Transcriptomic Basis of Serum Resistance and Virulence Related Traits in XDR P. aeruginosa Evolved Under Antibiotic Pressure in a Morbidostat Device.

Mumina Javed,Angel Angelov,Matthias Willmann,Viola Ueltzhoeffer,Ulrich Schoppmeier,Silke Peter,Sandra Schwarz,Maximilian Heinrich,Benedikt Jentzsch

doi:10.3389/fmicb.2020.619542

Abstract

Colistin is a last resort antibiotic against the critical status pathogen Pseudomonas aeruginosa. Virulence and related traits such as biofilm formation and serum resistance after exposure to sub-inhibitory levels of colistin have been underexplored. We cultivated P. aeruginosa in a semi-automated morbidostat device with colistin, metronidazole and a combination of the two antibiotics for 21 days, and completed RNA-Seq to uncover the transcriptional changes over time. Strains became resistant to colistin within this time period. Colistin-resistant strains show significantly increased biofilm formation: the cell density in biofilm increases under exposure to colistin, while the addition of metronidazole can remove this effect. After 7 days of colistin exposure, strains develop an ability to grow in serum, suggesting that colistin drives bacterial modifications conferring a protective effect from serum complement factors. Of note, strains exposed to colistin showed a decrease in virulence, when measured using the Galleria mellonella infection model. These phenotypic changes were characterized by a series of differential gene expression changes, particularly those related to LPS modifications, spermidine synthesis (via speH and speE) and the major stress response regulator rpoS. Our results suggest a clinically important bacterial evolution under sub-lethal antibiotic concentration leading to potential for significant changes in the clinical course of infection.

Highlights

Pseudomonas aeruginosa is a gram-negative, opportunistic bacterium and a frequent cause of healthcare acquired infections (HAIs)
The aim of this work was to investigate the specific contribution of metronidazole alongside the development of colistin resistance in P. aeruginosa and evaluate the effect of antibiotic stress and on bacterial fitness, geno- and phenotype
A number of assays were performed to elucidate the changes in the bacterial phenotype under distinct antibiotic pressure

Summary

Introduction

Pseudomonas aeruginosa is a gram-negative, opportunistic bacterium and a frequent cause of healthcare acquired infections (HAIs). It belongs to the group of ESKAPE pathogens, which consist of six microorganisms, namely Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp., with a high tendency for causing challenging, drugresistant, nosocomial infections (Rice, 2008). Antibiotics are often given in combination in standard therapeutic regimens, with metronidazole being a common drug partner for the treatment of infections by obligate and facultative anaerobic bacteria. It is effective for the management of intra-abdominal infections, gynecological infections, septicemia, endocarditis, bone, and joint infections, amongst several other types of infections (Löfmark et al, 2010). An interesting study found that despite metronidazole having no bactericidal effect on P. aeruginosa, in vitro exposure to a therapeutic concentration of metronidazole increased the number of mutations through induction of the SOS response, leading to emergence of antibiotic resistant bacteria (Hocquet and Bertrand, 2014)

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