A novel human skin chamber model to study wound infection ex vivo

Wound infections with multi-drug resistant bacteria increase morbidity and mortality and have considerable socioeconomic impact. They can lead to impaired wound healing, resulting in rising treatment costs. The aim of this study was to investigate an ex vivo human wound infection model. Human full-thickness skin from the operating room (OR) was placed into the Bo-Drum® and cultivated for 7 days in an air–liquid interphase. On day 8, the skin was inoculated with either (1) Pseudomonas aeruginosa, (2) Staphylococcus aureus (105 CFU, n = 3) or (3) carrier control. 1, 3 and 7 days after inoculation colony forming units in the tissue/media were determined and cytokine expression was quantified. A reliable and reproducible wound infection could be established for 7 days. At this timepoint, 1.8 × 108 CFU/g tissue of P. aeruginosa and 2 × 107 CFU/g tissue of S. aureus were detected. Immunohistochemical analysis demonstrated bacterial infection and epidermolysis in infected skin. RT-PCR analysis exhibited a significant induction of proinflammatory cytokines after infection. The BO-drum® is a robust, easy-to-use, sterilizable and reusable ex vivo full-skin culture system. For investigation of wound infection, treatment and healing, the BO-drum® presents a convenient model and may help to standardize wound research

Non-antibiotic quorum sensing inhibitors acting against N-acyl homoserine lactone synthase as druggable target

YesN-acylhomoserine lactone (AHL)-based quorum sensing (QS) is important for the regulation of proteobacterial virulence determinants. Thus, the inhibition of AHL synthases offers non-antibiotics-based therapeutic potentials against QS-mediated bacterial infections. In this work, functional AHL synthases of Pseudomonas aeruginosa LasI and RhlI were heterologously expressed in an AHL-negative Escherichia coli followed by assessments on their AHLs production using AHL biosensors and high resolution liquid chromatography–mass spectrometry (LCMS). These AHL-producing E. coli served as tools for screening AHL synthase inhibitors. Based on a campaign of screening synthetic molecules and natural products using our approach, three strongest inhibitors namely are salicylic acid, tannic acid and trans-cinnamaldehyde have been identified. LCMS analysis further confirmed tannic acid and trans-cinnemaldehyde efficiently inhibited AHL production by RhlI. We further demonstrated the application of trans-cinnemaldehyde inhibiting Rhl QS system regulated pyocyanin production in P. aeruginosa up to 42.06%. Molecular docking analysis suggested that trans-cinnemaldehyde binds to the LasI and EsaI with known structures mainly interacting with their substrate binding sites. Our data suggested a new class of QS-inhibiting agents from natural products targeting AHL synthase and provided a potential approach for facilitating the discovery of anti-QS signal synthesis as basis of novel anti-infective approach.University of Malaya High Impact Research (HIR) Grant (UM-MOHE HIR Grant UM.C/625/1/HIR/MOHE/CHAN/14/1, no. H-50001-A000027) given to K.G.C. and National Natural Science Foundation of China (no. 81260481) given to H.W

Broad Spectrum Pro-Quorum-Sensing Molecules as Inhibitors of Virulence in Vibrios

Quorum sensing (QS) is a bacterial cell-cell communication process that relies on the production and detection of extracellular signal molecules called autoinducers. QS allows bacteria to perform collective activities. Vibrio cholerae, a pathogen that causes an acute disease, uses QS to repress virulence factor production and biofilm formation. Thus, molecules that activate QS in V. cholerae have the potential to control pathogenicity in this globally important bacterium. Using a whole-cell high-throughput screen, we identified eleven molecules that activate V. cholerae QS: eight molecules are receptor agonists and three molecules are antagonists of LuxO, the central NtrC-type response regulator that controls the global V. cholerae QS cascade. The LuxO inhibitors act by an uncompetitive mechanism by binding to the pre-formed LuxO-ATP complex to inhibit ATP hydrolysis. Genetic analyses suggest that the inhibitors bind in close proximity to the Walker B motif. The inhibitors display broad-spectrum capability in activation of QS in Vibrio species that employ LuxO. To the best of our knowledge, these are the first molecules identified that inhibit the ATPase activity of a NtrC-type response regulator. Our discovery supports the idea that exploiting pro-QS molecules is a promising strategy for the development of novel anti-infectives

Utility of In Vivo Transcription Profiling for Identifying Pseudomonas aeruginosa Genes Needed for Gastrointestinal Colonization and Dissemination

Microarray analysis of Pseudomonas aeruginosa mRNA transcripts expressed in vivo during animal infection has not been previously used to investigate potential virulence factors needed in this setting. We compared mRNA expression in bacterial cells recovered from the gastrointestinal (GI) tracts of P. aeruginosa-colonized mice to that of P. aeruginosa in the drinking water used to colonize the mice. Genes associated with biofilm formation and type III secretion (T3SS) had markedly increased expression in the GI tract. A non-redundant transposon library in P. aeruginosa strain PA14 was used to test mutants in genes identified as having increased transcription during in vivo colonization. All of the Tn-library mutants in biofilm-associated genes had an attenuated ability to form biofilms in vitro, but there were no significant differences in GI colonization and dissemination between these mutants and WT P. aeruginosa PA14. To evaluate T3SS factors, we tested GI colonization and neutropenia-induced dissemination of both deletional (PAO1 and PAK) and insertional (PA14) mutants in four genes in the P. aeruginosa T3SS, exoS or exoU, exoT, and popB. There were no significant differences in GI colonization among these mutant strains and their WT counterparts, whereas rates of survival following dissemination were significantly decreased in mice infected by the T3SS mutant strains. However, there was a variable, strain-dependent effect on overall survival between parental and T3SS mutants. Thus, increased transcription of genes during in vivo murine GI colonization is not predictive of an essential role for the gene product in either colonization or overall survival following induction of neutropenia

Phenotypes of Non-Attached Pseudomonas aeruginosa Aggregates Resemble Surface Attached Biofilm

For a chronic infection to be established, bacteria must be able to cope with hostile conditions such as low iron levels, oxidative stress, and clearance by the host defense, as well as antibiotic treatment. It is generally accepted that biofilm formation facilitates tolerance to these adverse conditions. However, microscopic investigations of samples isolated from sites of chronic infections seem to suggest that some bacteria do not need to be attached to surfaces in order to establish chronic infections. In this study we employed scanning electron microscopy, confocal laser scanning microscopy, RT-PCR as well as traditional culturing techniques to study the properties of Pseudomonas aeruginosa aggregates. We found that non-attached aggregates from stationary-phase cultures have comparable growth rates to surface attached biofilms. The growth rate estimations indicated that, independently of age, both aggregates and flow-cell biofilm had the same slow growth rate as a stationary phase shaking cultures. Internal structures of the aggregates matrix components and their capacity to survive otherwise lethal treatments with antibiotics (referred to as tolerance) and resistance to phagocytes were also found to be strikingly similar to flow-cell biofilms. Our data indicate that the tolerance of both biofilms and non-attached aggregates towards antibiotics is reversible by physical disruption. We provide evidence that the antibiotic tolerance is likely to be dependent on both the physiological states of the aggregates and particular matrix components. Bacterial surface-attachment and subsequent biofilm formation are considered hallmarks of the capacity of microbes to cause persistent infections. We have observed non-attached aggregates in the lungs of cystic fibrosis patients; otitis media; soft tissue fillers and non-healing wounds, and we propose that aggregated cells exhibit enhanced survival in the hostile host environment, compared with non-aggregated bacterial populations

The Pseudomonas aeruginosa Transcriptome in Planktonic Cultures and Static Biofilms Using RNA Sequencing

In this study, we evaluated how gene expression differs in mature Pseudomonas aeruginosa biofilms as opposed to planktonic cells by the use of RNA sequencing technology that gives rise to both quantitative and qualitative information on the transcriptome. Although a large proportion of genes were consistently regulated in both the stationary phase and biofilm cultures as opposed to the late exponential growth phase cultures, the global biofilm gene expression pattern was clearly distinct indicating that biofilms are not just surface attached cells in stationary phase. A large amount of the genes found to be biofilm specific were involved in adaptation to microaerophilic growth conditions, repression of type three secretion and production of extracellular matrix components. Additionally, we found many small RNAs to be differentially regulated most of them similarly in stationary phase cultures and biofilms. A qualitative analysis of the RNA-seq data revealed more than 3000 putative transcriptional start sites (TSS). By the use of rapid amplification of cDNA ends (5′-RACE) we confirmed the presence of three different TSS associated with the pqsABCDE operon, two in the promoter of pqsA and one upstream of the second gene, pqsB. Taken together, this study reports the first transcriptome study on P. aeruginosa that employs RNA sequencing technology and provides insights into the quantitative and qualitative transcriptome including the expression of small RNAs in P. aeruginosa biofilms

The Extracellular Matrix Component Psl Provides Fast-Acting Antibiotic Defense in Pseudomonas aeruginosa Biofilms

Bacteria within biofilms secrete and surround themselves with an extracellular matrix, which serves as a first line of defense against antibiotic attack. Polysaccharides constitute major elements of the biofilm matrix and are implied in surface adhesion and biofilm organization, but their contributions to the resistance properties of biofilms remain largely elusive. Using a combination of static and continuous-flow biofilm experiments we show that Psl, one major polysaccharide in the Pseudomonas aeruginosa biofilm matrix, provides a generic first line of defense toward antibiotics with diverse biochemical properties during the initial stages of biofilm development. Furthermore, we show with mixed-strain experiments that antibiotic-sensitive “non-producing” cells lacking Psl can gain tolerance by integrating into Psl-containing biofilms. However, non-producers dilute the protective capacity of the matrix and hence, excessive incorporation can result in the collapse of resistance of the entire community. Our data also reveal that Psl mediated protection is extendible to E. coli and S. aureus in co-culture biofilms. Together, our study shows that Psl represents a critical first bottleneck to the antibiotic attack of a biofilm community early in biofilm development.National Institutes of Health (U.S.). National Institute of Environmental Health Sciences (Training Grant in Toxicology 5 T32 ES7020-37

Surface Hardness Impairment of Quorum Sensing and Swarming for Pseudomonas aeruginosa

The importance of rhamnolipid to swarming of the bacterium Pseudomonas aeruginosa is well established. It is frequently, but not exclusively, observed that P. aeruginosa swarms in tendril patterns—formation of these tendrils requires rhamnolipid. We were interested to explain the impact of surface changes on P. aeruginosa swarm tendril development. Here we report that P. aeruginosa quorum sensing and rhamnolipid production is impaired when growing on harder semi-solid surfaces. P. aeruginosa wild-type swarms showed huge variation in tendril formation with small deviations to the “standard” swarm agar concentration of 0.5%. These macroscopic differences correlated with microscopic investigation of cells close to the advancing swarm edge using fluorescent gene reporters. Tendril swarms showed significant rhlA-gfp reporter expression right up to the advancing edge of swarming cells while swarms without tendrils (grown on harder agar) showed no rhlA-gfp reporter expression near the advancing edge. This difference in rhamnolipid gene expression can be explained by the necessity of quorum sensing for rhamnolipid production. We provide evidence that harder surfaces seem to limit induction of quorum sensing genes near the advancing swarm edge and these localized effects were sufficient to explain the lack of tendril formation on hard agar. We were unable to artificially stimulate rhamnolipid tendril formation with added acyl-homoserine lactone signals or increasing the carbon nutrients. This suggests that quorum sensing on surfaces is controlled in a manner that is not solely population dependent