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

PLOS ONE, Dec 2019

Background Small 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 Findings One 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. Conclusions By 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.

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

et al. (2011) Phenotypic and Genome-Wide Analysis of an Antibiotic-Resistant Small Colony Variant (SCV) of Pseudomonas aeruginosa. PLoS ONE 6(12): e29276. doi:10.1371/journal.pone.0029276 Phenotypic and Genome-Wide Analysis of an Antibiotic- Resistant Small Colony Variant (SCV) of Pseudomonas aeruginosa Qing Wei 0 Saeed Tarighi 0 Andreas Do tsch 0 Susanne Ha ussler 0 Mathias Mu sken 0 Victoria J. Wright 0 Miguel Ca mara 0 Paul Williams 0 Steven Haenen 0 Bart Boerjan 0 Annelies Bogaerts 0 Evy 0 Vierstraete 0 Peter Verleyen 0 Liliane Schoofs 0 Ronnie Willaert 0 Vale rie N. De Groote 0 Jan Michiels 0 Ken Vercammen 0 Aure lie Crabbe 0 Pierre Cornelis 0 Jacques Ravel, Institute for Genome Sciences, University of Maryland School of Medicine, United States of America 0 Current address: Laboratory of Phytopathology, Department of Crop Protection, Ferdowsi University of Mashhad , Mashhad , Iran Background: Small 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 Findings: One 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 1025 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. Conclusions: By 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. - Funding: This work was supported by two research grants from the FWO Belgium (Fonds voor Wetenschappelijk Onderzoek Vlaanderen) and a grant from BBSRC UK (BBF0143921). QW has a CSC-VUB scholarship and got a FEMS student fellowship to perform experiments in the lab of Dr. Susanne Hau ssler. BB and AB are research assistants and PV is a postdoctoral researcher of the FWO-Vlaanderen. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Pseudomonas aeruginosa is a ubiquitous Gram-negative bacterium found in diverse ecological habitats such as soils, marshes and coastal marine waters. As an opportunistic pathogen, P. aeruginosa is able to infect humans, animals and plants [1,2,3]. P. aeruginosa is a primary nosocomial diseases causative agent and represents the major cause of morbidity and mortality in patients with cystic fibrosis (CF). P. aeruginosa produces a large panel of secreted virulence factors like the phenazine pyocyanin, the siderophore pyoverdine, elastase, and toxins. It is also characterized by its high level of drug resistance involving the formation of antibioticresistant biofilms resulting from the emergence of phenotypic variants [2,3]. During the course of infection, P. aeruginosa can efficiently adopt diverse strategies to evade antimicrobial stresses and the host immune system defenses, making it impossible to eradicate this bacterium permanently from CF lungs [2,4]. Important phenotypic variations can occur during chronic colonization, such as conversion to mucoidy [5], the emergence of persister cells after antibiotics treatment [6,7] or the occurrence of small colony variants with higher resistance to antibiotics [8,9,10,11,12]. Compared to wild-type P. aeruginosa, SCVs show increased antibiotic resistance, enhanced biofilm formation, reversion to wild-type-like morphotypes, reduced motility, and slow and auto-aggregative growth behavior [13,14]. SCVs hav (...truncated)


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Qing Wei, Saeed Tarighi, Andreas Dötsch, Susanne Häussler, Mathias Müsken, Victoria J. Wright, Miguel Cámara, Paul Williams, Steven Haenen, Bart Boerjan, Annelies Bogaerts, Evy Vierstraete, Peter Verleyen, Liliane Schoofs, Ronnie Willaert, Valérie N. De Groote, Jan Michiels, Ken Vercammen, Aurélie Crabbé, Pierre Cornelis. Phenotypic and Genome-Wide Analysis of an Antibiotic-Resistant Small Colony Variant (SCV) of Pseudomonas aeruginosa, PLOS ONE, 2011, Volume 6, Issue 12, DOI: 10.1371/journal.pone.0029276