Reverse Engineering Antibiotic Sensitivity in a Multidrug-Resistant Pseudomonas aeruginosa Isolate

Abstract

Antibiotic resistance is a pervasive and growing clinical problem. We describe an evaluation of a reverse engineering approach for identifying cellular mechanisms and genes that could be manipulated to increase antibiotic sensitivity in a resistant Pseudomonas aeruginosa isolate. We began by chemically mutating a broadly resistant isolate of P. aeruginosa and screening for mutants with increased sensitivity to the aminoglycoside amikacin, followed by performing whole-genome transcriptional profiling of the mutant and wild-type strains to characterize the global changes occurring as a result of the mutations. We then performed a series of assays to characterize the mechanisms involved in the increased sensitivity of the mutant strains. We report four primary results: (i) mutations that increase sensitivity occur at a high frequency (10 ؊2 ) relative to the frequency of those that increase resistance (10 ؊5 to 10 ؊10 ) and occur at a frequency 10 4 higher than the frequency of a single point mutation; (ii) transcriptional profiles were altered in sensitive mutants, resulting in overall expression patterns more similar to those of the sensitive laboratory strain PAO1 than those of the parental resistant strain; (iii) genes found from transcriptional profiling had the more dramatic changes in expression-encoded functions related to cellular membrane permeability and aminoglycoside modification, both of which are known aminoglycoside resistance mechanisms; and finally, (iv) even though we did not identify the specific sites of mutation, several different follow-up MIC assays suggested that the mutations responsible for increased sensitivity differed between sensitive mutants.Antibiotic resistance develops rapidly after the introduction of a new antibiotic and now exists, to some extent, for all antibiotics. Resistance has even evolved for drugs specifically designed to prevent selection for resistance (10,23,27,30,43,52,74,85,86). These factors underline the importance of understanding the genetic and phenotypic bases underlying antibiotic resistance and developing new strategies to combat the proliferation of resistant organisms. One approach has been to combine new and conventional antibiotics to simultaneously increase the sensitivities of resistant organisms and target essential genes (40,50,51,73). The success of such an approach is dependent upon the identification of genes and/or mechanisms that might be targeted to increase sensitivity. We report here on our efforts to evaluate a reverse engineering approach for identifying such genes and mechanisms. Specifically, we have identified aminoglycoside-sensitive mutants of a multiple-drug-resistant Pseudomonas aeruginosa isolate and characterized the global changes in gene expression associated with mutations that restored sensitivity in two of these mutants as well as the resistant parental strain and the sensitive laboratory strain PAO1.We chose to study a clinical isolate, named B1, that showed high levels of resistance to five aminoglycosides tested: amikacin,...

Genome-Scale Identification Method Applied to Find Cryptic Aminoglycoside Resistance Genes in Pseudomonas aeruginosa

“…We confirmed the increased resistance of the clones T2-C1, G16-C1, and G16-C2 to aminoglycosides in Luria-Bertani broth. We also confirmed that a previously characterized P. aeruginosa strain, B1 [30] , that has been found to have increased resistance to aminoglycosides in Luria-Bertani broth, maintained the elevated resistance levels in EZ RDM.…”

“…A definitive link between O-antigens and aminoglycoside resistance has yet to be established, though there have been studies supporting the idea of altered or increased amounts of O-antigen structures could increase resistance levels to aminoglycosides. The expression of O-antigen biosynthesis and assembly genes have been found to have a 2- to 4 log-fold decrease in expression between amikacin-sensitive mutants generated from an aminoglycoside-resistant clinical isolate of P. aeruginosa compared to the resistant parent isolate [30] .…”

Prophages are Infrequently Associated With Antibiotic Resistance inPseudomonas aeruginosaClinical Isolates

“…The CARD-RGI analysis supports these results, showing comparable gene presence and abundance. Genes such as mecA, mecR1, blaZ , and femA are consistently detected across all samples by both Solu Bio and CARD-RGI 47,48,49,50,51,52.…”

Antibiotic Resistance Determinants in a Pseudomonas putida Strain Isolated from a Hospital

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