Phenotypic and Genome-Wide Analysis of an Antibiotic-Resistant Small Colony Variant (SCV) of Pseudomonas aeruginosa

Qiaohua Wei ,Paul Williams,Ronnie Willaert,Bart Boerjan,Annelies Bogaerts,Evy Vierstraete,Mathias Müsken,Jan Michiels,Valérie N De Groote,Andreas Dötsch,Peter Verleyen,Aurélie Crabbé,Miguel Cámara,Saeed Tarighi,Ken Vercammen,Liliane Schoofs,Steven Haenen,Susanne Häußler ,Victoria Wright ,Pierre Cornélis

doi:10.1371/journal.pone.0029276

Abstract

BackgroundSmall colony variants (SCVs) are slow-growing bacteria, which often show increased resistance to antibiotics and cause latent or recurrent infections. It is therefore important to understand the mechanisms at the basis of this phenotypic switch.Methodology/Principal FindingsOne SCV (termed PAO-SCV) was isolated, showing high resistance to gentamicin and to the cephalosporine cefotaxime. PAO-SCV was prone to reversion as evidenced by emergence of large colonies with a frequency of 10−5 on media without antibiotics while it was stably maintained in presence of gentamicin. PAO-SCV showed a delayed growth, defective motility, and strongly reduced levels of the quorum sensing Pseudomonas quinolone signal (PQS). Whole genome expression analysis further suggested a multi-layered antibiotic resistance mechanism, including simultaneous over-expression of two drug efflux pumps (MexAB-OprM, MexXY-OprM), the LPS modification operon arnBCADTEF, and the PhoP-PhoQ two-component system. Conversely, the genes for the synthesis of PQS were strongly down-regulated in PAO-SCV. Finally, genomic analysis revealed the presence of mutations in phoP and phoQ genes as well as in the mexZ gene encoding a repressor of the mexXY and mexAB-oprM genes. Only one mutation occurred only in REV, at nucleotide 1020 of the tufA gene, a paralog of tufB, both encoding the elongation factor Tu, causing a change of the rarely used aspartic acid codon GAU to the more common GAC, possibly causing an increase of tufA mRNA translation. High expression of phoP and phoQ was confirmed for the SCV variant while the revertant showed expression levels reduced to wild-type levels.ConclusionsBy combining data coming from phenotypic, gene expression and proteome analysis, we could demonstrate that resistance to aminoglycosides in one SCV mutant is multifactorial including overexpression of efflux mechanisms, LPS modification and is accompanied by a drastic down-regulation of the Pseudomonas quinolone signal quorum sensing system.

Highlights

Pseudomonas aeruginosa is a ubiquitous Gram-negative bacterium found in diverse ecological habitats such as soils, marshes and coastal marine waters
By combining data coming from phenotypic, gene expression and proteome analysis, we could demonstrate that resistance to aminoglycosides in one Small Colony Variant (SCV) mutant is multifactorial including overexpression of efflux mechanisms, LPS modification and is accompanied by a drastic down-regulation of the Pseudomonas quinolone signal quorum sensing system
The ‘‘phenotypic variant regulator’’, PvrR, containing a conserved EAL domain of phosphodiesterase (PDE) involved in the hydrolysis of c-diGMP, has been identified to control the phenotypic switch from an antibiotic resistant and auto-aggregative rough SCV (RSCV) of P. aeruginosa strain PA14 to wild-type-like antibiotics susceptible revertants [15]. Another characteristic driven by the elevated level of c-di-GMP in SCVs is the contribution of two EPS-encoding loci in some P. aeruginosa strains (PA2231-PA2245 for psl and PA3058PA3064 for pel) to auto-aggregation and hyper adherence phenotypes characterized by increased Congo Red dye binding [27,28,29]

Summary

Introduction

Pseudomonas aeruginosa is a ubiquitous Gram-negative bacterium found in diverse ecological habitats such as soils, marshes and coastal marine waters. P. aeruginosa produces a large panel of secreted virulence factors like the phenazine pyocyanin, the siderophore pyoverdine, elastase, and toxins It is characterized by its high level of drug resistance involving the formation of antibioticresistant biofilms resulting from the emergence of phenotypic variants [2,3]. The ‘‘phenotypic variant regulator’’, PvrR, containing a conserved EAL domain of phosphodiesterase (PDE) involved in the hydrolysis of c-diGMP, has been identified to control the phenotypic switch from an antibiotic resistant and auto-aggregative rough SCV (RSCV) of P. aeruginosa strain PA14 to wild-type-like antibiotics susceptible revertants [15]. It is important to understand the mechanisms at the basis of this phenotypic switch

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Discussion

Conclusion