The resistome of Pseudomonas aeruginosa in relationship to phenotypic susceptibility

Abstract

Many clinical isolates of Pseudomonas aeruginosa cause infections that are difficult to eradicate due to their resistance to a wide variety of antibiotics. Key genetic determinants of resistance were identified through genome sequences of 390 clinical isolates of P. aeruginosa, obtained from diverse geographic locations collected between 2003 and 2012 and were related to microbiological susceptibility data for meropenem, levofloxacin, and amikacin. β-Lactamases and integron cassette arrangements were enriched in the established multidrug-resistant lineages of sequence types ST111 (predominantly O12) and ST235 (O11). This study demonstrates the utility of next-generation sequencing (NGS) in defining relevant resistance elements and highlights the diversity of resistance determinants within P. aeruginosa. This information is valuable in furthering the design of diagnostics and therapeutics for the treatment of P. aeruginosa infections.

FIG 1

(A) Temporal, geographic and anatomical association of isolates used in this study. The majority of isolates came from sites in Germany, Spain, France, China, and the United States, while others were procured from hospitals in Argentina, Canada, Colombia, Croatia, Brazil, Greece, Italy, Israel, India, Portugal, Philippines, Romania, and Taiwan (see Table S1 in the supplemental material). (B) Box plot depicting size of draft genomes of P. aeruginosa associated with different clades and different anatomical association. CF isolates are distinguished as a separate group. Percentiles are marked. (C) Unrooted maximum-likelihood tree of 405 P. aeruginosa genomes based on single nucleotide polymorphisms within the core genome of 1,278 genes as defined by alignment to the PAO1 genome. Isolates are colored according to their continental origin. Representative genomes from NCBI are marked and colored gray. Isolates from Asia are colored red, isolates from Europe are blue, isolates from North America are yellow, and isolates from South America are green. The impression of the true distance of the phylogenetic tree is also provided.

FIG 2

Summary of β-lactamase content. (A) Geographic association of β-lactamases among the collection of 390 P. aeruginosa isolates. Summary of class A (beige), B (yellow), and D (green) β-lactamase and cross-class combinations as marked: class D (2 types), gray; class AB, red; class AD, blue; class BD, orange; and class ABD, purple. Isolates that carried multiple β-lactamases from the same class are outlined in Table S1 in the supplemental material. Chromosomal ampC and bla_OXA-50 genes were accounted for in the isolates except where noted (see Fig. S2A in the supplemental material). (B) Phylogenetic association of class A (beige), B (yellow), and D (green) β-lactamases. Clades are colored accordingly (PAO1-like, PA14-like, PA7-like, and new). Reference isolates are highlighted as are the isolates corresponding to ST235 and ST111, which were the largest groups enriched with β-lactamases.

FIG 3

(A) Model identifying genetic determinants that were attributed to explaining the genetic susceptibility profile of the P. aeruginosa isolates. Meropenem susceptibility was genotypically assigned primarily based upon sequence modifications of OprD. Class B β-lactamases were also associated with a genotypic nonsusceptible phenotype when present in the absence of OprD modifications. Similarly, carbapenemases such as KPC-2 were considered to assign an isolate as nonsusceptible by genotype. For levofloxacin, combinations of mutations within the QRDR of gyrA/B and parC/E were primarily used to assign genotypic susceptibility. Amikacin genotypic susceptibility was assigned by identifying any of the contributing aminoglycoside resistance genes in any combination. Additional factors that may complicate the analysis and further contribute to resistance are also highlighted. (B) Comparison of the susceptibility profiles with the genetic determinants that classify an isolate as susceptible or nonsusceptible overlaid on the phylogenetic tree. The innermost ring (ring 1) illustrates the phenotypic susceptibility profile, where light red represents a nonsusceptible isolate and blue a susceptible isolate. Reference isolates are highlighted in gray as they serve for orientation and were not included in the analysis. Ring 2 highlights isolates that had changes in OprD (green) that would hinder the transport of meropenem through this porin. Isolates for which information could not be deduced for OprD due to incomplete sequence coverage are highlighted in gray. The third ring (ring 3) highlights the isolates that were found to carry class B MBL (purple) and those found to carry the carbapenemase KPC-2 (pink). The outermost ring (ring 4) highlights those isolates for which a discrepancy arose between the phenotypic susceptibility data and the genotypic analysis of the resistance-related components (black). (C) The 2-by-2 table summarizes the overlap in phenotypic (susceptibility) versus genotypic characterization for meropenem, levofloxacin, and amikacin.