Pseudomonas aeruginosa SST235 high-risk clone has unique adaptive response mechanisms to colistin stress

The dissemination of Pseudomonas aeruginosa (P.aeruginosa) ST235 high-risk clone is a public health threat. The emergence of antimicrobial resistance limits treatment options for P.aeruginosa infections. We aimed to identify new target sites for anti-virulent and antimicrobial therapy for multidrug-resistant ST235 P.aeruginosa infections by exploring metagenomic and transcriptomic responses of colistin-resistant P.aeruginosa. The study was performed in two stages. The twenty-seven patients diagnosed with colistin-resistant P.aeruginosa infection were included in the first resistant cohort. Secondly, two patients diagnosed with colistin-susceptible P.aeruginosa were included in the longitudinal study, and colistin tolerant/persister cells were induced under high colistin concentrations. The virulence genes and the genes related to persister formation were analyzed. Metagenomic analyses were performed to determine sequence type and the single nucleotide polymorphism (SNP). RNAseq was conducted to illuminate altered pathways. All assays were performed with Illumina HiseqXten/Novaseq sequencing platform. Outer membrane porin protein expression levels were studied with immunoblotting. The virulence scores of the isolates were determined by in vivo Caenorhabditis elegans (C. elegans) fertility model. We observed that the mortality rate was significantly higher in the cases infected with ST235 (81.8%) than in non-ST235 cases (36.4%) (p=0.0237). Molecular analysis showed that ST235 clones had amino acid substitutions in the pyoverdine biosynthesis genes of pvdA, pvdD, pvdJ, and pvdL, respectively. Besides, pvdD and pvdJ genes were downregulated in ST235 clones compared to NONST235 (p=0.0004). However, pyoverdine secretion was not significantly different in ST235 than the other clones (59.996 vs. 74.112 rfu/OD). In the longitudinal study, the metagenomic analysis presented that tolerant cell had genotypically the same missense mutations as susceptible and resistant isolates in persister-related genes. However, transcriptomic studies illuminated that tolerance cells expressed more DNA/repair/SOS response protein repair and stringent response genes than susceptible and resistant cells. Moreover, tolerance cells had the highest ((p)ppGpp synthesis gene relA expression compared to susceptible and resistant isolates. Colistin-resistant P.aeruginosa ST235 had completely different pyoverdine systems than other clones. These modifications in pyoverdine could contribute to the high virulence of the ST235 clone and should be considered as a target for anti-virulence therapy. The upregulation of the DNA repair/SOS response gene triggers the formation of colistin tolerant cells without alteration in genotype. Tolerome formation occurring in colistin susceptible isolates may play a significant role in the development of colistin resistance. Preventing the development of tolerance may offer a new strategy for delaying the emergence of resistance.