Biofilm formation - what we can learn from recent developments.

Although biofilms have been observed early in the history of microbial research, their impact has only recently been fully recognized. Biofilm infections, which contribute to up to 80% of human microbial infections, are associated with common human disorders, such as diabetes mellitus and poor dental hygiene, but also with medical implants. The associated chronic infections such as wound infections, dental caries and periodontitis significantly enhance morbidity, affect quality of life and can aid development of follow-up diseases such as cancer. Biofilm infections remain challenging to treat and antibiotic monotherapy is often insufficient, although some rediscovered traditional compounds have shown surprising efficiency. Innovative anti-biofilm strategies include application of anti-biofilm small molecules, intrinsic or external stimulation of production of reactive molecules, utilization of materials with antimicrobial properties and dispersion of biofilms by digestion of the extracellular matrix, also in combination with physical biofilm breakdown. Although basic principles of biofilm formation have been deciphered, the molecular understanding of the formation and structural organization of various types of biofilms has just begun to emerge. Basic studies of biofilm physiology have also resulted in an unexpected discovery of cyclic dinucleotide second messengers that are involved in interkingdom crosstalk via specific mammalian receptors. These findings even open up new venues for exploring novel anti-biofilm strategies.

Microbiota is a primary cause of pathogenesis of chronic wounds.

OBJECTIVE: Diverse microorganisms present on the surface of chronic wounds have been established to constitute wound microbiota. The aims of this study were to quantify the viability of wound microbiota, classify dispersal of viable microbes from the wound, and determine if human wound microbiota can produce a chronic wound in an animal model. METHOD: Wound microbiotas as units (multiple microbial species acting as one infectious agent) were obtained from well-defined human chronic wounds and seeded onto mouse surgical excision wounds to produce chronically infected wounds that closely resembled the chronic wounds observed in the original hosts. RESULTS: We found the wound microbiota harvested from 35 out of 43 (81%) patients could produce similar chronic wounds (producing slough and exudate) in a murine chronic wound model. The top 30 species present in patient wounds were identified in the mouse wounds by molecular sequencing. Koch's postulates could therefore be applied to establish wound microbiota as the cause of the original human chronic wound infections. Evidence-based medicine criteria such as Hill's criteria for causation can all be satisfied by what is currently known about wound microbiota. CONCLUSION: This study demonstrates that wound microbiota actively disseminates from the chronic wound by forces and mechanisms intrinsic to the wound. Koch's postulates and Hill's criteria for causation together suggest chronic wound microbiota to be the main cause underlying the pathogenesis of chronic wounds. DECLARATION OF INTEREST: RW has an equity interest in PathoGenius Labs. No funding was received for this study.

Host Responses to Biofilm.

From birth to death the human host immune system interacts with bacterial cells. Biofilms are communities of microbes embedded in matrices composed of extracellular polymeric substance (EPS), and have been implicated in both the healthy microbiome and disease states. The immune system recognizes many different bacterial patterns, molecules, and antigens, but these components can be camouflaged in the biofilm mode of growth. Instead, immune cells come into contact with components of the EPS matrix, a diverse, hydrated mixture of extracellular DNA (bacterial and host), proteins, polysaccharides, and lipids. As bacterial cells transition from planktonic to biofilm-associated they produce small molecules, which can increase inflammation, induce cell death, and even cause necrosis. To survive, invading bacteria must overcome the epithelial barrier, host microbiome, complement, and a variety of leukocytes. If bacteria can evade these initial cell populations they have an increased chance at surviving and causing ongoing disease in the host. Planktonic cells are readily cleared, but biofilms reduce the effectiveness of both polymorphonuclear neutrophils and macrophages. In addition, in the presence of these cells, biofilm formation is actively enhanced, and components of host immune cells are assimilated into the EPS matrix. While pathogenic biofilms contribute to states of chronic inflammation, probiotic Lactobacillus biofilms cause a negligible immune response and, in states of inflammation, exhibit robust antiinflammatory properties. These probiotic biofilms colonize and protect the gut and vagina, and have been implicated in improved healing of damaged skin. Overall, biofilms stimulate a unique immune response that we are only beginning to understand.

Biofilm maturity studies indicate sharp debridement opens a time- dependent therapeutic window.

OBJECTIVE: To investigate the hypothesis that newly formed wound biofilms (or bioburdens) are more susceptible to antimicrobial treatment. METHOD: Four separate and distinct models were performed by four separate biofilm research laboratories to evaluate the resistance of biofilms to antimicrobial treatments over time. These included a drip-flow biofilm model along with a hydrodebridement study, a porcine skin punch biopsy ex vivo model, a mouse chronic wound model and clinical longitudinal debridement study. RESULTS: All four models showed that, within the first 24 hours, the biofilm community was more susceptible to the selected antibiotics, and after maturing for up to 48 hours became increasingly tolerant. In each model, there was at least a 24-hour period in which the biofilms were more resistant to antibiotics. Each of the models utilised showed a significant decrease in the resistance of the biofilm/ burden to gentamicin for up to 24 hours with a confidence interval of at least 95%. The resistance increased in each of the models by 48 hours and reached original resistance levels by 72 hours. CONCLUSION: These data suggest the principles of biofilm-based wound care, along with the use of serial debridement to continually remove mature biofilm, followed by biofilm wound management strategies, including topical antibiotics while the bioburden is still immature and more susceptible, are valid.

Pseudomonas aeruginosa autoinducer modulates host cell responses through calcium signalling.

The opportunistic pathogen Pseudomonas aeruginosa utilizes a cell density-dependent signalling phenomenon known as quorum sensing (QS) to regulate several virulence factors needed for infection. Acylated homoserine lactones, or autoinducers, are the primary signal molecules that mediate QS in P. aeruginosa. The autoinducer N-3O-dodecanoyl-homoserine lactone (3O-C12) exerts effects on mammalian cells, including upregulation of pro-inflammatory mediators and induction of apoptosis. However, the mechanism(s) by which 3O-C12 affects mammalian cell responses is unknown. Here we report that 3O-C12 induces apoptosis and modulates the expression of immune mediators in murine fibroblasts and human vascular endothelial cells (HUVEC). The effects of 3O-C12 were accompanied by increases in cytosolic calcium levels that were mobilized from intracellular stores in the endoplasmic reticulum (ER). Calcium release was blocked by an inhibitor of phospholipase C, suggesting that release occurred through inositol triphosphate (IP3) receptors in the ER. Apoptosis, but not immunodulatory gene activation, was blocked when 3O-C12-exposed cells were co-incubated with inhibitors of calcium signalling. This study indicates that 3O-C12 can activate at least two independent signal transduction pathways in mammalian cells, one that involves increases in intracellular calcium levels and leads to apoptosis, and a second pathway that results in modulation of the inflammatory response.

The effects of infection of thermal injury by Pseudomonas aeruginosa PAO1 on the murine cytokine response.

Pseudomonas aeruginosa infection, one of the major complications of burn wounds, may lead to sepsis and death. Using the Multi-Probe Template/RNase protection assay, we have compared the expression of different cytokine genes within the skin and livers of thermally injured mice infected with P. aeruginosa PAO1. Thermal injury alone enhanced or up-regulated certain cytokines, including macrophage colony-stimulating factor (M-CSF), interleukin 1 (IL-1)RI, IL-1 beta, macrophage inflammatory protein (MIP)-1 beta and MIP-2; while PAO1 challenge alone up-regulated tumour necrosis factor alpha (TNF-alpha) and transforming growth factor beta (TGF-beta) expression. The combination of thermal injury plus PAO1 infection enhanced the expression of several pro-inflammatory and haematopoietic cytokines [stem cell factor (SCF), leukocyte inhibitory factor (LIF), IL-6 and TNF-alpha]; induced the expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) and G-CSF by 5 h and the expression of additional cytokines, including TGF-beta, TNF-beta, lymphotoxin beta (LT-beta), interferon gamma (IFN-gamma), and IFN-beta by 40 h post-burn/infection. While the most intense cytokine expression occurred in the skin, the majority of cytokines tested were also expressed in the liver by 40 h post-burn/infection. These results suggest that in P. aeruginosa infection of burn wounds: (1) up-regulation of the expression of different cytokines, locally and within the livers of burned mice, is an indication of P. aeruginosa -induced sepsis; and (2) IL-6 and G-CSF play an important role in the host response mechanism.

The role of quorum sensing in the in vivo virulence of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen that causes a wide variety of infections. The cell-density-dependent signaling mechanisms known as quorum sensing play a role in several of these infections including corneal, lung and burn wound infections. In addition, the quorum-sensing systems contribute to the ability of P. aeruginosa to form biofilms on medically important devices. The quorum-sensing systems accomplish their effect by controlling the production of different virulence factors and by manipulating the host immune response.

Pseudomonas aeruginosa strains obtained from patients with tracheal, urinary tract and wound infection: variations in virulence factors and virulence genes.

Pseudomonas aeruginosa produces several virulence factors including exotoxin A, exoenzyme S and elastase. In previous reports we have analysed several clinical isolates for the production of these three virulence factors and for possible heterogeneity within the genes that code for these factors (toxA, lasB and the exoS genes). The isolates were obtained from three specific sites (trachea, urinary tract and wounds). Although the isolates produced variable levels of these factors, isolates that were obtained specifically from urinary tract and wound infections produced increased levels of exotoxin A and exoenzyme S. In addition, a prolonged infection with P. aeruginosa appears to enhance exoenzyme S production. Restriction site polymorphism was very limited within the toxA, lasB, and exoS structural genes; however, the upstream region of toxA showed restriction site polymorphisms between the different isolates. The observed polymorphisms did not correlate with any variations in the levels of the virulence factors. In this article, we provide a short review of these studies.

Contribution of quorum sensing to the virulence of Pseudomonas aeruginosa in burn wound infections.

The Pseudomonas aeruginosa quorum-sensing systems, las and rhl, control the production of numerous virulence factors. In this study, we have used the burned-mouse model to examine the contribution of quorum-sensing systems to the pathogenesis of P. aeruginosa infections in burn wounds. Different quorum-sensing mutants of P. aeruginosa PAO1 that were defective in the lasR, lasI, or rhlI gene or both the lasI and rhlI genes were utilized. The following parameters of the P. aeruginosa infection were examined: (i) lethality to the burned mouse, (ii) dissemination of the P. aeruginosa strain within the body of the infected mouse (by determining the numbers of CFU of P. aeruginosa within the liver and spleen), and (iii) spread of the P. aeruginosa strain within the burned skin (by determining the numbers of CFU of P. aeruginosa at the inoculation site and at a site about 15 mm from the inoculation site [distant site]). In comparison with that of PAO1, the in vivo virulence of lasI, lasR, and rhlI mutants was significantly reduced. However, the most significant reduction in in vivo virulence was seen with the lasI rhlI mutant. The numbers of CFU that were recovered from the livers, spleens, and skin of mice infected with different mutants were significantly lower than those of PAO1. At 8 and 16 h post burn infection, comparable numbers of CFU of PAO1 and lasI and rhlI mutants were obtained from both the inoculation and distant sites of the burned skin of infected mice. In contrast, CFU of the lasR mutant and the lasI rhlI double mutant were recovered only from the inoculation site of infected mice at 8 and 16 h post burn infection. The ability of a plasmid carrying either the lasI or rhlI gene or the lasI and rhlI genes to complement the defect of the lasI rhlI double mutant was also examined. The presence of any of these plasmids within the lasI rhlI double mutant significantly enhanced its in vivo virulence, as well as its ability to spread within the burned skin. These results suggest that the quorum-sensing systems play an important role in the horizontal spread of P. aeruginosa within burned skin and in the dissemination of P. aeruginosa within the bodies of burned-and-infected mice and contributed to the overall virulence of P. aeruginosa in this animal model.

Analysis of Pseudomonas aeruginosa clinical isolates for possible variations within the virulence genes exotoxin A and exoenzyme S.

We have previously characterized several Pseudomonas aeruginosa isolates that were obtained from patients with tracheal, urinary tract, or wound infections (A. H. Hamood, J. A. Griswold, and C. M. Duhan, 1996, J. Surg. Res. 61: 425). Analysis of additional isolates showed that regardless of the isolation site, some isolates produced significantly higher or significantly lower levels of either exotoxin A or exoenzyme S proteins. These variations did not correlate with the mucoid phenotype of the isolates. One aim of this study was to determine if the variations in the level of exotoxin A or exoenzyme S are due to DNA rearrangements within either the toxA or the exoS gene. This was accomplished by Southern blot hybridization experiments using a toxA internal probe, a toxA upstream probe, or an exoS internal probe. Hybridization with the toxA internal probe produced a 0.8-kb hybridizing fragment, whereas hybridization with the exoS internal probe produced either a 2.0- or a 2.3-kb hybridizing fragment. Hybridization with the toxA upstream probe, however, produced hybridizing fragments of varying sizes, regardless of their isolation site. Isolates that showed a similar hybridization fragment with either the toxA upstream probe or the exoS internal probe produced variable levels of exotoxin A or exoenzyme S. These results suggest that: [1] specific location within the host has no effect on either the mucoid phenotype of the isolate or the level of exotoxin A or exoenzyme S produced by the isolates; [2] although restriction polymorphism exists within the toxA upstream region, both the toxA and the exoS structural genes are relatively conserved; and [3] variations in the level of exoenzyme S and exotoxin A produced by different isolates do not correlate with either the observed heterogeneity within the toxA upstream region or the mucoid phenotype of the isolates.

Contribution of the regulatory gene lasR to the pathogenesis of Pseudomonas aeruginosa infection of burned mice.

Pseudomonas aeruginosa is a gram-negative opportunistic pathogen that causes severe infections in patients with burns. The P aeruginosa regulatory gene, lasR, regulates the expression of several virulence factors. The specific lasR isogenic mutant, PAO-R1, is defective in the synthesis of the 2 elastases (LasB and LasA) and produces low levels of exotoxin A and alkaline proteases. In this study, we used a burned mouse model to examine the role of lasR in the pathogenesis of P aeruginosa infections. We have examined the following aspects of P aeruginosa infections: 1) lethality to the burned mouse, 2) the dissemination within the body of the burned mouse, and 3) the local spread within the burned skin. In comparison with its parent strain, PAO1, PAO-R1 was less lethal. In addition, the numbers of PAO-R1 microorganisms recovered from the livers and spleens of the burned mice were less than those of PAO1. Furthermore, at 8 hours postinfection, equivalent numbers of PAO1 and PAO-R1 were detected at the inoculation site of the burned skin. However, only PAO1 microorganisms were detected at other sites of the burned skin. These results suggest that the lasR gene contributes (directly and indirectly) to the dissemination of P aeruginosa within the body of burned mice and its horizontal spread within the burned skin.