Development of carbapenem resistance in Pseudomonas aeruginosa is associated with OprD polymorphisms, particularly the amino acid substitution at codon 170.

Objectives: Pan-susceptible Pseudomonas aeruginosa (PSPA) clinical isolates carrying an OprD with loop 7 shortening (the group-1A allele) were found to rapidly develop carbapenem resistance under continuous selection pressure. We further studied whether OprD polymorphisms are associated with the potential to develop carbapenem resistance. Methods: OprD amino acid sequences of 126 PSPA clinical isolates were analysed to determine their STs using P. aeruginosa strain PAO1 as the control strain. Site-directed mutagenesis was performed in PAO1 to generate polymorphisms of interest. A disc diffusion method was used to select carbapenem-resistant variants from the mutant strains. Expression levels of oprD were determined by quantitative RT-PCR. MICs of carbapenems were determined by Etest. Results: Forty-eight (38.1%) of the tested isolates carried the group-1A allele. Another two major STs, C1 and C2, both of which harboured an F170L polymorphism, were found in 21 (16.7%) and 39 (31.0%) isolates, respectively. The PAO1 type was also found in 14 (11.1%) isolates. Under continuous selective pressure, isolates of most STs developed carbapenem resistance at different numbers of passaging events; only those belonging to the PAO1 type remained susceptible. However, PAO1 mutants carrying either the oprD group-1A allele or the OprD-F170L polymorphism were able to develop carbapenem resistance. Reduced oprD expression triggered by continuous imipenem challenge was found in PAO1 mutants, but not in the PAO1 WT strain. Conclusions: OprD polymorphisms, particularly the F170L substitution and the specific shortening in loop 7, appear to determine the potential for P. aeruginosa to develop carbapenem resistance.

Reduced production of OprM may promote oprD mutations and lead to imipenem resistance in Pseudomonas aeruginosa carrying an oprD-group 1A allele.

Resistance mechanisms in a group of carbapenemase-negative Pseudomonas aeruginosa that were susceptible to all antibiotics except carbapenems (carbapenem resistance-only P. aeruginosa [CROPA]) were studied. Ten genetically nonrelated CROPA isolates and their carbapenem-susceptible counterparts were further investigated. OprD production was demonstrated by protein electrophoresis in only 1 of the 10 carbapenem-susceptible isolates, while the other 9 isolates showed hyperproduction of OprM. DNA sequencing of oprD revealed a shortened loop 7 domain (group 1A allele) in eight carbapenem-susceptible isolates. Various oprD mutations, leading to early terminations, were found in 9 of the 10 CROPA isolates. RNA analysis demonstrated hyperexpression of oprM with normal expression of mexA in eight of the carbapenem-susceptible isolates, while in seven of their CROPA counterparts, the oprM expression was significantly reduced. Deletion of oprM was performed in two pairs of representative isolates. Selection of imipenem resistant variants by a disc assay indicated that the lost-of-function mutations in oprD occurred relatively faster in the ΔoprM mutants compared with their corresponding parent strains. Under selection pressure, reduced production of OprM may promote the selection of spontaneous changes in oprD, resulting in the carbapenem resistance in a group of pan-susceptible P. aeruginosa isolates characterized by harboring an oprD-group 1A allele.

Vancomycin promotes the bacterial autolysis, release of extracellular DNA, and biofilm formation in vancomycin-non-susceptible Staphylococcus aureus.

Staphylococcus aureus, an important human pathogen, is particularly adept at producing biofilms on implanted medical devices. Although antibiotic treatment of nonsusceptible bacteria will not kill these strains, the consequences should be studied. The present study focuses on investigating the effect of vancomycin on biofilm formation by vancomycin-non-susceptible S. aureus. Biofilm adherence assays and scanning electron microscopy demonstrated that biofilm formation was significantly enhanced following vancomycin treatment. Bacterial autolysis of some subpopulations was observed and was confirmed by the live/dead staining and confocal laser scanning microscopy. A significant increase in polysaccharide intercellular adhesin (PIA) production was observed by measuring icaA transcript levels and in a semi-quantitative PIA assay in one resistant strain. We show that the release of extracellular DNA (eDNA) via cidA-mediated autolysis is a major contributor to vancomycin-enhanced biofilm formation. The addition of xenogeneic DNA could also significantly enhance biofilm formation by a PIA-overproducing S. aureus strain. The magnitude of the development of the biofilm depends on a balance between the amounts of eDNA and PIA. In conclusion, sublethal doses of cell wall-active antibiotics like vancomycin induce biofilm formation through an autolysis-dependent mechanism in vancomycin-non-susceptible S. aureus.