Genome-scale identification method applied to find cryptic aminoglycoside resistance genes in Pseudomonas aeruginosa

Abstract

Background: The ability of bacteria to rapidly evolve resistance to antibiotics is a critical public health problem. Resistance leads to increased disease severity and death rates, as well as imposes pressure towards the discovery and development of new antibiotic therapies. Improving understanding of the evolution and genetic basis of resistance is a fundamental goal in the field of microbiology.

Results: We have applied a new genomic method, Scalar Analysis of Library Enrichments (SCALEs), to identify genomic regions that, given increased copy number, may lead to aminoglycoside resistance in Pseudomonas aeruginosa at the genome scale. We report the result of selections on highly representative genomic libraries for three different aminoglycoside antibiotics (amikacin, gentamicin, and tobramycin). At the genome-scale, we show significant (p<0.05) overlap in genes identified for each aminoglycoside evaluated. Among the genomic segments identified, we confirmed increased resistance associated with an increased copy number of several genomic regions, including the ORF of PA5471, recently implicated in MexXY efflux pump related aminoglycoside resistance, PA4943-PA4946 (encoding a probable GTP-binding protein, a predicted host factor I protein, a delta 2-isopentenylpyrophosphate transferase, and DNA mismatch repair protein mutL), PA0960-PA0963 (encoding hypothetical proteins, a probable cold shock protein, a probable DNA-binding stress protein, and aspartyl-tRNA synthetase), a segment of PA4967 (encoding a topoisomerase IV subunit B), as well as a chimeric clone containing two inserts including the ORFs PA0547 and PA2326 (encoding a probable transcriptional regulator and a probable hypothetical protein, respectively).

Conclusions: The studies reported here demonstrate the application of new a genomic method, SCALEs, which can be used to improve understanding of the evolution of antibiotic resistance in P. aeruginosa. In our demonstration studies, we identified a significant number of genomic regions that increased resistance to multiple aminoglycosides. We identified genetic regions that include open reading frames that encode for products from many functional categories, including genes related to O-antigen synthesis, DNA repair, and transcriptional and translational processes.

Figure 1. Enriched genomic regions plotted along the P. aeruginosa PAO1 genome.

The genomic regions highlighted were were enriched during aminoglycoside resistance selections and identified via microarray. The aminoglycoside used in the selection and concentration are given in the labels of the concentric rings. The unselected library sample shows that signal was present all along the genome, indicating proper genomic coverage.

Figure 2. Hierarchical clustering of selection population microarray data, displaying most enriched genetic loci.

The selection populations are represented with different columns, with names given across the top. Enriched loci are shown in the rows. Regions colored in yellow are less enriched. Regions colored in blue are more enriched. Loci are listed as genomic position given in the center base pair of the locus, ORF number, or gene name. Colored boxes on the far right correspond to genomic regions identified in conventional sequencing and later confirmed to increase aminoglycoside Resistance. The following abbreviations were used to title arrays: A16 (amikacin 16 µg/ml population), A8 (amikacin 8 µg/ml population), G16 (gentamicin 16 µg/ml population), G8 (gentamicin 8 µg/ml population), T2 (tobramycin 2 µg/ml population), and Libr (unselected mixed libraries population).

Figure 3. Contours of microarray signal intensity plotted according to genomic position for the genomic regions identified during sequencing, listed in Table 1 .

The regions highlighted are those of (A) clone T2-C1, (B) clone T2-C3, (C) clone G16-C1, (D) clone G16-C2, and the two regions contained within clone A8-C1 (E and F). The Y-axis on each individual graph depicts the relative microarray signal intensity. The X-axis depicts the genomic position in base pairs. Below the X-axis are the ORFs for which the depicted genomic region encompasses.