Genomewide identification of genetic determinants of antimicrobial drug resistance in Pseudomonas aeruginosa

Abstract

The emergence of antimicrobial drug resistance is of enormous public concern due to the increased risk of delayed treatment of infections, the increased length of hospital stays, the substantial increase in the cost of care, and the high risk of fatal outcomes. A prerequisite for the development of effective therapy alternatives is a detailed understanding of the diversity of bacterial mechanisms that underlie drug resistance, especially for problematic gram-negative bacteria such as Pseudomonas aeruginosa. This pathogen has impressive chromosomally encoded mechanisms of intrinsic resistance, as well as the potential to mutate, gaining resistance to current antibiotics. In this study we have screened the comprehensive nonredundant Harvard PA14 library for P. aeruginosa mutants that exhibited either increased or decreased resistance against 19 antibiotics commonly used in the clinic. This approach identified several genes whose inactivation sensitized the bacteria to a broad spectrum of different antimicrobials and uncovered novel genetic determinants of resistance to various classes of antibiotics. Knowledge of the enhancement of bacterial susceptibility to existing antibiotics and of novel resistance markers or modifiers of resistance expression may lay the foundation for effective therapy alternatives and will be the basis for the development of new strategies in the control of problematic multiresistant gram-negative bacteria.

FIG. 1.

AST of the Harvard P. aeruginosa PA14 transposon mutant library using the Vitek 2 system. Min and Max, minimum and maximum MICs (in micrograms per milliliter) that can be determined with the Vitek 2 system. Calling ranges are also indicated by a box in each column. The MIC of each antibiotic for wild-type PA14 is given by aligning the number of wild-type PA14 isolates (shaded) horizontally with the applicable MIC on the left. Similarly, the distribution of the MICs of a particular antibiotic across all PA14 mutants is given by aligning the numbers of mutant isolates horizontally with the applicable MICs on the left. A MIC below the calling range should be read as “less than or equal to” the value given, and a MIC above the calling range should be read as “greater than or equal to” the value given. Note that the MICs for SXT are shown in the rightmost column. Failed, number of failed measurements.

FIG. 2.

SDS-polyacrylamide gel electrophoresis of membrane proteins. In contrast to wild-type PA14 (lane 1), the PA14 oprF mutant (lane 2) is missing the OprF band at 37.6 kDa (arrow). Lane M, prestained PageRuler protein ladder (Fermentas).