Biosensor-guided detection of outer membrane-specific antimicrobial activity against Pseudomonas aeruginosa from fungal cultures and medicinal plant extracts.

IMPORTANCE: New approaches are needed to discover novel antimicrobials, particularly antibiotics that target the Gram-negative outer membrane. By exploiting bacterial sensing and responses to outer membrane (OM) damage, we used a biosensor approach consisting of polymyxin resistance gene transcriptional reporters to screen natural products and a small drug library for biosensor activity that indicates damage to the OM. The diverse antimicrobial compounds that cause induction of the polymyxin resistance genes, which correlates with outer membrane damage, suggest that these LPS and surface modifications also function in short-term repair to sublethal exposure and are required against broad membrane stress conditions.

Pseudomonas aeruginosa displays a dormancy phenotype during long-term survival in water.

Pseudomonas aeruginosa is capable of long-term survival in water, which may serve as a reservoir for infection. Although viable cell counts of PAO1 incubated in water remain stable throughout 8 weeks, LIVE/DEAD staining indicated a high proportion of cells stained with propidium iodide (PI). The proportion of PI-stained cells increased by 4 weeks, then decreased again by 8 weeks, suggesting an adaptive response. This was also evident in an observed shift in cell morphology from a rod to a coccoid shape after 8 weeks. Fluorescence-activated cell sorting (FACS) was used to recover PI-stained cells, which were plated and shown to be viable, indicating that PI-stained cells were membrane-compromised but still cultivable. PAO1 mid-log cells in water were labeled with the dsDNA-binding dye PicoGreen to monitor viability as well as DNA integrity, which demonstrated that the population remains viable and transitions towards increased dsDNA staining. Metabolic activity was found to decrease significantly in water by 4 weeks. The PAO1 outer membrane became less permeable and more resistant to polymyxin B damage in water, and the profile of total membrane lipids changed over time. Among the ~1400 transcriptional lux fusions, gene expression in water revealed that the majority of genes were repressed, but subsets of genes were induced at particular time points. In summary, these results indicate that P. aeruginosa is dormant in water and this adaptation involves a complex pattern of gene regulation and changes to the cell to promote long-term survival and antibiotic tolerance. The approach of P. aeruginosa incubated in water may be useful to study antibiotic tolerance and the mechanisms of dormancy and survival in nutrient limiting conditions.

Exopolysaccharide-Repressing Small Molecules with Antibiofilm and Antivirulence Activity against Pseudomonas aeruginosa.

Biofilm formation is a universal virulence strategy in which bacteria grow in dense microbial communities enmeshed within a polymeric extracellular matrix that protects them from antibiotic exposure and the immune system. Pseudomonas aeruginosa is an archetypal biofilm-forming organism that utilizes a biofilm growth strategy to cause chronic lung infections in cystic fibrosis (CF) patients. The extracellular matrix of P. aeruginosa biofilms is comprised mainly of exopolysaccharides (EPS) and DNA. Both mucoid and nonmucoid isolates of P. aeruginosa produce the Pel and Psl EPS, each of which have important roles in antibiotic resistance, biofilm formation, and immune evasion. Given the central importance of the EPS for biofilms, they are attractive targets for novel anti-infective compounds. In this study, we used a high-throughput gene expression screen to identify compounds that repress expression of the pel genes. The pel repressors demonstrated antibiofilm activity against microplate and flow chamber biofilms formed by wild-type and hyperbiofilm-forming strains. To determine the potential role of EPS in virulence, pel/psl mutants were shown to have reduced virulence in feeding behavior and slow killing virulence assays in Caenorhabditis elegans The antibiofilm molecules also reduced P. aeruginosa PAO1 virulence in the nematode slow killing model. Importantly, the combination of antibiotics and antibiofilm compounds increased killing of P. aeruginosa biofilms. These small molecules represent a novel anti-infective strategy for the possible treatment of chronic P. aeruginosa infections.

D-enantiomeric peptides that eradicate wild-type and multidrug-resistant biofilms and protect against lethal Pseudomonas aeruginosa infections.

In many infections, bacteria form surface-associated communities known as biofilms that are substantially more resistant to antibiotics than their planktonic counterparts. Based on the design features of active antibiofilm peptides, we made a series of related 12-amino acid L-, D- and retro-inverso derivatives. Specific D-enantiomeric peptides were the most potent at inhibiting biofilm development and eradicating preformed biofilms of seven species of wild-type and multiply antibiotic-resistant Gram-negative pathogens. Moreover, these peptides showed strong synergy with conventional antibiotics, reducing the antibiotic concentrations required for complete biofilm inhibition by up to 64-fold. As shown previously for 1018, these D-amino acid peptides targeted the intracellular stringent response signal (p)ppGpp. The most potent peptides DJK-5 and DJK-6 protected invertebrates from lethal Pseudomonas aeruginosa infections and were considerably more active than a previously described L-amino acid peptide 1018. Thus, the protease-resistant peptides produced here were more effective both in vitro and in vivo.

Current Research Approaches to Target Biofilm Infections.

This review will focus on strategies to develop new treatments that target the biofilm mode of growth and that can be used to treat biofilm infections. These approaches aim to reduce or inhibit biofilm formation, or to increase biofilm dispersion. Many antibiofilm compounds are not bactericidal but render the cells in a planktonic growth state, which are more susceptible to antibiotics and more easily cleared by the immune system. Novel compounds are being developed with antibiofilm activity that includes antimicrobial peptides, natural products, small molecules and polymers. Bacteriophages are being considered for use in treating biofilms, as well as the use of enzymes that degrade the extracellular matrix polymers to dissolve biofilms. There is great potential in these new approaches for use in treating chronic biofilm infections.

Anti-Biofilm and Immunomodulatory Activities of Peptides That Inhibit Biofilms Formed by Pathogens Isolated from Cystic Fibrosis Patients.

Cystic fibrosis (CF) patients often acquire chronic respiratory tract infections due to Pseudomonas aeruginosa and Burkholderia cepacia complex (Bcc) species. In the CF lung, these bacteria grow as multicellular aggregates termed biofilms. Biofilms demonstrate increased (adaptive) resistance to conventional antibiotics, and there are currently no available biofilm-specific therapies. Using plastic adherent, hydroxyapatite and flow cell biofilm models coupled with confocal and scanning electron microscopy, it was demonstrated that an anti-biofilm peptide 1018 prevented biofilm formation, eradicated mature biofilms and killed biofilms formed by a wide range of P. aeruginosa and B. cenocepacia clinical isolates. New peptide derivatives were designed that, compared to their parent peptide 1018, showed similar or decreased anti-biofilm activity against P. aeruginosa biofilms, but increased activity against biofilms formed by the Gram-positive bacterium methicillin resistant Staphylococcus aureus. In addition, some of these new peptide derivatives retained the immunomodulatory activity of 1018 since they induced the production of the chemokine monocyte chemotactic protein-1 (MCP-1) and suppressed lipopolysaccharide-mediated tumor necrosis factor-α (TNF-α) production by human peripheral blood mononuclear cells (PBMC) and were non-toxic towards these cells. Peptide 1018 and its derivatives provide promising leads for the treatment of chronic biofilm infections and hyperinflammatory lung disease in CF patients.

Characterization of the type III capsular polysaccharide produced by Burkholderia pseudomallei.

Burkholderia pseudomallei has been shown to produce more than one capsular polysaccharide (CPS). Analysis of the B. pseudomallei genome has revealed that the organism contains four CPS operons (I-IV). One of these operons (CPS III) was selected for further study. Comparative sequencing analysis revealed that the genes encoding CPS III are present in B. pseudomallei and Burkholderia thailandensis but not in Burkholderia mallei. In this study, CPS III was not found to contribute to the virulence of B. pseudomallei. Strains containing mutations in CPS III had the same LD(50) value as the wild-type when tested in an animal infection model. Production of CPS III was shown to be induced in water but inhibited in 30% normal human serum using a lux reporter fusion assay. Microarray analysis of capsule gene expression in infected hamsters revealed that the genes encoding CPS III were not significantly expressed in vivo compared with the genes encoding the previously characterized mannoheptose capsule (CPS I), which is an important virulence factor in B. pseudomallei. Glycosyl-composition analysis by combined GC/MS indicated that the CPS III genes are involved in the synthesis of a capsule composed of galactose, glucose, mannose and xylose.

Genetics and function of the capsules of Burkholderia pseudomallei and their potential as therapeutic targets.

Burkholderia pseudomallei is the causative agent of melioidosis, a fatal disease that is endemic to Southeast Asia and northern Australia. The clinical manifestations of melioidosis may range from an acute pneumonia or acute septicemia, to chronic and latent infections. B. pseudomallei is inherently resistant to a number of antibiotics, and even with aggressive antibiotic therapy, the mortality rate remains high, and the incidence of relapse is common. The resistance of this organism to a number of antibiotics has created a need for the development of other therapeutic strategies, including the identification of novel therapeutic targets. B. pseudomallei has been shown to produce a number of capsular polysaccharides, one of which has been shown to contribute to the virulence of the organism. The structures of these polysaccharides have been determined and the genes encoding for the biosynthesis of one of the capsular polysaccharides (CPS I) have been identified. Analysis of the genome sequence of this organism has revealed the presence of three other capsule gene clusters that may encode for the chemical structures previously identified. Since one of the capsules produced by B. pseudomallei has been shown to be important in virulence, the genes encoding for the proteins responsible for its biosynthesis may be considered as potential targets.

The capsular polysaccharide of Burkholderia pseudomallei contributes to survival in serum by reducing complement factor C3b deposition.

Burkholderia pseudomallei produces an extracellular polysaccharide capsule -3)-2-O-acetyl-6-deoxy-beta-D-manno-heptopyranose-(1- which has been shown to be an essential virulence determinant. The addition of purified capsule was shown to increase the virulence of a capsule mutant strain in the Syrian hamster model of acute melioidosis. An increase in the number of wild-type B. pseudomallei cells in the blood was seen by 48 h, while the number of capsule mutant cells in the blood declined by 48 h. Capsule expression was shown to be induced in the presence of serum using a lux reporter fusion to the capsule gene wcbB. The addition of purified B. pseudomallei capsule to serum bactericidal assays increased the survival of B. pseudomallei SLR5, a serum-sensitive strain, by 1,000-fold in normal human serum. Capsule production by B. pseudomallei contributed to reduced activation of the complement cascade by reducing the levels of complement factor C3b deposition. An increase in phagocytosis of the capsule mutant compared to the wild type was observed in the presence of normal human serum. These results suggest that the production of this capsule contributes to resistance to phagocytosis by reducing C3b deposition on the surface of the bacterium, thereby contributing to the persistence of bacteria in the blood of the infected host. Continued studies to characterize this capsule are essential for understanding the pathogenesis of B. pseudomallei infections and the development of preventive strategies for treatment of this disease.

Contribution of gene loss to the pathogenic evolution of Burkholderia pseudomallei and Burkholderia mallei.

Burkholderia pseudomallei is the causative agent of melioidosis. Burkholderia thailandensis is a closely related species that can readily utilize l-arabinose as a sole carbon source, whereas B. pseudomallei cannot. We used Tn5-OT182 mutagenesis to isolate an arabinose-negative mutant of B. thailandensis. Sequence analysis of regions flanking the transposon insertion revealed the presence of an arabinose assimilation operon consisting of nine genes. Analysis of the B. pseudomallei chromosome showed a deletion of the operon from this organism. This deletion was detected in all B. pseudomallei and Burkholderia mallei strains investigated. We cloned the B. thailandensis E264 arabinose assimilation operon and introduced the entire operon into the chromosome of B. pseudomallei 406e via homologous recombination. The resultant strain, B. pseudomallei SZ5028, was able to utilize l-arabinose as a sole carbon source. Strain SZ5028 had a significantly higher 50% lethal dose for Syrian hamsters compared to the parent strain 406e. Microarray analysis revealed that a number of genes in a type III secretion system were down-regulated in strain SZ5028 when cells were grown in l-arabinose, suggesting a regulatory role for l-arabinose or a metabolite of l-arabinose. These results suggest that the ability to metabolize l-arabinose reduces the virulence of B. pseudomallei and that the genes encoding arabinose assimilation may be considered antivirulence genes. The increase in virulence associated with the loss of these genes may have provided a selective advantage for B. pseudomallei as these organisms adapted to survival in animal hosts.