Whole genome and transcriptome analyses of environmental antibiotic sensitive and multi-resistant Pseudomonas aeruginosa isolates exposed to waste water and tap water.
Bottom Line: The expression of resistance genes, genetic mobile elements in PA49 was independent from the water matrix.Consistently, the antibiotic sensitive strain PA30 did not show any difference in expression of the intrinsic resistance determinants and genetic mobile elements.However, changes in environmental milieus resulted in rather unspecific transcriptional responses than selected and stimuli-specific gene regulation.
Affiliation: Institute of Functional Interfaces (IFG), Campus North, Karlsruhe Institute of Technology (KIT), Hermann von Helmholtz Platz 1, Eggenstein-Leopoldshafen, D-76344, Germany.Show MeSH
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Mentions: The prediction of protein coding sequences (CDS) yielded for both strains a comparatively large number of genes, about 99% of which were successfully annotated according to their best-hit BLAST alignments (Table S1). The vast majority of the predicted genes were found in both strains and also in the PAO1 reference genome (5262 genes), representing the conserved core genome of P. aeruginosa. Regarding the development of multi-drug resistance, P. aeruginosa is known for its high intrinsic resistance that is caused by a combination of low membrane permeability, efflux pumps and resistance genes encoded in the core genome (Nikaido, 2001; Schweizer, 2003), together with the potential to develop high-level resistance by accumulation of small mutations (Fajardo et al., 2008; Dötsch et al., 2009; Martinez et al., 2009; Alvarez-Ortega et al., 2010; Breidenstein et al., 2011; Bruchmann et al., 2013). However, the most obvious cause of multi-drug resistance is the acquisition of resistance genes by horizontal gene transfer (Davies and Davies, 2010). Therefore, we performed a blast search of the predicted genes of the two strains in the Comprehensive Antibiotic Resistance Database (CARD) (McArthur et al., 2013) and scanned both genomes for genetic variations of intrinsic resistance determinants. In a previous work, strain PA49 was found to be resistant towards the antibiotics gentamicin (GM), amikacin (AN), azlocillin (AZ), ceftazidime (CAZ), piperacillin/tazobactam (PT), ciprofloxacin (CIP) and imipenem (IPM) (Schwartz et al., 2006). Searching its genome for resistance determinants revealed the presence of one aminoglycoside acetyltransferase of the AAC(6’)-type, two aminoglycoside adenylyltransferases of type ANT(2'’) and ANT(3'’) and one VIM metallo-beta-lactamase (Table 1). Two additional genes were annotated as beta-lactamases in PA49 only by the BLAST search in the National Center for Biotechnology Information (NCBI) non-redundant (nr) protein database but not found in the CARD database (Fig. 3A). Taken together, these genes confer resistance towards a wide range of aminoglycosides and beta-lactam antibiotics, explaining the resistance towards GM, AN, AZ, CAZ and PT. Fluoroquinolones like CIP target the DNA gyrase and Topoisomerase IV enzyme complexes, and high-level resistance towards these antibiotics is often caused by sequence variations of the two subunits GyrA (gyrase) and ParC (topoisomerase) (Ruiz, 2003) and indeed, both proteins contained a single amino acid exchange in the resistance determining region (Table 1). These two mutations represent the most common type of variations found in fluoroquinolone resistant isolates of P. aeruginosa and have recently been shown to be sufficient for the development of high-level resistance towards CIP (Bruchmann et al., 2013). Finally, a frameshift mutation in the outer membrane porin OprD was found that is likely to cause misfolding or decreased functionality of the protein. Defective mutations of OprD are known to cause resistance towards carbapenems including IPM in combination with intrinsic beta-lactamases and efflux pumps (Pirnay et al., 2002). Of note, the strain PA30 that is sensitive towards all these antibiotics did not contain any known horizontally acquired resistance genes and harboured wild-type alleles of the target genes gyrA, parC and oprD (Table 1). In summary, these results provide a comprehensive explanation for the resistance phenotype covering all the antibiotics that were tested, since all resistance determining genes and alleles (besides the ones intrinsic to P. aeruginosa) were exclusively found in PA49 (Fig. 3A; Table 2).
Affiliation: Institute of Functional Interfaces (IFG), Campus North, Karlsruhe Institute of Technology (KIT), Hermann von Helmholtz Platz 1, Eggenstein-Leopoldshafen, D-76344, Germany.