Genomic heterogeneity underlies multidrug resistance in Pseudomonas aeruginosa: A population-level analysis beyond susceptibility testing

Abstract

Background Pseudomonas aeruginosa is a persistent and difficult-to-treat pathogen in many patients, especially those with Cystic Fibrosis (CF). Herein, we describe a longitudinal analysis of a series of multidrug resistant (MDR) P. aeruginosa isolates recovered in a 17-month period, from a young female CF patient who underwent double lung transplantation. Our goal was to understand the genetic basis of the observed resistance phenotypes, establish the genomic population diversity, and define the nature of sequence evolution over time. Methods Twenty-two sequential P. aeruginosa isolates were obtained within a 17-month period, before and after a double-lung transplant. At the end of the study period, antimicrobial susceptibility testing, whole genome sequencing (WGS), phylogenetic analyses and RNAseq were performed in order to understand the genetic basis of the observed resistance phenotypes, establish the genomic population diversity, and define the nature of sequence changes over time. Results The majority of isolates were resistant to almost all tested antibiotics. A phylogenetic reconstruction revealed 3 major clades representing a genotypically and phenotypically heterogeneous population. The pattern of mutation accumulation and variation of gene expression suggested that a group of closely related strains was present in the patient prior to transplantation and continued to change throughout the course of treatment. A trend toward accumulation of mutations over time was observed. Different mutations in the DNA mismatch repair gene mutL consistent with a hypermutator phenotype were observed in two clades. RNAseq performed on 12 representative isolates revealed substantial differences in the expression of genes associated with antibiotic resistance and virulence traits. Conclusions The overwhelming current practice in the clinical laboratories setting relies on obtaining a pure culture and reporting the antibiogram from a few isolated colonies to inform therapy decisions. Our analyses revealed significant underlying genomic heterogeneity and unpredictable evolutionary patterns that were independent of prior antibiotic treatment, highlighting the need for comprehensive sampling and population-level analysis when gathering microbiological data in the context of CF P. aeruginosa chronic infection. Our findings challenge the applicability of antimicrobial stewardship programs based on single-isolate resistance profiles for the selection of antibiotic regimens in chronic infections such as CF.

The effects of antibiotics and illness on gut microbial composition in the fawn-footed mosaic-tailed rat (Melomys cervinipes)

“…Neither species was identified in the microbiome of mosaic-tailed rats assessed over a year later. Pseudomonas aeruginosa is an opportunistic multi-drug resistant pathogen that produces redox-active phenazines (e.g., pyocyanin and phenazine 1-carboxylic acid or PCA) involved in several biological pathways, including quorum sensing [84], biofilm formation and virulence [85,86], iron acquisition [84,85] and exopolysaccharide biosynthesis [87]. Pseudomonas aeruginosa converts PCA to pyocyanin via the phenazine-modifying genes, phzM and phzS [88] and is also known to suppress host immunity by activating the DAF-2-Insulin-like signalling pathway [89].…”

Bows and swords: why bacteria carry short and long-range weapons

“…We assume toxins to be freely-diffusible solutes that kill susceptible cells when their local concentration uT exceeds a lethal threshold TC (controlled by Nhits; see below). To represent natural variability in toxin susceptibility [66][67][68] , lethal toxin threshold is drawn for each cell from a normal distribution, N(1, 0.2) at birth (we ignore this stochasticity in the discrete contact toxin model, since with mean Nhits = 1, the chances of any cell surviving more than one hit are approximately 1:130,000). To model the toxin concentration field uT = uT(x, y) [kgTm -3 ] for a given cell configuration, we use the reaction-diffusion equation ∂uT/∂t = DT∇ 2 uT + kTɑ⍴ɸ(x, y) 69 .…”

Genomic diversity and molecular epidemiology of a multidrug resistantPseudomonas aeruginosaDMC30b isolated from hospitalized burn patient in Bangladesh

“…The increasingly frequent infections caused by MDR and XDR strains with limited therapeutic options are associated with high morbidity and mortality worldwide [13,14]. Particularly the emergence of MDR and XDR strains due to bacterial expression of resistance genes such as βlactamases, 16S rRNA methylases, and carbapenemases in recent years leading to severe infections with serious global threats to human health, emphasizing the need for novel (antibiotics-independent) treatment strategies.…”

“…Moreover, P. aeruginosa is a persistent and difficult-to-treat pathogen in many patients, and possesses a versatile arsenal of antimicrobial resistance determinants and virulence factors that enable survival, adaptation, and consequent persistence within the complex milieu of infections [13]. The increasingly frequent infections caused by MDR and XDR strains with limited therapeutic options are associated with high morbidity and mortality worldwide [13, 14]. Particularly the emergence of MDR and XDR strains due to bacterial expression of resistance genes such as β-lactamases, 16S rRNA methylases, and carbapenemases in recent years leading to severe infections with serious global threats to human health, emphasizing the need for novel (antibiotics-independent) treatment strategies.…”

“…The World Health Organization and Centers for Disease Control and Prevention have both designated P. aeruginosa as one of the major (critical) pathogens for which new antibiotics are desperately needed [11, 12]. Moreover, P. aeruginosa is a persistent and difficult-to-treat pathogen in many patients, and possesses a versatile arsenal of antimicrobial resistance determinants and virulence factors that enable survival, adaptation, and consequent persistence within the complex milieu of infections [13]. The increasingly frequent infections caused by MDR and XDR strains with limited therapeutic options are associated with high morbidity and mortality worldwide [13, 14].…”