Antimicrobial activity of a bioelectric dressing using an in vitro wound pathogen colony drip-flow reactor biofilm model.

OBJECTIVE: We performed in vitro antibiofilm testing of a silver and zinc containing microcurrent generating bioelectric dressing (BED) against clinical wound pathogens to determine its efficacy in preventing biofilm formation under low shear and continuous flow conditions, simulating wound infection environments. METHOD: We customised an in vitro colony drip-flow reactor (DFR) biofilm model for efficacy evaluation of BED. Each bacterial pathogen was diluted to 104CFU/ml and inoculated on the polycarbonate filter membrane as an abiotic support. BED and controls (no treatment, gauze, and blank polyester with no silver and zinc) were applied directly on the membranes where bacterial cultures were inoculated. Biofilms were continuously developed onto the membranes for 72 hours at room temperature. Biofilm formation was confirmed by crystal violet staining and microscopic observation. Through vigorous shaking and sonication, the released bacteria were serially diluted, plated, and incubated for 24 hours at 37°C to determine the numbers of surviving bacteria. RESULTS: Biofilms were well developed onto blank polyesters, but not the BED after 72 hours incubation. Crystal violet staining from the blank polyesters showed large and fully grown biofilms. We observed an inhibition in bacterial growth on BED treatment. The antibiofilm activity of the BED against each of eight monospecies biofilms showed a 1- or 2 log10 (or 10- or 100-fold) reduction in bacterial numbers compared with those of controls. CONCLUSION: Our results demonstrated that colony DFR biofilm model was an appropriate for testing the antibiofilm efficacy of BED under low shear and continuous flow conditions for simulating clinical wound environments. The bioelectric currents generated from the silver and zinc active ingredients in the dressing resulted in antibiofilm activity of this wound care device. DECLARATION OF INTEREST: The opinions or assertions contained herein are the private views of the authors, based on scientific investigation, and are not to be construed as official or as reflecting the views of the Department of Defense, the United States Government or any of the authors' employers. Dr. Mina Izadjoo has served as a consultant to Vomaris Innovations Inc.

Antibiofilm efficacy evaluation of a bioelectric dressing in mono- and multi-species biofilms.

OBJECTIVE: Chronic infections are often related to the formation of single or polymicrobial biofilms, which lead to hard-to-treat infections, difficult wound management, and recurrent infections. In this study, we evaluated in vitro antibiofilm properties of a silver and zinc bioelectric dressing (Procellera). METHOD: We customised and established a poloxamer biofilm model using glass coverslips for the efficacy evaluation of the bioelectric dressing. For antimicrobial susceptibility testing, each bacterial pathogen was diluted to 10(5)CFUs/ml, mixed with 30% poloxamer hydrogels and dropped onto round glass coverslips (25mm diameter) as an abiotic support. Additionally, we mixed four bacterial strains and developed multi-species biofilms in the poloxamer model to examine anti-biofilm efficacy testing against polymicrobial biofilms. RESULTS: After 24 hours incubation, we observed significant inhibition of bacterial growth in nine pathogens (∼2- or 3-fold log10 reduction) compared to controls (no treatment, gauze, and blank polyester with no silver and zinc). The smallest effect was seen with Enterococcus faecalis strain where there was approximately a 1-fold log10 reduction of microbial growth. The antibiofilm efficacy against multi-species (four pathogens) biofilms, evaluated on chromogenic agar plates, was 1- or 2-fold log10 reduction compared to controls. CONCLUSION: This poloxamer biofilm model was easy to set up, simple to apply, and demonstrated appropriate biofilm formation. The data showed the formation of biofilms inoculated with either single or polymicrobial bacteria under no shear condition, and the bioelectric dressing tested in this study showed effective antibiofilm activity against both the mono- and multi-species biofilms. DECLARATION OF INTEREST: None. The opinions or assertions contained here are the private views of the authors, based on scientific investigation, and are not to be construed as official or as reflecting the views of the US Department of the Army, the Department of Defense, the Department of Veterans Affairs, or the Henry M. Jackson Foundation for the Advancement of Military Medicine.

Measurement of microelectric potentials in a bioelectrically-active wound care device in the presence of bacteria.

OBJECTIVE: Wound healing is enhanced in the presence of an external electrical field. The purpose of this study was first to investigate whether microelectric potentials (EPs) can be generated when the innovative design of a silver (Ag)- and zinc (Zn)-printed wound care device was exposed to saline solution which is commonly used to clean wound sites; and second to measure the generated EPs while the device was exposed to bacterial culture suspensions to mimic infection. METHOD: An Ag/Zn-printed test specimen as a wound care device was designed to accommodate Ag and Zn half-cell potentials by alternatively printing them on a woven polyester material in a well-characterised dot matrix pattern. A well-calibrated high impedance EPs measuring system was used to measure any EPs generated. Ultrasensitive inductively coupled plasma analysis was performed to determine whether the device induced any increase in trace metals in rabbit blood following implantation for 2-4 weeks. RESULTS: EPs were consistently generated under various conductive solutions at the levels of 120.4±26.3mV (average±standard deviation) on Ag dots and -506.5±76.3mV over Zn dots to form microcircuits with EPs of 626.7±86.3mV between the Ag and Zn metallic elements of the dressing. Interestingly, the patterns of EPs generated with stable polarities were consistent when the device was exposed to bacterial suspensions for mimicking wound infection. Implantation of the device did not cause any increase in Ag or Zn in rabbit blood. CONCLUSION: The Ag/Zn-printed wound device generated sustained EPs successfully in the presence of various conductive fluids without changing EPs including polarities. Consistently generated EPs at each battery couple with Ag/Zn-based wound device would restore disrupted physiologic bioelectric signals on wound sites, which could lead to improved wound healing. DECLARATION OF INTEREST: The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the US Department of the Army, the US Department of Defense, the Department of Veterans Affairs, or the Henry M. Jackson Foundation for Advancement of Military Medicine. The research was supported by the US Air Force medical support agency (AFMSA), office of the Air Force Surgeon General, USA.

Wound infections and healing: are they contributing factors for carcinogenesis?

The link between inflammation and tumourisation has long been considered as a key event in clinical cancer development. Inflammation and inflammatory diseases can be caused by many factors including infectious agents, altered genetics and various degrees of injuries from simple cuts to traumatic wounds, such as those suffered in battlefield. Improved management of all wound types is critical in protecting affected individuals against the development of tumourisation cues, which may potentially lead to cancer development. There have been numerous studies on the mechanism of inflammation-induced tumourisation. Thus, in this mini review, we summarised evidence demonstrating the potential link between infectious agents and their moonlight proteins, wounding, trauma, overactive repair mechanisms, and carcinogenesis.

Distinctive stages and strain variations of A. baumannii biofilm development under shear flow.

OBJECTIVE: To examine and characterise the process of Acinetocbacter baumannii biofilm development under shear flow. METHOD: Using an automated flow-cell system with microfluidic channels connected to an imaging system, I 0 clinical wound isolates of A. baumannii were examined for their ability to form biofilms under shear flow, on the glass surface of the flow through device. Flow biofilm development by this organism was observed to comprise four stages: attachment, microcolony formation, maturation with three dimensional structures, and dispersion. RESULTS: A. baumannii adheres readily to glass surfaces and quickly forms biofilm. Significant variations were observed among these I 0 clinical strains in their ability to attach and form flow biofilms. CONCLUSION: These specific characteristics in biofilm formation may contribute to this organism's ability to disseminate within health-care environments.

An overview of biofilm and its detection in clinical samples.

In many instances, wound colonising-bacteria are thought to be capable of forming biofilms, a significant factor contributing to delays or failure in wound healing. This review summarises the accumulated knowledge of biofilm and addresses the unmet need of biofilm detection in clinical wound samples.

Aberrant expression of chromogranin A, miR-146a, and miR-146b-5p in prostate structures with focally disrupted basal cell layers: an early sign of invasion and hormone-refractory cancer?

Our recent studies have suggested that prostate tumor invasion is triggered by autoimmunoreactions induced focal basal cell layer disruptions (FBCLD) that selectively favor monoclonal proliferation of the overlying progenitors or of a biologically more aggressive cell clone. As circulating chromogranin-A (CgA) levels are found to correlate with tumor progression and the status of hormone refractoriness, our current study attempted to assess whether CgA-positive cells would be preferentially distributed in epithelial structures with FBCLD. Paraffin-embedded specimens from 50 patients with organ-confined prostate cancer were subjected to double immunohistochemical analysis with monoclonal antibodies to basal cells and CgA. From each case, 3-5 randomly selected fields were digitally photographed and the photos were magnified 400% and the numbers of CgA-positive cells in epithelial structures with non-disrupted, focally disrupted, and lost basal cell layer were separately counted. The averaged number of cell for each category was statistically compared with the Pearson's Chi-square test. In addition, morphologically similar structures with and without CgA-positive cell clusters were microdissected from four selected cases and subjected to a comparison of differential micro-RNA expression levels. Our study revealed that, although isolated CgA-positive cells were seen in both the basal cell layer and the luminal cell population in all cases, only 8 cases (16%) harbored large clusters of CgA-positive cells that were concentrated in a given area, in which all or nearly all cells appeared to share a similar morphological and immunohistochemical profile. Microdissected epithelial structures with CgA-positive cell clusters exhibited a more than 5- and 7-fold lower expression of miR-146a and miR-146b-5p than their CgA-negative counterparts. As focal basal cell layer disruptions and the reduction or loss of miR-146a and miR-146b-5p has been documented to correlate with prostate tumor invasion and hormone refractoriness, our findings suggest that aberrant CgA expression in epithelial structures with FBCLD may represent an early sign of these events.

Novel model of peripheral tissue trauma-induced inflammation and gastrointestinal dysmotility.

BACKGROUND: Trauma is a leading cause of death and although the gut is recognized as the 'motor' of post-traumatic systemic inflammatory response syndrome and multiple organ failure, studies on the gastrointestinal (GI) tract are few. Our objectives were to create a precisely controllable tissue injury model in which GI motility, systemic inflammation and wound fluid can be analyzed. METHODS: A non-narcotic murine trauma model was developed by the subcutaneous dorsal trans-implantation of a devitalized donor syngeneic harvested tissue-bone matrix (TBX), which was precisely adjusted to % total body weight and studied after 21 h. Gastrointestinal transit histograms were plotted after the oral administration of non-digestible FITC-dextran and geometric centers calculated. Organ bath evaluated jejunal circular muscle contractility. Multiplex electrochemiluminescence measurements of serum and TBX wound fluid inflammatory mediators were performed. KEY RESULTS: Increasing TBX amounts progressively delayed transit, whereas TBX heat denaturation or decellularization prevented ileus and death. In the TBX(17.5%) model, jejunal muscle contractility was suppressed and a systemic inflammatory response developed as significant serum elevations in IL-6, keratinocyte cytokine and IL-10 compared to sham. In addition, inflammatory responses within the wound fluid showed elevated levels of preformed IL-1ß and TNF-α, whereas, 21 h after implantation IL-1ß, IL-6 and keratinocyte cytokine were significantly increased in the wound. CONCLUSIONS & INFERENCES: A novel donor tissue-bone matrix trauma model was developed that is precisely adjustable and recapitulates important clinical phenomena. The non-narcotic model demonstrated that increasing tissue injury progressively caused ileus, initiated a systemic inflammatory response and developed inflammatory changes within the wound.

Resistance carrying plasmid in a traumatic wound.

OBJECTIVE: To isolate and identify antibiotic-resistant bacteria from the exudate of a complex wound and determine if antibiotic resistance genes are chromosomal or plasmid borne. METHOD: Antibiotic resistant bacteria from wound exudate of a single clinical sample were selected on agar media with ampicillin. A single colony was further screened for resistance to kanamycin by antibiotic-supplemented agar and to other antibiotics by an automated Phoenix instrument. Identification of the isolate was carried out by biochemical profiling and by 16S rDNA analysis. RESULTS: Approximately 51% of total bacteria in the wound exudate with identical colony morphotype were resistant to 100 microg/ml of ampicillin. A single colony from this population also demonstrated resistance to 50 microg/ml of kanamycin on kanamycin-supplemented agar. Further antimicrobial sensitivity testing by the Phoenix instrument indicated resistance to inhibitory concentrations of amoxicillin-clavulanate, ampicillin-sulbactam, cefazolin, gentamicin, nitrofurantoin, tobramycin, and trimethoprim-sulfamethoxazole. Biochemical and 16S rDNA analysis identified this bacterial isolate as a member of genus Enterobacter. A plasmid preparation from this isolate successfully transferred ampicillin and kanamycin resistance to E. coli competent cells. E. coli transformants displayed two resistance phenotypes and the plasmids from these transformants displayed two different restriction type patterns, with one correlating to ampicillin and kanamycin resistance and the other only to ampicillin resistance. CONCLUSION: A multiple antibiotic-resistant Enterobacter spp. from the wound fluid of a clinical sample was found to carry an antibiotic-resistant plasmid in a closely related species E. coli. The presence of antibiotic resistance plasmid in Enterobacteria that are part of the normal microbial flora of the human gut and skin could lead to the spread of resistance phenotype and emergence of antibiotic resistant pathogens. This study suggests normal human microbial fl ora could be a potential reservoir for resistance genes.

A study on the use of male animal models for developing a live vaccine for brucellosis.

To study the safety of Brucella melitensis WR201, a live vaccine candidate, we compared the course of infection of this strain with that of virulent 16M in male BALB/c mice. At various times after oral immunization with strains WR201 or 16M, lungs, liver, spleen, testis, epididymis, inguinal and cervical lymph nodes were removed. Tissues were divided for microbiologic culture and histopathological examination. WR201 infection in male BALB/c mice had lower intensity and shorter duration than infection caused by virulent 16M. Pathological examination of testis and epididymis revealed no inflammation following strain WR201 immunization. In contrast, animals given virulent 16M strain had substantial inflammation in infected tissues. These data confirm the marked attenuation of WR201 relative to 16M. In addition, these studies suggest that male mice may be useful to assess the safety of live, attenuated Brucella vaccine candidates.

Impaired control of Brucella melitensis infection in Rag1-deficient mice.

After intranasal inoculation, Brucella melitensis chronically infects the mononuclear phagocyte system in BALB/c mice, but it causes no apparent illness. Adaptive immunity, which can be transferred by either T cells or antibody from immune to naive animals, confers resistance to challenge infection. The role of innate, non-B-, non-T-cell-mediated immunity in control of murine brucellosis, however, is unknown. In the present study, we documented that BALB/c and C57BL/6 mice had a similar course of infection after intranasal administration of 16M, validating the usefulness of the model in the latter mouse strain. We then compared the course of infection in Rag1 knockout mice (C57BL/6 background) (referred to here as RAG-1 mice) which have no B or T cells as a consequence of deletion of Rag1 (recombination-activating gene 1), with infection in normal C57BL/6 animals after intranasal administration of B. melitensis 16M. C57BL/6 mice cleared brucellae from their lungs by 8 to 12 weeks and controlled infection in the liver and spleen at a low level. In contrast, RAG-1 mice failed to reduce the number of bacteria in any of these organs. From 1 to 4 weeks after inoculation, the number of splenic bacteria increased from 2 to 4.5 logs and remained at that level. In contrast to the consistently high numbers of brucellae observed in the spleens, the number of bacteria rose in the livers sampled for up to 20 weeks. Immunohistologic examination at 8 weeks after infection disclosed foci of persistent pneumonia and large amounts of Brucella antigen in macrophages in lung, liver, and spleen in RAG-1, but not C57BL/6, mice. These studies indicate that T- and B-cell-independent immunity can control Brucella infection at a high level in the murine spleen, but not in the liver. Immunity mediated by T and/or B cells is required for clearance of bacteria from spleen and lung and for control of bacterial replication in the liver.

Protection of mice against brucellosis by vaccination with Brucella melitensis WR201(16MDeltapurEK).

Human brucellosis can be acquired from infected animal tissues by ingestion, inhalation, or contamination of the conjunctiva or traumatized skin by infected animal products. A vaccine to protect humans from occupational exposure or from zoonotic infection in areas where the disease is endemic would reduce an important cause of morbidity worldwide. Vaccines currently used in animals are unsuitable for human use. We tested a live, attenuated, purine-auxotrophic mutant strain of Brucella melitensis, WR201, for its ability to elicit cellular and humoral immune responses and to protect mice against intranasal challenge with B. melitensis 16M. Mice inoculated intraperitoneally with WR201 made serum antibody to lipopolysaccharide and non-O-polysaccharide antigens. Splenocytes from immunized animals released interleukin-2 (IL-2), gamma interferon, and IL-10 when cultured with Brucella antigens. Immunization led to protection from disseminated infection but had only a slight effect on clearance of the challenge inoculum from the lungs. These studies suggest that WR201 should be further investigated as a vaccine to prevent human brucellosis.

Acquisition of Salmonella flora by turtle hatchlings on commercial turtle farms.

A commercial turtle pond in South Louisiana was studied to identify the mechanism by which turtle hatchlings acquire Salmonella flora. The visceral organs and mature eggs removed from 31 adult gravid female turtles over the course of two egg-laying seasons and from 37 adult females during one winter dormant period were examined bacteriologically for Salmonella. Pond water, egg nest soil, and hatchlings produced by eggs removed from the oviducts and nest soil were also tested. Eighty-eight turtles hatched from eggs removed from the oviducts of 15 turtles at necropsy did not excrete or harbor systemically Salmonella, nor were these pathogens isolated from ovarian tissue or immature eggs. The findings suggest transovarian transmission of these pathogens does not occur frequently. Turtles hatched from eggs retrieved from soil nests 1 to 2 h after deposition harbor and excrete these organisms. This result coupled with the isolation of these pathogens from the cloaca, colon contents, and bursal fluid from 18 females captured in the act of egg laying supports the cloaca to egg and nest soil to egg mode for salmonellae infection in the resultant hatchling. Salmonella arizonae and Salmonella serogroups B, C2, and E1 were isolated from the cloaca, colon contents, pond water, and nest soil, and were excreted by hatchlings produced from eggs removed from the soil nests. These same serogroups were isolated from the colon contents of 19 of 37 females tested during the dormant period, suggesting the salmonellae persist in the pond environment in the adult throughout the year.