The Development of Ciprofloxacin Resistance in Pseudomonas aeruginosa Involves Multiple Response Stages and Multiple Proteins

Abstract

Microbes have developed resistance to nearly every antibiotic, yet the steps leading to drug resistance remain unclear. Here we report a multistage process by which Pseudomonas aeruginosa acquires drug resistance following exposure to ciprofloxacin at levels ranging from 0.5؋ to 8؋ the initial MIC. In stage I, susceptible cells are killed en masse by the exposure. In stage II, a small, slow to nongrowing population survives antibiotic exposure that does not exhibit significantly increased resistance according to the MIC measure. In stage III, exhibited at 0.5؋ to 4؋ the MIC, a growing population emerges to reconstitute the population, and these cells display heritable increases in drug resistance of up to 50 times the original level. We studied the stage III cells by proteomic methods to uncover differences in the regulatory pathways that are involved in this phenotype, revealing upregulation of phosphorylation on two proteins, succinate-semialdehyde dehydrogenase (SSADH) and methylmalonate-semialdehyde dehydrogenase (MMSADH), and also revealing upregulation of a highly conserved protein of unknown function. Transposon disruption in the encoding genes for each of these targets substantially dampened the ability of cells to develop the stage III phenotype. Considering these results in combination with computational models of resistance and genomic sequencing results, we postulate that stage III heritable resistance develops from a combination of both genomic mutations and modulation of one or more preexisting cellular pathways.In the ongoing war between bug and drug, Pseudomonas aeruginosa is a frequent victor because it rapidly develops new defenses to any drug that is generated (31,41). This is of grave concern for prevention and spread of infectious disease and is a significant mystery to bacteriologists. While there are a number of known resistance mechanisms that develop in P. aeruginosa, the mystery stems from how these are rapidly generated and accumulate in a population to quickly form high-level resistance to an antimicrobial drug after exposure. Finding solutions to inhibit the rise of resistance in P. aeruginosa is important because the organism is responsible for chronic lung infection in individuals with cystic fibrosis (CF) (6) or chronic obstructive pulmonary disease (COPD) (13,15,28), and it also accounts for nearly 10% of hospital-acquired infections (47, 52).There is a small set of drugs commonly used to treat P. aeruginosa infection, including ciprofloxacin, tobramycin, gentamicin, ceftazidime, and imipenem. While P. aeruginosa has developed various levels of resistance to each of these, its response to ciprofloxacin is of particular interest because the drug is initially very effective, but P. aeruginosa rapidly acquires high-level resistance, rendering the drug impotent. In clinical isolates, approximately 30% of strains now present high-level ciprofloxacin resistance (31).While there have been many studies of factors involved in ciprofloxacin resistance in P. aeruginosa (e.g., references 17,...

Comparative Genome Analysis of Ciprofloxacin-ResistantPseudomonas aeruginosaReveals Genes Within Newly Identified High Variability Regions Associated With Drug Resistance Development

“…26 These two identified proteins were not located in any of our suggested high variability regions in PA; however, they may be associated with the variation candidates we discovered in this study since some of them showed a close relationship with signal transduction and energy-related pathways.…”

“…This idea is supported by the fact that when we used transposons to truncate a single gene in these positions, this increased fitness advantage was eliminated, seemingly because the variants involved in the pathways might act together to develop resistance. Resistance in PA is a multiregulation process involving preexisting cellular pathways 26 and accumulated gene mutations (the results shown in this study). By performing comparative genome analysis with 454 pyrosequencing technology, we found three main potential high variability regions in PA, and from the transposon gene functional assay, we found that the physical locations of the genomic variations impacted the development of ciprofloxacin resistance differently.…”

“…In a previous study, we found that when PA was continuously exposed to ciprofloxacin during its stationary growth phase (i.e., up to 48 hr), PA acquired the drug resistance in a multistage process. 26 Our data indicated that resistance development in PA is not the result of a preexisting resistance in the population, nor is it developed in a single gene mutation event. Instead, the data suggested that preexisting cellular pathways, protein modification regulation, and single nucleotide gene mutations each support the resistance development process, such that highlevel resistance in PA is a stepwise, ratcheting up process.…”

“…The cell culture inoculation and ciprofloxacin exposure were performed following our previously established methods. 26 Minimal inhibitory concentrations (MICs) were determined by Etest (AB bioMérieux, AB Biodisk), using the prescribed protocol. The ciprofloxacin-resistant P. aeruginosa PAO1-CipR (in our previous paper 26 we named it PAO1-6) was a derivative strain of the susceptible wild type PAO1 after 48 hr of exposure.…”

“…Most of the variants located in Zone C showed an interesting linkage between energy-dependent transportation and signal transduction systems, which provides further support of the results found in our previous study. 26 …”