Biofilm Associated Genotypes of Multidrug-Resistant Pseudomonas aeruginosa

Abstract

Pseudomonas aeruginosa is a ubiquitous environmental microorganism that is also a common cause of nosocomial infections that vary in severity from chronic wound infections to pneumonia, bloodstream infections and sepsis. Its ability to survive in many different environments and persistently colonize human tissue is linked to its presence within biofilms that form on indwelling device surfaces such as plastics and stainless steel. Biofilm promotes bacterial adhesion and survival on surfaces, reduces susceptibility to desiccation, and the actions of antibiotics and disinfectants. Recent genome sequencing studies demonstrate that P. aeruginosa is a highly diverse species with a very large pan-genome consistent with its adaptability to differing environments. However, most MDR infections are caused by a small number of “high-risk” clones or lineages that recently emerged and spread globally. In our 2017 study of the resistome of P. aeruginosa we confirmed the power of genome-wide association (GWAS) techniques to explore the genetic basis of several antibiotic resistance phenotypes and discovered 46 novel putative resistance polymorphisms. In this study we sought to examine genetic associations within a subset of these isolates with simple biofilm phenotypes. We examined the genetic basis for biofilm production on polystyrene at room temperature (22°C) and body temperature (37°C) within a total of 280 isolates. 69% of isolates (n=193) produced more biofilm mass at 22°C, whilst those producing more biofilm at 37°C had reduced optical density 540 variation. We found statistically significant associations with IpxO and other genes associated with arsenic resistance to be significantly associated with this trait. IpxO which encodes a lipid A hydroxylase and arsenic reduction genes have previously been found to be associated with biofilm production in this species. We analyzed 260 ST111 and ST235 genomes and found considerable genetic variation between isolates in their content of genes previously found associated with biofilm production. This is indicative of a highly variable and flexible population within these clades with frequent emergence of successful sub-lineages. Analysis of 48 of these isolates’ ability to form biofilm on stainless steel surfaces showed that a ‘good’ biofilm-forming phenotype had significant intra-clone variation, independent of core genome phylogeny with pan-genome analysis, suggesting a possible association and involvement of components of the type IV secretion system. However, GWAS and pan-GWAS analyses yielded weaker statistical significance. This study confirms GWAS and pan-GWAS trait associations can be performed for biofilm phenotype and produce data in agreement with each other. This panel of 280 study isolates, matched to genomic data has potential for the investigation of other phenotypes in P. aeruginosa perhaps as part of a growing database / collection. A representative, curated, genome sequenced collection should increase in usefulness as it grows offering increasing statistical power. Importance P. aeruginosa is a major cause of multiply antibiotic infections worldwide but it is also found in many hospital and natural environments, especially aquatic ones. In this study we examined genetic polymorphism associated with biofilm production at room temperature and at body temperature, the biofilm associated gene repertoire of two major MDR clones and also genetic polymorphisms associated with biofilm production on stainless steel. Using these genome-wide and pan-genome wide association methods we identified / confirmed potential key genes involved in biofilm production and survival of P. aeruginosa. The study demonstrates the potential usefulness of large, genome sequenced isolate collections such as ours, to better understand the genetics underlying phenotypic diversity in this species.