Increased mutability of Pseudomonas aeruginosa in biofilms.

OBJECTIVES: Isolates of Pseudomonas aeruginosa from cystic fibrosis (CF) patients are frequently hypermutable due to selection of mutants with defects in DNA repair genes such as mutS. Since P. aeruginosa grows as a biofilm within the infected CF lung, it is possible that this mode of growth enhances the mutability of the organism thereby increasing the opportunity to derive permanent hypermutators through mutation in DNA repair genes. We have now conducted experiments to examine this possibility. METHODS: Using established procedures, we examined the mutability of P. aeruginosa PA01 in planktonic cultures and in biofilm cultures generated by growth in a Sorbarod system. Transcriptional profiling by DNA microarray was used to compare gene expression in planktonic and biofilm cells. RESULTS: Mutation frequency determinations for resistance to rifampicin and ciprofloxacin demonstrated that biofilm cultures of P. aeruginosa displayed up to a 105-fold increase in mutability compared with planktonic cultures. Several genes (ahpC, katA, sodB and PA3529, a probable peroxidase) that encode enzymes conferring protection against oxidative DNA damage were down-regulated in biofilm cells. In particular, katA, which encodes the major pseudomonal antioxidant catalase, was down-regulated 7.7-fold. CONCLUSIONS: Down-regulation of antioxidant enzymes in P. aeruginosa biofilms may enhance the rate of mutagenic events due to the accumulation of DNA damage. Since P. aeruginosa forms biofilms in the CF lung, this mode of growth may enhance the direct selection of antibiotic-resistant organisms in CF patients and also increase the opportunity to derive permanent hypermutators thereby providing a further source of antibiotic-resistant mutants in the CF lung.

A DHA14 drug efflux gene from Xanthomonas albilineans confers high-level albicidin antibiotic resistance in Escherichia coli.

AIMS: Identification of a gene for self-protection from the antibiotic-producing plant pathogen Xanthomonas albilineans, and functional testing by heterologous expression. METHODS AND RESULTS: Albicidin antibiotics and phytotoxins are potent inhibitors of prokaryote DNA replication. A resistance gene (albF) isolated by shotgun cloning from the X. albilineans albicidin-biosynthesis region encodes a protein with typical features of DHA14 drug efflux pumps. Low-level expression of albF in Escherichia coli increased the MIC of albicidin 3000-fold, without affecting tsx-mediated albicidin uptake into the periplasm or resistance to other tested antibiotics. Bioinformatic analysis indicates more similarity to proteins involved in self-protection in polyketide-antibiotic-producing actinomycetes than to multi-drug resistance pumps in other gram-negative bacteria. A complex promoter region may co-regulate albF with genes for hydrolases likely to be involved in albicidin activation or self-protection. CONCLUSIONS: AlbF is the first apparent single-component antibiotic-specific efflux pump from a gram-negative antibiotic producer. It shows extraordinary efficiency as measured by resistance level conferred upon heterologous expression. SIGNIFICANCE AND IMPACT OF THE STUDY: Development of the clinical potential of albicidins as potent bactericidial antibiotics against diverse bacteria has been limited because of low yields in culture. Expression of albF with recently described albicidin-biosynthesis genes may enable large-scale production. Because albicidins are X. albilineans pathogenicity factors, interference with AlbF function is also an opportunity for control of the associated plant disease.

Antimicrobial activity and mechanisms of resistance to cephalosporin P1, an antibiotic related to fusidic acid.

The antimicrobial properties of cephalosporin P1, an antibiotic structurally related to fusidic acid, were examined. Cephalosporin P1 exhibited potent activity against methicillin-sensitive Staphylococcus aureus, methicillin-resistant S. aureus and vancomycin-intermediate S. aureus. Mutants of S. aureus resistant to cephalosporin P1 arose with a frequency of 1.6 x 10(-6) for selections at 4 x MIC, a frequency similar to that for fusidic acid. The mutations conferred cross-resistance to fusidic acid and mapped in fusA, the gene encoding elongation factor G. Cross-resistance between cephalosporin P1 and fusidic acid also occurred for S. aureus fusA mutants selected with fusidic acid, and in fusidic acid-resistant clinical isolates. Plasmid pUB101, which mediates resistance to fusidic acid in S. aureus, also conferred resistance to cephalosporin P1. Escherichia coli was intrinsically resistant to both fusidic acid and cephalosporin P1, but deletion of the AcrAB efflux pump resulted in susceptibility to both antibiotics. Although complete cross-resistance between fusidic acid and cephalosporin P1 was demonstrated, the nature and location of fusA mutations in S. aureus when cephalosporin P1 was the selective agent frequently differed from those selected with fusidic acid. This may reflect differences in the interaction of the two antibiotics with the translational apparatus, which results in the selection of separate mutation classes for each antibiotic. Furthermore, in three of 14 mutants selected with fusidic acid, resistance was attributed to mutations lying outside fusA. In contrast, mutations in 10 mutants selected with cephalosporin P1 were all located in fusA.