Pyochelin biotransformation by Staphylococcus aureus shapes bacterial competition with Pseudomonas aeruginosa in polymicrobial infections.

Pseudomonas aeruginosa and Staphylococcus aureus are among the most frequently isolated bacterial species from polymicrobial infections of patients with cystic fibrosis and chronic wounds. We apply mass spectrometry guided interaction studies to determine how chemical interaction shapes the fitness and community structure during co-infection of these two pathogens. We demonstrate that S. aureus is equipped with an elegant mechanism to inactivate pyochelin via the yet uncharacterized methyltransferase Spm (staphylococcal pyochelin methyltransferase). Methylation of pyochelin abolishes the siderophore activity of pyochelin and significantly lowers pyochelin-mediated intracellular reactive oxygen species (ROS) production in S. aureus. In a murine wound co-infection model, an S. aureus mutant unable to methylate pyochelin shows significantly lower fitness compared with its parental strain. Thus, Spm-mediated pyochelin methylation is a mechanism to increase S. aureus survival during in vivo competition with P. aeruginosa.

The Bactericidal Tandem Drug, AB569: How to Eradicate Antibiotic-Resistant Biofilm Pseudomonas aeruginosa in Multiple Disease Settings Including Cystic Fibrosis, Burns/Wounds and Urinary Tract Infections.

The life-threatening pandemic concerning multi-drug resistant (MDR) bacteria is an evolving problem involving increased hospitalizations, billions of dollars in medical costs and a remarkably high number of deaths. Bacterial pathogens have demonstrated the capacity for spontaneous or acquired antibiotic resistance and there is virtually no pool of organisms that have not evolved such potentially clinically catastrophic properties. Although many diseases are linked to such organisms, three include cystic fibrosis (CF), burn/blast wounds and urinary tract infections (UTIs), respectively. Thus, there is a critical need to develop novel, effective antimicrobials for the prevention and treatment of such problematic infections. One of the most formidable, naturally MDR bacterial pathogens is Pseudomonas aeruginosa (PA) that is particularly susceptible to nitric oxide (NO), a component of our innate immune response. This susceptibility sets the translational stage for the use of NO-based therapeutics during the aforementioned human infections. First, we discuss how such NO therapeutics may be able to target problematic infections in each of the aforementioned infectious scenarios. Second, we describe a recent discovery based on years of foundational information, a novel drug known as AB569. AB569 is capable of forming a "time release" of NO from S-nitrosothiols (RSNO). AB569, a bactericidal tandem consisting of acidified NaNO2 (A-NO2 -) and Na2-EDTA, is capable of killing all pathogens that are associated with the aforementioned disorders. Third, we described each disease state in brief, the known or predicted effects of AB569 on the viability of PA, its potential toxicity and highly remote possibility for resistance to develop. Finally, we conclude that AB569 can be a viable alternative or addition to conventional antibiotic regimens to treat such highly problematic MDR bacterial infections for civilian and military populations, as well as the economical burden that such organisms pose.

Antimicrobial Susceptibility of Corynebacterium bovis Isolates from Immunodeficient Rodents.

Corynebacterium bovis, the causative agent of hyperkeratotic dermatitis in immunodeficient mice, is a significant problem in preclinical oncology research. Infection results in lifelong skin colonization and a decrease in successful engraftment of patient-derived xenograft tumor models. The use of antimicrobial agents for C. bovis is controversial in light of reports of poor efficacy and the possibility of selection for resistant strains. The purpose of this study was to describe the antimicrobial susceptibilities of C. bovis isolates obtained exclusively from immunodeficient rodents in order to aid in antimicrobial dose determination. Between 1995 and 2018, 15 isolates were collected from 11 research institutions across the United States. Antimicrobial susceptibility testing was performed for 24 antimicrobials commonly used against gram-positive bacteria. Our results provide an updated understanding of the susceptibility profiles of rodent C. bovis isolates, indicating little variability between geographically and temporally distant isolates. These results will facilitate appropriate antimicrobial use to prevent and treat C. bovis infections in immunodeficient rodents.

The Overall Poor Specificity of MRCP in the Preoperative Evaluation of the Jaundiced Patient Will Increase the Incidence of Nontherapeutic ERCP.

Laparoscopic cholecystectomy remains one of the most common surgical operations. Common bile duct stones (CBDS) are estimated to be present in 10%-20% of individuals with symptomatic gallstones. Preoperative magnetic resonance cholangiopancreatography (MRCP) and intraoperative cholangiography (IOC) remain the most common methods of evaluation, with subsequent endoscopic retrograde cholangiopancreatography (ERCP) for stone extraction if positive for CBDS. We examined our experience with preoperative MRCP versus IOC for the management of the jaundiced patient with cholelithiasis. This is a retrospective single-institution study that examined all laparoscopic cholecystectomies performed over a 15-month period between 2017 and 2018. Outpatient elective cases were excluded from the analysis. Charts were reviewed for demographics, operative details, and whether an MRCP, IOC, or ERCP was performed. Data were evaluated using a 2-sample t-test. A total of 460 patients underwent laparoscopic cholecystectomy over a 15-month period. Of those, 147 underwent either an MRCP or an IOC for clinical suspicion for CBDS. ERCP after MRCP was nontherapeutic in 11/32 (34%) compared with 2/12 (17%) of patients following IOC. The sensitivity and specificity of MRCP were 91% and 80%, respectively, with a positive predictive value of 66% and a negative predictive value of 96%. The sensitivity and specificity of IOC were 83% and 97%, respectively, with a positive predictive value of 83% and a negative predictive value of 97%. MRCP and IOC have unique advantages and disadvantages. MRCP has greater sensitivity, but poor specificity, resulting in unnecessary ERCPs with associated morbidity and increased costs to the patient.

Early Comfort Care Following Operative Intervention for Traumatic Injury.

BACKGROUND: Several studies have described the population of adult trauma patients who undergo withdrawal of life-sustaining treatments (WLST); however, no study has looked specifically at trauma patients who undergo WLST following surgery. METHODS: This was a retrospective chart review of all trauma patients who underwent surgery at our trauma center between January 1 and December 31, 2017. Demographics were collected along with injury patterns and advance directives. Charts of all patients who died or who were discharged to hospice were analyzed to determine whether WLST occurred. Statistics included Fisher's exact test and Mann-Whitney U test. RESULTS: Three thousand and twenty-five adult trauma patients received care and 1495 (49.4%) had operations. Thirty (2.0%) patients underwent WLST, 15 (50.0%) of whom died in the hospital and 15 (50.0%) of whom were discharged to hospice. Twenty-six (86.7%) patients had a palliative care consult and 12 (40.0%) had prior advance directives. The most common injuries were femur fractures and subdural hematomas. Adjusting for age, white race, and age-adjusted CCI, femur fracture patients had, on average, 8.8 more hours between presentation and surgery (95% CI 2.1-15.4, P = .01) and 39 fewer hours between surgery and WLST (95% CI -107-29, P = .26) than traumatic brain injury patients. DISCUSSION: The short time between surgery and WLST in this cohort of patients may demonstrate that surgery was not aligned with patients' goals of care. A patient-centered approach that includes surgeon-driven palliative care discussions may help avoid nonbeneficial surgery in the last few days of life.

Into the wild and on to the table: A Western Trauma Association multicenter analysis and comparison of wilderness falls in rock climbers and nonclimbers.

BACKGROUND: Wilderness activities expose outdoor enthusiasts to austere environments with injury potential, including falls from height. The majority of published data on falls while climbing or hiking are from emergency departments. We sought to more accurately describe the injury pattern of wilderness falls that lead to serious injury requiring trauma center evaluation and to further distinguish climbing as a unique pattern of injury. METHODS: Data were collected from 17 centers in 11 states on all wilderness falls (fall from cliff: International Classification of Diseases, Ninth Revision, e884.1; International Classification of Diseases, 10th Revision, w15.xx) from 2006 to 2018 as a Western Trauma Association multicenter investigation. Demographics, injury characteristics, and care delivery were analyzed. Comparative analyses were performed for climbing versus nonclimbing mechanisms. RESULTS: Over the 13-year study period, 1,176 wilderness fall victims were analyzed (301 climbers, 875 nonclimbers). Fall victims were male (76%), young (33 years), and moderately injured (Injury Severity Score, 12.8). Average fall height was 48 ft, and average rescue/transport time was 4 hours. Nineteen percent were intoxicated. The most common injury regions were soft tissue (57%), lower extremity (47%), head (40%), and spine (36%). Nonclimbers had a higher incidence of severe head and facial injuries despite having equivalent overall Injury Severity Score. On multivariate analysis, climbing remained independently associated with increased need for surgery but lower odds of composite intensive care unit admission/death. Contrary to studies of urban falls, height of fall in wilderness falls was not independently associated with mortality or Injury Severity Score. CONCLUSION: Wilderness falls represent a unique population with distinct patterns of predominantly soft tissue, head, and lower extremity injury. Climbers are younger, usually male, more often discharged home, and require more surgery but less critical care. LEVEL OF EVIDENCE: Epidemiological, Level IV.

Pseudomonas aeruginosa Alginate Benefits Staphylococcus aureus?

In this issue of Journal of Bacteriology, Price et al. show that the Pseudomonas aeruginosa-produced exopolysaccharide alginate protects Staphylococcus aureus by dampening the expression of P. aeruginosa virulence products that usually inhibit S. aureus respiration and cell membrane integrity when the two organisms compete in other environments (C. E. Price, D. G. Brown, D. H. Limoli, V. V. Phelan, and G. A. O'Toole, J Bacteriol 202:e00559-19, 2020, https://doi.org/10.1128/jb.00559-19). This is the first report that exogenously added alginate affects P. aeruginosa competition and provides a partial explanation for S. aureus and P. aeruginosa coinfections in cystic fibrosis.

Low-cost, Small-scale Decontamination of Laboratory Equipment by Using Chlorine Dioxide Gas.

A significant concern in laboratory animal medicine is contamination due to pathogen outbreaks and how to adequately decontaminate small equipment. Many factors play a role in the selection of the decontamination method including cost, efficacy, personnel time and safety. Chlorine dioxide (ClO2) gas is an effective method, but decontamination often requires a ClO2 gas generator with a specialized air-tight exposure chamber. Although this method works well for large-scale decon- tamination, the use of a gas generator may be impractical and too costly for smaller-scale decontamination. The goal of this study was to create and validate an effective, small-scale decontamination method that uses ClO2 gas and which is an affordable, efficient, safe, and reproducible. First, we identified a product that generates ClO2 gas after the combination of 2 dry reagents. To find an affordable exposure chamber, we evaluated the ability of 4 household totes with gasket-seal lid systems to retain ClO2 gas and relative humidity (RH). The efficacy of decontamination was validated by concurrently using 2 different biologic indicators (BI), Bacillus atrophaeus (B.a.) and Geobacillus stearothermophilus (G.s.). All household totes evaluated held sufficient gas and RH for a 15-h cycle, providing adequate contact time to inactivate both BI evaluated. Our results suggest that a total exposure dose of 71 ± 42 ppm-h of ClO2 gas over 15 h at 90% or greater RH is adequate to inactivate both B.a. and G.s. There was no statistical significance between the 2 BI as indicators for decontamination; 65 of 230 (28.3%) B.a. and 75 of 230 (32.6%) G.s spore strips were positive for growth (P = 0.36). In conclusion, we successfully combined a variety of low-cost materials to establish an effective, small-scale method to decontaminate laboratory equipment. Depending on the size of the tote and whether BI are used, the cost of our method is roughly 1% that of large-scale ClO2 gas generators used with specialized air-tight exposure chambers.

The anti-sigma factor MucA of Pseudomonas aeruginosa: Dramatic differences of a mucA22 vs. a ΔmucA mutant in anaerobic acidified nitrite sensitivity of planktonic and biofilm bacteria in vitro and during chronic murine lung infection.

Mucoid mucA22 Pseudomonas aeruginosa (PA) is an opportunistic lung pathogen of cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) patients that is highly sensitive to acidified nitrite (A-NO2-). In this study, we first screened PA mutant strains for sensitivity or resistance to 20 mM A-NO2- under anaerobic conditions that represent the chronic stages of the aforementioned diseases. Mutants found to be sensitive to A-NO2- included PA0964 (pmpR, PQS biosynthesis), PA4455 (probable ABC transporter permease), katA (major catalase, KatA) and rhlR (quorum sensing regulator). In contrast, mutants lacking PA0450 (a putative phosphate transporter) and PA1505 (moaA2) were A-NO2- resistant. However, we were puzzled when we discovered that mucA22 mutant bacteria, a frequently isolated mucA allele in CF and to a lesser extent COPD, were more sensitive to A-NO2- than a truncated ΔmucA deletion (Δ157-194) mutant in planktonic and biofilm culture, as well as during a chronic murine lung infection. Subsequent transcriptional profiling of anaerobic, A-NO2--treated bacteria revealed restoration of near wild-type transcript levels of protective NO2- and nitric oxide (NO) reductase (nirS and norCB, respectively) in the ΔmucA mutant in contrast to extremely low levels in the A-NO2--sensitive mucA22 mutant. Proteins that were S-nitrosylated by NO derived from A-NO2- reduction in the sensitive mucA22 strain were those involved in anaerobic respiration (NirQ, NirS), pyruvate fermentation (UspK), global gene regulation (Vfr), the TCA cycle (succinate dehydrogenase, SdhB) and several double mutants were even more sensitive to A-NO2-. Bioinformatic-based data point to future studies designed to elucidate potential cellular binding partners for MucA and MucA22. Given that A-NO2- is a potentially viable treatment strategy to combat PA and other infections, this study offers novel developments as to how clinicians might better treat problematic PA infections in COPD and CF airway diseases.

Defining the criteria for intubation of the patient with thermal burns.

OBJECTIVES: Recent studies demonstrate that burn patients are undergoing unnecessary intubations. We sought to determine the clinical criteria that predict intubations with benefit. METHODS: This was a retrospective review of intubated adults admitted to our center with thermal burns 2008-2013. Criteria for intubation were defined as traditional criteria (suspected smoke inhalation, oropharynx soot, hoarseness, dysphagia, singed facial hair, oral edema, oral burn, non-full thickness facial burns), or ABA criteria as defined by the 2011 ABA guidelines (full thickness facial burns, stridor, respiratory distress, swelling on laryngoscopy, upper airway trauma, altered mentation, hypoxia/hypercarbia, hemodynamic instability). Patients with <26days free from mechanical ventilation (ventilator-free days (VFD)) out of 28, were deemed indicated long-term intubations. Those with ≥26 VFD were deemed unnecessary short-term intubations. RESULTS: Of 218 patients, 151 had long-term and 67 had short-term intubations. Long-term intubation was strongly associated with ABA criteria (77.5%) compared to traditional criteria (22.5%) (p<0.001). Sensitivity of ABA criteria for long-term intubation was 77% and specificity 46%. Traditional criteria associated with long-term intubation included suspected smoke inhalation (OR 2.45 [95% CI, 1.18-5.11]), and singed facial hair (OR 2.53 [95% CI, 1.25-5.09]). The addition of these to ABA criteria created the Denver criteria, which exhibited an increased sensitivity for long-term intubations (95%), but decreased specificity (24%). CONCLUSIONS: Intubation should be considered for patients displaying the Denver criteria, which includes full thickness facial burns, stridor, respiratory distress, swelling on laryngoscopy, upper airway trauma, altered mentation, hypoxia/hypercarbia, hemodynamic instability, suspected smoke inhalation, and singed facial hair. Patients lacking these criteria should not be intubated.

PDGF-BB does not accelerate healing in diabetic mice with splinted skin wounds.

Topical application of platelet-derived growth factor-BB (PDGF-BB) is considered to accelerate tissue repair of impaired chronic wounds. However, the vast literature is plagued with conflicting reports of its efficacy in animal models and this is often influenced by a wide array of experimental variables making it difficult to compare the results across the studies. To mitigate the confounding variables that influence the efficacy of topically applied PDGF-BB, we used a controlled full thickness splinted excisional wound model in db/db mice (type 2 diabetic mouse model) for our investigations. A carefully-defined silicone-splinted wound model, with reduced wound contraction, controlled splint and bandage maintenance, allowing for healing primarily by reepithelialization was employed. Two splinted 8 mm dorsal full thickness wounds were made in db/db mice. Wounds were topically treated once daily with either 3 µg PDGF-BB in 30 µl of 5% PEG-PBS vehicle or an equal volume of vehicle for 10 days. Body weights, wound contraction, wound closure, reepithelialization, collagen content, and wound bed inflammation were evaluated clinically and histopathologically. The bioactivity of PDGF-BB was confirmed by in vitro proliferation assay. PDGF-BB, although bioactive in vitro, failed to accelerate wound healing in vivo in the db/db mice using the splinted wound model. Considering that the predominant mechanism of wound healing in humans is by re-epithelialization, the most appropriate model for evaluating therapeutics is one that uses splints to prevent excessive wound contraction. Here, we report that PDGF-BB does not promote wound closure by re-epithelialization in a murine splinted wound model. Our results highlight that the effects of cytoactive factors reported in vivo ought to be carefully interpreted with critical consideration of the wound model used.

The Pseudomonas aeruginosa AlgZR two-component system coordinates multiple phenotypes.

Pseudomonas aeruginosa is an opportunistic pathogen that causes a multitude of infections. These infections can occur at almost any site in the body and are usually associated with a breach of the innate immune system. One of the prominent sites where P. aeruginosa causes chronic infections is within the lungs of cystic fibrosis patients. P. aeruginosa uses two-component systems that sense environmental changes to differentially express virulence factors that cause both acute and chronic infections. The P. aeruginosa AlgZR two component system is one of its global regulatory systems that affects the organism's fitness in a broad manner. This two-component system is absolutely required for two P. aeruginosa phenotypes: twitching motility and alginate production, indicating its importance in both chronic and acute infections. Additionally, global transcriptome analyses indicate that it regulates the expression of many different genes, including those associated with quorum sensing, type IV pili, type III secretion system, anaerobic metabolism, cyanide and rhamnolipid production. This review examines the complex AlgZR regulatory network, what is known about the structure and function of each protein, and how it relates to the organism's ability to cause infections.

Raman spectroscopy enables noninvasive biochemical characterization and identification of the stage of healing of a wound.

Accurate and rapid assessment of the healing status of a wound in a simple and noninvasive manner would enable clinicians to diagnose wounds in real time and promptly adjust treatments to hasten the resolution of nonhealing wounds. Histologic and biochemical characterization of biopsied wound tissue, which is currently the only reliable method for wound assessment, is invasive, complex to interpret, and slow. Here we demonstrate the use of Raman microspectroscopy coupled with multivariate spectral analysis as a simple, noninvasive method to biochemically characterize healing wounds in mice and to accurately identify different phases of healing of wounds at different time-points. Raman spectra were collected from "splinted" full thickness dermal wounds in mice at 4 time-points (0, 1, 5, and 7 days) corresponding to different phases of wound healing, as verified by histopathology. Spectra were deconvolved using multivariate factor analysis (MFA) into 3 "factor score spectra" (that act as spectral signatures for different stages of healing) that were successfully correlated with spectra of prominent pure wound bed constituents (i.e., collagen, lipids, fibrin, fibronectin, etc.) using non-negative least squares (NNLS) fitting. We show that the factor loadings (weights) of spectra that belonged to wounds at different time-points provide a quantitative measure of wound healing progress in terms of key parameters such as inflammation and granulation. Wounds at similar stages of healing were characterized by clusters of loading values and slowly healing wounds among them were successfully identified as "outliers". Overall, our results demonstrate that Raman spectroscopy can be used as a noninvasive technique to provide insight into the status of normally healing and slow-to-heal wounds and that it may find use as a complementary tool for real-time, in situ biochemical characterization in wound healing studies and clinical diagnosis.

Iron-regulated expression of alginate production, mucoid phenotype, and biofilm formation by Pseudomonas aeruginosa.

UNLABELLED: Pseudomonas aeruginosa strains of non-cystic fibrosis (non-CF) origin do not produce significant amounts of extracellular alginate and are nonmucoid. In CF, such isolates can become mucoid through mutation of one of the genes (mucA, mucB, mucC, or mucD) that produce regulatory factors that sequester AlgU, required for increased expression of alginate genes. Mutation of the muc genes in the nonmucoid PAO1, PA14, PAKS-1, and Ps388 strains led to increased levels of extracellular alginate and an obvious mucoid phenotype, but only under iron-limiting growth conditions (≤5 µM), not under iron-replete conditions (≥10 µM). In contrast, >50% of P. aeruginosa isolates from chronic CF pulmonary infections expressed increased levels of alginate and mucoidy both under iron-limiting and iron-replete conditions (i.e., iron-constitutive phenotype). No single iron regulatory factor (e.g., Fur, PvdS) was associated with this loss of iron-regulated alginate expression and mucoidy in these CF isolates. However, the loss of only pyoverdine production, or its uptake, abrogated the ability of P. aeruginosa to produce a robust biofilm that represents the Psl-type of biofilm. In contrast, we show that mutation of the pyoverdine and pyochelin biosynthesis genes and the pyoverdine receptor (FpvA) lead to iron-constitutive expression of the key alginate biosynthesis gene, algD, and an explicitly mucoid phenotype in both iron-limiting and iron-replete conditions. These data indicate that alginate production and mucoidy, in contrast to other types of biofilms produced by P. aeruginosa, are substantially enhanced under iron limitation. These results also have compelling implications in relation to the use of iron chelators in the treatment of P. aeruginosa CF infections. IMPORTANCE: Pseudomonas aeruginosa is a leading model for the investigation of biofilms. While data have been generated about the role of iron in alginate-independent (Psl/Pel) biofilm development, there is a paucity of data regarding the role of iron in alginate production and its associated mucoid phenotype. We demonstrate that biologically relevant levels of iron that exist in the airway mucus of cystic fibrosis (CF) patients have a substantial influence on production of alginate and the overt mucoid phenotype, pathognomonic of P. aeruginosa infections in CF. Mucoid mutants of non-CF P. aeruginosa isolates are mucoid only under iron limitation and do not express increased levels of alginate under iron-replete growth conditions. However, a significant number of long-term CF isolates lost their iron-regulated expression of increased alginate production and mucoidy and became iron constitutive for these properties. In contrast to the formation of Psl-type biofilms, increasing iron limitation ultimately leads to an iron-constitutive expression of alginate and mucoidy.

Perioperative visual loss after excision and autografting of a thermal burn to the back.

Peri-operative visual loss is an uncommon and poorly understood entity whose severity launched a Practice Advisory to identify peri-operative risk factors including prone positioning, anemia, hypotension, blood loss >44.7% of EBV, and surgical time >4-6.5 h. Contributing co-morbidities are obesity, tobacco, malnutrition, and PAD, which reduce blood flow to the optic nerve. We describe a patient with POVL focusing on the peri-operative course defined as the immediate preoperative assessment through discharge to compare the hospital course with previous reports of POVL in cardiac and spine operations.ss A middle-aged man admitted to the burn unit with 10% deep partial and full thickness burns to the back and neck underwent excision and autografting while prone. He was subsequently diagnosed with ischemic optic neuropathy and blindness. Co-morbidities were tobacco, malnutrition (albumin of 2.6 g/dl), and obesity (BMI 30.1). Preoperative risk assessment included anemia and prone positioning. Intra-operative hypotension to SBP 75 mmHg was noted. Operative duration was 5 h. Blood loss was estimated to be 43.7% of EBV. Risk factors for POVL are present in many prone burn operations as these patients have long operative times and significant blood loss. Thus, minimization of these factors where possible is advised.

Expression of mucoid induction factor MucE is dependent upon the alternate sigma factor AlgU in Pseudomonas aeruginosa.

BACKGROUND: Alginate overproduction in P. aeruginosa, also referred to as mucoidy, is a poor prognostic marker for patients with cystic fibrosis (CF). We previously reported the construction of a unique mucoid strain which overexpresses a small envelope protein MucE leading to activation of the protease AlgW. AlgW then degrades the anti-sigma factor MucA thus releasing the alternative sigma factor AlgU/T (σ(22)) to initiate transcription of the alginate biosynthetic operon. RESULTS: In the current study, we mapped the mucE transcriptional start site, and determined that P(mucE) activity was dependent on AlgU. Additionally, the presence of triclosan and sodium dodecyl sulfate was shown to cause an increase in P(mucE) activity. It was observed that mucE-mediated mucoidy in CF isolates was dependent on both the size of MucA and the genotype of algU. We also performed shotgun proteomic analysis with cell lysates from the strains PAO1, VE2 (PAO1 with constitutive expression of mucE) and VE2ΔalgU (VE2 with in-frame deletion of algU). As a result, we identified nine algU-dependent and two algU-independent proteins that were affected by overexpression of MucE. CONCLUSIONS: Our data indicates there is a positive feedback regulation between MucE and AlgU. Furthermore, it seems likely that MucE may be part of the signal transduction system that senses certain types of cell wall stress to P. aeruginosa.

Antibacterial efficacy of silver-impregnated polyelectrolyte multilayers immobilized on a biological dressing in a murine wound infection model.

OBJECTIVE: To investigate the antibacterial effect of augmenting a biological dressing with polymer films containing silver nanoparticles. BACKGROUND: Biological dressings, such as Biobrane, are commonly used for treating partial-thickness wounds and burn injuries. Biological dressings have several advantages over traditional wound dressings. However, as many as 19% of wounds treated with Biobrane become infected, and, once infected, the Biobrane must be removed and a traditional dressing approach should be employed. Silver is a commonly used antimicrobial in wound care products, but current technology uses cytotoxic concentrations of silver in these dressings. We have developed a novel and facile technology that allows immobilization of bioactive molecules on the surfaces of soft materials, demonstrated here by augmentation of Biobrane with nanoparticulate silver. Surfaces modified with nanometer-thick polyelectrolyte multilayers (PEMs) impregnated with silver nanoparticles have been shown previously to result in in vitro antibacterial activity against Staphylococcus epidermidis at loadings of silver that are noncytotoxic. METHODS: We demonstrated that silver-impregnated PEMs can be nondestructively immobilized onto the surface of Biobrane (Biobrane-Ag) and determined the in vitro antibacterial activity of Biobrane-Ag with Staphylococcus aureus. In this study, we used an in vivo wound infection model in mice induced by topical inoculation of S aureus onto full-thickness 6-mm diameter wounds. After 72 hours, bacterial quantification was performed. RESULTS: Wounds treated with Biobrane-Ag had significantly (P < 0.001) fewer colony-forming units than wounds treated with unmodified Biobrane (more than 4 log10 difference). CONCLUSIONS: The results of our study indicate that immobilizing silver-impregnated PEMs on the wound-contact surface of Biobrane significantly reduces bacterial bioburden in full-thickness murine skin wounds. Further research will investigate whether this construct can be considered for human use.

Analysis of the Pseudomonas aeruginosa regulon controlled by the sensor kinase KinB and sigma factor RpoN.

Alginate overproduction by Pseudomonas aeruginosa, also known as mucoidy, is associated with chronic endobronchial infections in cystic fibrosis. Alginate biosynthesis is initiated by the extracytoplasmic function sigma factor (σ(22); AlgU/AlgT). In the wild-type (wt) nonmucoid strains, such as PAO1, AlgU is sequestered to the cytoplasmic membrane by the anti-sigma factor MucA that inhibits alginate production. One mechanism underlying the conversion to mucoidy is mutation of mucA. However, the mucoid conversion can occur in wt mucA strains via the degradation of MucA by activated intramembrane proteases AlgW and/or MucP. Previously, we reported that the deletion of the sensor kinase KinB in PAO1 induces an AlgW-dependent proteolysis of MucA, resulting in alginate overproduction. This type of mucoid induction requires the alternate sigma factor RpoN (σ(54)). To determine the RpoN-dependent KinB regulon, microarray and proteomic analyses were performed on a mucoid kinB mutant and an isogenic nonmucoid kinB rpoN double mutant. In the kinB mutant of PAO1, RpoN controlled the expression of approximately 20% of the genome. In addition to alginate biosynthetic and regulatory genes, KinB and RpoN also control a large number of genes including those involved in carbohydrate metabolism, quorum sensing, iron regulation, rhamnolipid production, and motility. In an acute pneumonia murine infection model, BALB/c mice exhibited increased survival when challenged with the kinB mutant relative to survival with PAO1 challenge. Together, these data strongly suggest that KinB regulates virulence factors important for the development of acute pneumonia and conversion to mucoidy.

StrataGraft skin substitute is well-tolerated and is not acutely immunogenic in patients with traumatic wounds: results from a prospective, randomized, controlled dose escalation trial.

OBJECTIVE: The goal of this study was to assess the immunogenicity and antigenicity of StrataGraft skin tissue in a randomized phase I/II clinical trial for the temporary management of full-thickness skin loss. BACKGROUND: StrataGraft skin tissue consists of a dermal equivalent containing human dermal fibroblasts and a fully stratified, biologically active epidermis derived from Near-diploid Immortalized Keratinocyte S (NIKS) cells, a pathogen-free, long-lived, consistent, human keratinocyte progenitor. METHODS: Traumatic skin wounds often require temporary allograft coverage to stabilize the wound bed until autografting is possible. StrataGraft and cadaveric allograft were placed side by side on 15 patients with full-thickness skin defects for 1 week before autografting. Allografts were removed from the wound bed and examined for allogeneic immune responses. Immunohistochemistry and indirect immunofluorescence were used to assess tissue structure and cellular composition of allografts. In vitro lymphocyte proliferation assays, chromium-release assays, and development of antibodies were used to examine allogeneic responses. RESULTS: One week after patient exposure to allografts, there were no differences in the numbers of T or B lymphocytes or Langerhans cells present in StrataGraft skin substitute compared to cadaver allograft, the standard of care. Importantly, exposure to StrataGraft skin substitute did not induce the proliferation of patient peripheral blood mononuclear cells to NIKS keratinocytes or enhance cell-mediated lysis of NIKS keratinocytes in vitro. Similarly, no evidence of antibody generation targeted to the NIKS keratinocytes was seen. CONCLUSIONS: These findings indicate that StrataGraft tissue is well-tolerated and not acutely immunogenic in patients with traumatic skin wounds. Notably, exposure to StrataGraft did not increase patient sensitivity toward or elicit immune responses against the NIKS keratinocytes. We envision that this novel skin tissue technology will be widely used to facilitate the healing of traumatic cutaneous wounds.This study was registered at www.clinicaltrials.gov (NCT00618839).

Functional implications of long-term pain following outpatient inguinal herniorrhaphy--a prospective evaluation.

BACKGROUND: The purpose of the present study is to assess pain and functional outcomes at 1 y following inguinal herniorrhaphy in which patients were randomized to receive a continuous wound infusion of bupivacaine to receiving a saline infusion. METHODS: Patients received saline or bupivacaine prior to incision and then for 60 h postoperatively. The incidence, severity, and functional interference of pain were assessed for five postoperative days, and at 1 y. RESULTS: Seventy patients completed a survey 1 y following herniorrhaphy. Four percent (3/72) of patients were in moderate to severe pain "almost always" or "often". Twenty-one percent (15/72) of patients experienced pain with ambulation. There was no difference between groups at 1 y. CONCLUSIONS: The incidence of moderate or severe pain is concerning 1 y following surgery. Functional aberrations associated with pain should be assessed in all studies evaluating long-term pain after herniorrhaphy.