PMC10235614_01
Timeline depicting the different antibiotic and phage therapies, the isolation of Pseudomonas aeruginosa strains from blood cultures and their antibiotic susceptibility and genomic analyses. PFGE of these strains in described in Supplementary Figure S1. Antimicrobial information: 1MEM, Meropenem. 2CZA, Ceftazidime-avibactam. 3MIC, Minimum inhibitory concentration. 4TZP, Piperacillin-tazobactam. The five isolates that underwent Whole Genome Sequencing (WGS) are shown with the "target" symbols; of those, the two isolates that are further analyzed in the manuscript, P. aeruginosa isolate HE2011471 (pre-phage therapy) and P. aeruginosa isolate HE2105886 (post-phage therapy) are highlighted with the red boxes.
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P. aeruginosa. Antimicrobial susceptibility testing was performed by broth microdilution (Microscan, Beckman Coulter) and interpreted using European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria. Pulsed-field gel electrophoresis (PFGE) revealed that four representative isolates, from 2020 and 2021, showed the same pattern and therefore were clonal (Supplementary Figure S1). HE2105886, recovered 1 month after phage treatment, exhibited a decrease in the Minimum Inhibitory Concentration (MICs) of ss-lactam and quinolone antibiotics (Figure 1) probably due to the mutations of the membrane receptors, regulators and efflux pumps. However, we did not test any of these isolates against the individual phages in the cocktail to track the sensitivity or resistance to each of them.
PMC10235614_02
Timeline depicting the different antibiotic and phage therapies, the isolation of Pseudomonas aeruginosa strains from blood cultures and their antibiotic susceptibility and genomic analyses. PFGE of these strains in described in Supplementary Figure S1. Antimicrobial information: 1MEM, Meropenem. 2CZA, Ceftazidime-avibactam. 3MIC, Minimum inhibitory concentration. 4TZP, Piperacillin-tazobactam. The five isolates that underwent Whole Genome Sequencing (WGS) are shown with the "target" symbols; of those, the two isolates that are further analyzed in the manuscript, P. aeruginosa isolate HE2011471 (pre-phage therapy) and P. aeruginosa isolate HE2105886 (post-phage therapy) are highlighted with the red boxes.
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F1
P. aeruginosa. Antimicrobial susceptibility testing was performed by broth microdilution (Microscan, Beckman Coulter) and interpreted using European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria. Pulsed-field gel electrophoresis (PFGE) revealed that four representative isolates, from 2020 and 2021, showed the same pattern and therefore were clonal (Supplementary Figure S1). HE2105886, recovered 1 month after phage treatment, exhibited a decrease in the Minimum Inhibitory Concentration (MICs) of ss-lactam and quinolone antibiotics (Figure 1) probably due to the mutations of the membrane receptors, regulators and efflux pumps. However, we did not test any of these isolates against the individual phages in the cocktail to track the sensitivity or resistance to each of them.
Circular chromosomic view of the bacterial genomes of two Pseudomonas aeruginosa isolates: P. aeruginosa isolate HE2105886 (after phage treatment) in comparison with P. aeruginosa isolate HE2011471 (prior to phage administration). The SNPs found in P. aeruginosa isolate HE2105886 respect to P. aeruginosa isolate HE2011471 are indicated.
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MexCD-OprJ, in which multiple amino acid changes were observed in the last 3 isolates (Supplementary Table S1). Moreover, comparison of the genomes of P. aeruginosa isolates HE2011471 (previous to phage administration) and HE2105886 (1 month after phage treatment) revealed important mutations (Figure 2).
Graphic representation of the proteins coded by the P. aeruginosa HE2105886 genome (post-phage therapy isolate), that presented mutations when compared to pre-phage therapy isolate, with emphasis of changes associated with mechanisms of resistance and response to phage infection. Among them we highlight phage infection by adsorption as well as Quorum Sensing activation (1A, 1B) alteration of the antibiotic susceptibility (receptors and efflux pumps) (2A), inhibition of the phage infection (filamentous prophages, TA (toxin-antitoxin) systems and small molecules) (2B) and finally, virulence (oxidative stress and secretion systems) (2C). aSmall molecules such as aminoglycosides which participate in the inhibition of the phage infection well as bacterial antimicrobial profile.
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Interestingly, several detected genomic changes could be involved in phage resistance (Supplementary Table S2, Figure 3).
Comparison of the biofilm production (A) and the frequency of occurrence of phage resistant mutants (B) between the Pseudomonas aeruginosa isolates HE2011471 (pre-phage therapy) and HE2105886 (post-phage therapy). Statistical analysis is t-test with Graphpad 9.0.0 ****<0.0001; **0.0030.
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These two tests were deemed relevant, as bacteria can form biofilms as a response to environmental stress, such as the presence of antibiotics, and this, alongside the emergence of phage-resistance could explain the observed failure to eradicate the infection with phage therapy (Figures 4A, B).