Insulin treatment modulates the host immune system to enhance Pseudomonas aeruginosa wound biofilms.

Diabetes affects 25.8 million people in the United States, or 8.3% of the population, and these numbers are even higher in developing countries. Diabetic patients are more susceptible to the development of chronic wounds with debilitating bacterial infections than nondiabetics. Previously, we compared the ability of the opportunistic pathogen Pseudomonas aeruginosa to cause biofilm-associated infections in chronic wounds of diabetic and nondiabetic mice (C. Watters, K. DeLeon, U. Trivedi, J. A. Griswold, M. Lyte, K. J. Hampel, M. J. Wargo, and K. P. Rumbaugh, Med. Microbiol. Immunol. 202:131-141, 2013). Unexpectedly, we observed that insulin-treated diabetic mice had significantly more biofilm in their wounds, which correlated with higher antibiotic tolerance. Here, we investigated whether insulin treatment modulates the diabetic immune system to favor P. aeruginosa biofilm formation. Utilizing a murine chronic wound model, we found that DNA protected P. aeruginosa in the wounds of insulin-treated diabetic mice from antibiotic treatment. We also observed increased numbers of neutrophils, reduced numbers of macrophages, and increased cell death in the wounds of diabetic mice on insulin therapy. Taken together, these data suggest that high levels of lysed neutrophils in the wounds of diabetic mice on insulin, combined with fewer macrophages to remove the cellular debris, contribute to increased DNA levels, which enhance P. aeruginosa biofilms.

A novel virulence strategy for Pseudomonas aeruginosa mediated by an autotransporter with arginine-specific aminopeptidase activity.

The opportunistic human pathogen, Pseudomonas aeruginosa, is a major cause of infections in chronic wounds, burns and the lungs of cystic fibrosis patients. The P. aeruginosa genome encodes at least three proteins exhibiting the characteristic three domain structure of autotransporters, but much remains to be understood about the functions of these three proteins and their role in pathogenicity. Autotransporters are the largest family of secreted proteins in Gram-negative bacteria, and those characterised are virulence factors. Here, we demonstrate that the PA0328 autotransporter is a cell-surface tethered, arginine-specific aminopeptidase, and have defined its active site by site directed mutagenesis. Hence, we have assigned PA0328 with the name AaaA, for arginine-specific autotransporter of P. aeruginosa. We show that AaaA provides a fitness advantage in environments where the sole source of nitrogen is peptides with an aminoterminal arginine, and that this could be important for establishing an infection, as the lack of AaaA led to attenuation in a mouse chronic wound infection which correlated with lower levels of the cytokines TNFα, IL-1α, KC and COX-2. Consequently AaaA is an important virulence factor playing a significant role in the successful establishment of P. aeruginosa infections.

Pseudomonas aeruginosa biofilms perturb wound resolution and antibiotic tolerance in diabetic mice.

Diabetic patients are more susceptible to the development of chronic wounds than non-diabetics. The impaired healing properties of these wounds, which often develop debilitating bacterial infections, significantly increase the rate of lower extremity amputation in diabetic patients. We hypothesize that bacterial biofilms, or sessile communities of bacteria that reside in a complex matrix of exopolymeric material, contribute to the severity of diabetic wounds. To test this hypothesis, we developed an in vivo chronic wound, diabetic mouse model to determine the ability of the opportunistic pathogen, Pseudomonas aeruginosa, to cause biofilm-associated infections. Utilizing this model, we observed that diabetic mice with P. aeruginosa-infected chronic wounds displayed impaired bacterial clearing and wound closure in comparison with their non-diabetic littermates. While treating diabetic mice with insulin improved their overall health, it did not restore their ability to resolve P. aeruginosa wound infections or speed healing. In fact, the prevalence of biofilms and the tolerance of P. aeruginosa to gentamicin treatment increased when diabetic mice were treated with insulin. Insulin treatment was observed to directly affect the ability of P. aeruginosa to form biofilms in vitro. These data demonstrate that the chronically wounded diabetic mouse appears to be a useful model to study wound healing and biofilm infection dynamics, and suggest that the diabetic wound environment may promote the formation of biofilms. Further, this model provides for the elucidation of mechanistic factors, such as the ability of insulin to influence antimicrobial effectiveness, which may be relevant to the formation of biofilms in diabetic wounds.

Skin Swabbing for Staphylococcus aureus-Targeting Phages.

INTRODUCTION: Staphylococcus aureus (SA) is a major human bacterial pathogen increasingly refractory to antibiotics. Given the dearth of novel antibiotics in the developmental pipeline, we require concerted efforts at optimizing novel antimicrobial approaches. One promising option is the utilization of bacteriophage (phage) therapy, which has been resurrected as a viable clinical therapeutic. Specifically, an expanded library of phages targeting SA is desired. We surmised that SA-targeting phages would be readily accessible as a major component of the cutaneous microbiome. Specifically, we sought to discern if easily accessible (convenient) and discrete anatomic locations, including the nares, axilla, fingernails, toenails, and web spaces, could provide intact phages via a noninvasive, expedient procedure involving swabbing. METHODS: One hundred subjects participated in systematic skin swab specimen collections. Pooled samples were subject to phage harvesting utilizing the soft agar overlay technique. The approval was secured from the Naval Medical Research Center Institutional Review Board (NMRC 2018.0004 FWA00000152). We utilized the same procedures from known samples containing SA-targeting phages. As another positive control, we employed the same swab and acquired samples from an active wound infection. RESULTS: As anticipated, there were no adverse events, and the procedure was successfully implemented within the projected 10-minute duration. No phages were identified exploiting this methodology. Positive controls from various environmental samples identified SA-targeting phages as did the wound effluent sample. CONCLUSIONS: Skin swabbing at multiple anatomic sites from 100 adults yielded insufficient biomass for phage recovery. The negative results provide helpful information for future phage isolation attempts. The lessons learned on why this study failed to isolate phages can be easily utilized by others. With a desire to increase our SA-targeting phage library in pursuit of future clinical trials, and acknowledging the paucity of these phages accessible via traditional recovery from environmental sources, we will next acquire large volumes of wound effluent from confirmed infected wounds with SA to optimize the biomass for phage recovery.

Kin selection, quorum sensing and virulence in pathogenic bacteria.

Bacterial growth and virulence often depends upon the cooperative release of extracellular factors excreted in response to quorum sensing (QS). We carried out an in vivo selection experiment in mice to examine how QS evolves in response to variation in relatedness (strain diversity), and the consequences for virulence. We started our experiment with two bacterial strains: a wild-type that both produces and responds to QS signal molecules, and a lasR (signal-blind) mutant that does not release extracellular factors in response to signal. We found that: (i) QS leads to greater growth within hosts; (ii) high relatedness favours the QS wild-type; and (iii) low relatedness favours the lasR mutant. Relatedness matters in our experiment because, at relatively low relatedness, the lasR mutant is able to exploit the extracellular factors produced by the cells that respond to QS, and hence increase in frequency. Furthermore, our results suggest that because a higher relatedness favours cooperative QS, and hence leads to higher growth, this will also lead to a higher virulence, giving a relationship between relatedness and virulence that is in the opposite direction to that usually predicted by virulence theory.

Evaluation of Pseudomonas aeruginosa pathogenesis and therapeutics in military-relevant animal infection models.

Modern combat-related injuries are often associated with acute polytrauma. As a consequence of severe combat-related injuries, a dysregulated immune response results in serious infectious complications. The gram-negative bacterium Pseudomonas aeruginosa is an opportunistic pathogen that often causes life-threatening bloodstream, lung, bone, urinary tract, and wound infections following combat-related injuries. The rise in the number of multidrug-resistant P. aeruginosa strains has elevated its importance to civilian clinicians and military medicine. Development of novel therapeutics and treatment options for P. aeruginosa infections is urgently needed. During the process of drug discovery and therapeutic testing, in vivo testing in animal models is a critical step in the bench-to-bedside approach, and required for Food and Drug Administration approval. Here, we review current and past literature with a focus on combat injury-relevant animal models often used to understand infection development, the interplay between P. aeruginosa and the host, and evaluation of novel treatments. Specifically, this review focuses on the following animal infection models: wound, burn, bone, lung, urinary tract, foreign body, and sepsis.

Topical Delivery of Lactobacillus Culture Supernatant Increases Survival and Wound Resolution in Traumatic Acinetobacter baumannii Infections.

Species of Lactobacillus have been proposed as potential candidates for treating wound infections due to their ability to lower pH, decrease inflammation, and release antimicrobial compounds. This study investigated the impact of lactobacilli (Lactobacillus acidophilus ATCC 4356, Lactobacillus casei ATCC 393, Lactobacillus reuteri ATCC 23272) secreted products on wound pathogens in vitro and in a murine wound infection model. Evaluation of 1-5 day lactobacilli conditioned media (CM) revealed maximal inhibition against wound pathogens using the 5-day CM. The minimum inhibitory concentration (MIC) of 5-day Lactobacillus CMs was tested by diluting CM in Mueller-Hinton (MH) broth from 0 to 25% and was found to be 12.5% for A. baumannii. Concentrating the CM to 10× with a 3 kDa centrifuge filter decreased the CM MIC to 6.25-12.5% for A. baumannii planktonic cells. Minimal impact of 5-day CMs was observed against bacterial biofilms. No toxicity was observed when these Lactobacillus CMs were injected into Galleria melonella waxworms. For the murine A. baumannii wound infection studies, improved survival was observed following topical treatment with L. acidophilus ATCC 4356 or L. reuteri ATCC 23272, while L. reuteri ATCC 23272 treatment alone improved wound resolution. Overall, this study suggests that the topical application of certain Lactobacillus species byproducts could be effective against gram-negative multi-drug resistant (MDR) wound pathogens, such as A. baumannii.

Gallium maltolate treatment eradicates Pseudomonas aeruginosa infection in thermally injured mice.

Gallium (Ga) is a semimetallic element that has demonstrated therapeutic and diagnostic-imaging potential in a number of disease settings, including cancer and infectious diseases. Gallium's biological actions stem from its ionic radius being almost the same as that of ferric iron (Fe(3+)), whereby it can replace iron (Fe) in Fe(3+)-dependent biological systems, such as bacterial and mammalian Fe transporters and Fe(3+)-containing enzymes. Unlike Fe(3+), ionic gallium (Ga(3+)) cannot be reduced, and when incorporated, it inactivates Fe(3+)-dependent reduction and oxidation processes that are necessary for bacterial and mammalian cell proliferation. Most pathogenic bacteria require Fe for growth and function, and the availability of Fe in the host or environment can greatly enhance virulence. We examined whether gallium maltolate (GaM), a novel formulation of Ga, had antibacterial activity in a thermally injured acute infection mouse model. Dose-response studies indicated that a GaM dose as low as 25 mg/kg of body weight delivered subcutaneously was sufficient to provide 100% survival in a lethal P. aeruginosa-infected thermally injured mouse model. Mice treated with 100 mg/kg GaM had undetectable levels of Pseudomonas aeruginosa in their wounds, livers, and spleens, while the wounds of untreated mice were colonized with over 10(8) P. aeruginosa CFU/g of tissue and their livers and spleens were colonized with over 10(5) P. aeruginosa CFU/g of tissue. GaM also significantly reduced the colonization of Staphylococcus aureus and Acinetobacter baumannii in the wounds of thermally injured mice. Furthermore, GaM was also therapeutically effective in preventing preestablished P. aeruginosa infections at the site of the injury from spreading systemically. Taken together, our data suggest that GaM is potentially a novel antibacterial agent for the prevention and treatment of wound infections following thermal injury.

Application of Lactobacillus gasseri 63 AM supernatant to Pseudomonas aeruginosa-infected wounds prevents sepsis in murine models of thermal injury and dorsal excision.

Introduction. Severely burned patients are susceptible to bacterial infection within their burn wounds, which frequently leads to sepsis, multiple organ failure and death. The opportunistic pathogen Pseudomonas aeruginosa, an organism inherently resistant to multiple antibiotics, is a common cause of sepsis in these patients.Aim. Development of a topical treatment unrelated to conventional antibiotics is essential for prevention of P. aeruginosa infection and sepsis, leading to a role for the direct application of probiotics or their by-products.Methodology. We examined the effectiveness of 20× concentrated supernatant from Lactobacillus gasseri strain 63 AM (LgCS) grown in de Man, Rogosa and Sharpe broth in inhibiting P. aeruginosa biofilms in vitro, as well as in reducing wound bioburden and P. aeruginosa sepsis in vivo.Results. LgCS inhibited the growth of P. aeruginosa strain PAO1, prevented its biofilm development and eliminated partially developed PAO1 biofilms. In the murine model of thermal injury, a single injection of LgCS following injury and PAO1 infection reduced mortality to 0 % and prevented systemic spread (sepsis). Furthermore, a second injection of LgCS 24 h after the first eliminated PAO1 from the wound. In the murine dorsal excision infection model, either LgCS or ceftazidime treatment of the PAO1-infected wound significantly reduced the mortality rate among infected mice, while combining LgCS with ceftazidime eliminated mortality.Conclusion. These results suggest the potential of LgCS in preventing sepsis from P. aeruginosa infection in severely burned and other immunocompromised patients.

Peroxisome proliferator-activated receptors mediate host cell proinflammatory responses to Pseudomonas aeruginosa autoinducer.

The pathogenic bacterium Pseudomonas aeruginosa utilizes the 3-oxododecanoyl homoserine lactone (3OC(12)-HSL) autoinducer as a signaling molecule to coordinate the expression of virulence genes through quorum sensing. 3OC(12)-HSL also affects responses in host cells, including the upregulation of genes encoding inflammatory cytokines. This proinflammatory response may exacerbate underlying disease during P. aeruginosa infections. The specific mechanism(s) through which 3OC(12)-HSL influences host responses is unclear, and no mammalian receptors for 3OC(12)-HSL have been identified to date. Here, we report that 3OC(12)-HSL increases mRNA levels for a common panel of proinflammatory genes in murine fibroblasts and human lung epithelial cells. To identify putative 3OC(12)-HSL receptors, we examined the expression patterns of a panel of nuclear hormone receptors in these two cell lines and determined that both peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) and PPARgamma were expressed. 3OC(12)-HSL functioned as an agonist of PPARbeta/delta transcriptional activity and an antagonist of PPARgamma transcriptional activity and inhibited the DNA binding ability of PPARgamma. The proinflammatory effect of 3OC(12)-HSL in lung epithelial cells was blocked by the PPARgamma agonist rosiglitazone, suggesting that 3OC(12)-HSL and rosiglitazone are mutually antagonistic negative and positive regulators of PPARgamma activity, respectively. These data identify PPARbeta/delta and PPARgamma as putative mammalian 3OC(12)-HSL receptors and suggest that PPARgamma agonists may be employed as anti-inflammatory therapeutics for P. aeruginosa infections.

Enzymatic degradation of in vitro Staphylococcus aureus biofilms supplemented with human plasma.

Enzymatic debridement is a therapeutic strategy used clinically to remove necrotic tissue from wounds. Some of the enzymes utilized for debridement have been tested against bacterial pathogens, but the effectiveness of these agents in dispersing clinically relevant biofilms has not been fully characterized. Here, we developed an in vitro Staphylococcus aureus biofilm model that mimics wound-like conditions and employed this model to investigate the antibiofilm activity of four enzymatic compounds. Human plasma at concentrations of 0%-50% was supplemented into growth media and used to evaluate biofilm biomass accumulation over 24 hours and 48 hours in one methicillin-sensitive and five methicillin-resistant strains of S. aureus. Supplementation of media with 10% human plasma resulted in the most robust biofilms in all six strains. The enzymes α-amylase, bromelain, lysostaphin, and papain were then tested against S. aureus biofilms cultured in 10% human plasma. Quantification of biofilms after 2 hours and 24 hours of treatment using the crystal violet assay revealed that lysostaphin decreased biomass by up to 76%, whereas α-amylase, bromelain, and papain reduced biomass by up to 97%, 98%, and 98%, respectively. Scanning electron microscopy confirmed that the dispersal agents detached the biofilm exopolysaccharide matrix and bacteria from the growth surface. Lysostaphin caused less visible dispersal of the biofilms, but unlike the other enzymes, induced morphological changes indicative of bacterial cell damage. Overall, our results indicate that use of enzymes may be an effective means of eradicating biofilms and a promising strategy to improve treatment of multidrug-resistant bacterial infections.

An in vivo polymicrobial biofilm wound infection model to study interspecies interactions.

Chronic wound infections are typically polymicrobial; however, most in vivo studies have focused on monospecies infections. This project was designed to develop an in vivo, polymicrobial, biofilm-related, infected wound model in order to study multispecies biofilm dynamics and in relation to wound chronicity. Multispecies biofilms consisting of both Gram negative and Gram positive strains, as well as aerobes and anaerobes, were grown in vitro and then transplanted onto the wounds of mice. These in vitro-to-in vivo multi-species biofilm transplants generated polymicrobial wound infections, which remained heterogeneous with four bacterial species throughout the experiment. We observed that wounded mice given multispecies biofilm infections displayed a wound healing impairment over mice infected with a single-species of bacteria. In addition, the bacteria in the polymicrobial wound infections displayed increased antimicrobial tolerance in comparison to those in single species infections. These data suggest that synergistic interactions between different bacterial species in wounds may contribute to healing delays and/or antibiotic tolerance.

Human transcriptome analysis reveals a potential role for active transport in the metabolism of Pseudomonas aeruginosa autoinducers.

The opportunistic pathogen Pseudomonas aeruginosa employs acyl homoserine lactones (AHL) as signaling compounds to regulate virulence gene expression via quorum sensing. The AHL N-3-oxo-dodecanoyl-l-homoserine lactone (3OC(12)-HSL) also induces mammalian cell responses, including apoptosis and immune modulation. In certain cell types the apoptotic effects of 3OC(12)-HSL are mediated via a calcium-dependent signaling pathway, while some pro-inflammatory effects involve intracellular transcriptional regulators. However, the mechanisms by which mammalian cells perceive and respond to 3OC(12)-HSL are still not completely understood. Here we used microarray analysis to investigate the transcriptional response of human lung epithelial cells after exposure to 3OC(12)-HSL. These data revealed that mRNA levels for several genes involved in xenobiotic sensing and drug transport were increased in cells exposed to 3OC(12)-HSL, which led us to examine the intracellular fate of 3OC(12)-HSL. Using radiolabeled autoinducer uptake assays, we discovered that intracellular 3OC(12)-HSL levels increased after exposure and achieved maximal levels after 20-30 min. Intracellular 3OC(12)-HSL decreased to background levels over the next 90 min and this process was blocked by pre-treatment with an inhibitor of the ABC transporter ABCA1. Taken together, these data suggest that mammalian cells detect 3OC(12)-HSL and activate protective mechanisms to expel it from the cell.

Quorum sensing and the social evolution of bacterial virulence.

The ability of pathogenic bacteria to exploit their hosts depends upon various virulence factors, released in response to the concentration of small autoinducer molecules that are also released by the bacteria [1-5]. In vitro experiments suggest that autoinducer molecules are signals used to coordinate cooperative behaviors and that this process of quorum sensing (QS) can be exploited by individual cells that avoid the cost of either producing or responding to signal [6, 7]. However, whether QS is an exploitable social trait in vivo, and the implications for the evolution of virulence [5, 8-10], remains untested. We show that in mixed infections of the bacterium Pseudomonas aeruginosa, containing quorum-sensing bacteria and mutants that do not respond to signal, virulence in an animal (mouse) model is reduced relative to that of an infection containing no mutants. We show that this is because mutants act as cheats, exploiting the cooperative production of signal and virulence factors by others, and hence increase in frequency. This supports the idea that the invasion of QS mutants in infections of humans [11-13] is due to their social fitness consequences [6, 7, 14] and predicts that increased strain diversity will select for lower virulence.