Colistin-degrading proteases confer collective resistance to microbial communities during polymicrobial infections

Abstract

Background: The increasing prevalence of resistance against the last-resort antibiotic colistin is a significant threat to global public health. Here, we discovered a novel colistin resistance mechanism via enzymatic inactivation of the drug and proposed its clinical importance in microbial communities during polymicrobial infections.

Results: A bacterial strain of the Gram-negative opportunistic pathogen Stenotrophomonas maltophilia capable of degrading colistin and exhibiting a high-level colistin resistance was isolated from the soil environment. A colistin-degrading protease (Cdp) was identified in this strain, and its contribution to colistin resistance was demonstrated by growth inhibition experiments using knock-out (Δcdp) and complemented (Δcdp::cdp) mutants. Coculture and coinfection experiments revealed that S. maltophilia carrying the cdp gene could inactivate colistin and protect otherwise susceptible Pseudomonas aeruginosa, which may seriously affect the clinical efficacy of the drug for the treatment of cystic fibrosis patients with polymicrobial infection.

Conclusions: Our results suggest that Cdp should be recognized as a colistin resistance determinant that confers collective resistance at the microbial community level. Our study will provide vital information for successful clinical outcomes during the treatment of complex polymicrobial infections, particularly including S. maltophilia and other colistin-susceptible Gram-negative pathogens such as P. aeruginosa. Video abstract.

Keywords: Antimicrobial resistance; Colistin; Colistin-degrading protease; Collective resistance; Polymicrobial infection; Stenotrophomonas maltophilia.

Fig. 1

Cleavage and inactivation of colistin by S. maltophilia strain Col1. a HPLC chromatograms and disk diffusion assay results (inset) from culture supernatants. b A proposed mechanism of the cleavage of colistin by S. maltophilia strain Col1. DAB, MO, and MH indicate l-diaminobutyric acid, 6-methyloctanoyl, and 6-methylheptanoyl, respectively

Fig. 2

Inhibition of growth by colistin and colistin degradation in the cultures of S. maltophilia strains. Colistin was added to 12-h cultures at a concentration of 128 mg/L. The number of viable cells was determined as colony-forming units (CFU). Circle, triangle, and square indicate strains Col1 (wild type), Col2 (Δcdp), and Col3 (Δcdp::cdp), respectively. Closed and open symbols indicate viable cell count and concentration of residual colistin in the cultures, respectively

Fig. 3

Colistin exposure protection to P. aeruginosa provided by Cdp-producing S. maltophilia strains during cocultures. a Planktonic coculture assay. Changes in viable cell number were estimated for P. aeruginosa strain PAO1 (red) and S. maltophilia strains (black). The concentration of residual colistin in cocultures was measured after colistin spike (32 mg/L). b Solid agar coculture assay. The bacterial lawn of S. maltophilia strains was positioned at the bottom. Red arrows indicate the spreading growth of P. aeruginosa strain PAO1 toward S. maltophilia strains

Fig. 4

Antibacterial efficacy of colistin influenced by Cdp-producing S. maltophilia strains during Drosophila coinfection. Antibacterial efficacy of colistin was determined by the survival rate of Drosophila animals infected by P. aeruginosa PA14. S. maltophilia cells (strains Col1, Col2, and Col3) were coinfected with P. aeruginosa as described in the “Methods” section. The infected flies were fed with either 1 mg/mL colistin, and their survival rates were determined over time. The dotted lines represent the time required to reach 50% mortality. The statistical significance based on a log-rank test is indicated (*p = 0.0142; **p = 0.0058). Closed and open symbols indicate animals treated with and without colistin, respectively

Fig. 5

Phylogeny of S. maltophilia genomes associated with colistin-degrading proteases. a A genome-based phylogenetic tree was reconstructed by the maximum likelihood method using the concatenated alignments of 1073 core genes among S. maltophilia genomes. S. rhizophila was used as an outgroup. Shaded colors on strain names indicate amino acid sequence identity compared to that of the Cdp of strain Col1. Colors in the inner circle indicate the isolation source of strains: human (blue), environment (brown), animal (pink), plant (green), and unknown (gray). Colors in the outer circle indicate the different sources of human specimens: respiratory tract (cyan), blood and bodily fluid (red), skin (orange), minor specimen (purple), and unknown (gray). Strains tested for colistin-degrading activity are marked with numbers. b Characteristics of S. maltophilia strains tested for colistin-degrading activity