S11.K. pneumoniae antibiotic interaction FIC indices (FICi) (n=53).The x axis for graphs in both columns denotes the isolate number.

Fig S14 .
Fig S14.Principal component analysis of all species interaction profile against each other following the parallel Monte Carlo analysis.a) Eigen values calculated for each of the principal component (PC).b).The percentage proportion of variance calculated for each of the PC for the