Irith Wiegand

As multidrug resistance increases alarmingly, polymyxin B and colistin are increasingly being used in the clinic to treat serious Pseudomonas aeruginosa infections. In this opportunistic pathogen, subinhibitory levels of polymyxins and certain antimicrobial peptides induce resistance toward higher, otherwise lethal, levels of these antimicrobial agents. It is known that the modification of lipid A of lipopolysaccharide (LPS) is a key component of this adaptive peptide resistance, but to date, the regulatory mechanism underlying peptide regulation in P. aeruginosa has remained elusive. The PhoP-PhoQ and PmrA-PmrB two-component systems, which control this modification under low-Mg2+ conditions, do not appear to play a major role in peptide-mediated adaptive resistance, unlike in Salmonella where PhoQ is a peptide sensor. Here we describe the identification and characterization of a novel P. aeruginosa two-component regulator affecting polymyxin-adaptive resistance, ParR-ParS (PA1799-PA1798). This system was required for activation of the arnBCADTEF LPS modification operon in the presence of subinhibitory concentrations of polymyxin, colistin, or the bovine peptide indolicidin, leading to increased resistance to various polycationic antibiotics, including aminoglycosides. This study highlights the complexity of the regulatory network controlling resistance to cationic antibiotics and host peptides in P. aeruginosa, which has major relevance in the development and deployment of cationic antimicrobials.

Pseudomonas aeruginosa exhibits swarming motility on semisolid surfaces (0.5 to 0.7% agar). Swarming is a more than just a form of locomotion and represents a complex adaptation resulting in changes in virulence gene expression and antibiotic resistance. In this study, we used a comprehensive P. aeruginosa PA14 transposon mutant library to investigate how the complex swarming adaptation process is regulated. A total of 233 P. aeruginosa PA14 transposon mutants were verified to have alterations in swarming motility. The swarming-associated genes functioned not only in flagellar or type IV pilus biosynthesis but also in processes as diverse as transport, secretion, and metabolism. Thirty-three swarming-deficient and two hyperswarming mutants had transposon insertions in transcriptional regulator genes, including genes encoding two-component sensors and response regulators; 27 of these insertions were newly identified. Of the 25 regulatory mutants whose swarming motility was highly impaired (79 to 97%), only 1 (a PA1458 mutant) had a major defect in swimming, suggesting that this regulator might influence flagellar synthesis or function. Twitching motility, which requires type IV pili, was strongly affected in only two regulatory mutants (pilH and PA2571 mutants) and was moderately affected in three other mutants (algR, ntrB, and nosR mutants). Microarray analyses were performed to compare the gene expression profile of a swarming-deficient PA3587 mutant to that of the wild-type PA14 strain under swarming conditions. PA3587 showed 63% homology to metR, which encodes a regulator of methionine biosynthesis in Escherichia coli. The observed dysregulation in the metR mutant of nine different genes required for swarming motility provided a possible explanation for the swarming-deficient phenotype of this mutant.

In this study, we analyzed the mechanisms of multiresistance for 22 clinical multiresistant and clonally different Pseudomonas aeruginosa strains from Germany. Twelve and 10 strains originated from cystic fibrosis (CF) and non-CF patients, respectively. Overproduction of the efflux systems MexAB-OprM, MexCD-OprJ, MexEF-OprN, and MexXY-OprM was studied. Furthermore, loss of OprD, alterations in type II topoisomerases, AmpC overproduction, and the presence of 25 acquired resistance determinants were investigated. The presence of a hypermutation phenotype was also taken into account. Besides modifications in GyrA (91%), the most frequent mechanisms of resistance were MexXY-OprM overproduction (82%), OprD loss (82%), and AmpC overproduction (73%). Clear differences between strains from CF and non-CF patients were found: numerous genes coding for aminoglycoside-modifying enzymes and located, partially in combination with beta-lactamase genes, in class 1 integrons were found only in strains from non-CF patients. Furthermore, multiple modifications in type II topoisomerases conferring high quinolone resistance levels and overexpression of MexAB-OprM were exclusively detected in multiresistant strains from non-CF patients. Correlations of the detected phenotypes and resistance mechanisms revealed a great impact of efflux pump overproduction on multiresistance in P. aeruginosa. Confirming previous studies, we found that additional, unknown chromosomally mediated resistance mechanisms remain to be determined. In our study, 11 out of 12 strains and 3 out of 10 strains from CF patients and non-CF patients, respectively, were hypermutable. This extremely high proportion of mutator strains should be taken into consideration for the treatment of multiresistant P. aeruginosa.