CrkII/Abl phosphorylation cascade is critical for NLRC4 inflammasome activity and is blocked by Pseudomonas aeruginosa ExoT.

Type 3 Secretion System (T3SS) is a highly conserved virulence structure that plays an essential role in the pathogenesis of many Gram-negative pathogenic bacteria, including Pseudomonas aeruginosa. Exotoxin T (ExoT) is the only T3SS effector protein that is expressed in all T3SS-expressing P. aeruginosa strains. Here we show that T3SS recognition leads to a rapid phosphorylation cascade involving Abl / PKCδ / NLRC4, which results in NLRC4 inflammasome activation, culminating in inflammatory responses that limit P. aeruginosa infection in wounds. We further show that ExoT functions as the main anti-inflammatory agent for P. aeruginosa in that it blocks the phosphorylation cascade through Abl / PKCδ / NLRC4 by targeting CrkII, which we further demonstrate to be important for Abl transactivation and NLRC4 inflammasome activation in response to T3SS and P. aeruginosa infection.

Presence of CrkI-containing microvesicles in squamous cell carcinomas could have ramifications on tumor biology and cancer therapeutics.

Recently, we described a phenomenon whereby apoptotic cells generate and release CrkI-containing microvesicles, which stimulate proliferation in surrounding cells upon contact to compensate for their own demise. We termed these microvesicles "ACPSVs" for Apoptotic Compensatory Proliferation Signaling microvesicles. As immune cells and a majority of current cancer therapeutics destroy tumor cells primarily by apoptosis, we conducted a small pilot study to assess the possibility that ACPSVs may also be generated in squamous cell carcinomas. We first evaluated a primary and a metastatic squamous cell carcinoma cancer cell lines for their ability to produce ACPSVs under normal and apoptotic conditions. We next conducted a pilot study to assess the occurrence of ACPSVs in solid tumors extracted from 20 cancer patients with squamous cell carcinomas. Both cancer cell lines produced copious amounts of ACPSVs under apoptotic conditions. Interestingly, the metastatic squamous cell carcinoma cancer cell line also produced high levels of ACPSVs under healthy condition, suggesting that the ability to generate ACPSVs may be hijacked by these cells. Importantly, ACPSVs were also abundant in the solid tumors of all squamous cell carcinoma cancer patients. Detection of ACPSVs in cancer has potentially important ramifications in tumor biology and cancer therapeutics which warrants further investigation.

Overriding impaired FPR chemotaxis signaling in diabetic neutrophil stimulates infection control in murine diabetic wound.

Infection is a major co-morbidity that contributes to impaired healing in diabetic wounds. Although impairments in diabetic neutrophils have been blamed for this co-morbidity, what causes these impairments and whether they can be overcome, remain largely unclear. Diabetic neutrophils, isolated from diabetic individuals, exhibit chemotaxis impairment but this peculiar functional impairment has been largely ignored because it appears to contradict the clinical findings which blame excessive neutrophil influx as a major impediment to healing in chronic diabetic ulcers. Here, we report that exposure to glucose in diabetic range results in impaired chemotaxis signaling through the formyl peptide receptor (FPR) in neutrophils, culminating in reduced chemotaxis and delayed neutrophil trafficking in the wound of Leprdb (db/db) type two diabetic mice, rendering diabetic wound vulnerable to infection. We further show that at least some auxiliary receptors remain functional under diabetic conditions and their engagement by the pro-inflammatory cytokine CCL3, overrides the requirement for FPR signaling and substantially improves infection control by jumpstarting the neutrophil trafficking toward infection, and stimulates healing in diabetic wound. We posit that CCL3 may have therapeutic potential for the treatment of diabetic foot ulcers if it is applied topically after the surgical debridement process which is intended to reset chronic ulcers into acute fresh wounds.

IL-10 Dysregulation Underlies Chemokine Insufficiency, Delayed Macrophage Response, and Impaired Healing in Diabetic Wounds.

Persistent inflammation is a major contributor to healing impairment in diabetic chronic wounds. Paradoxically, diabetic wound environment during the acute phase of healing is completely different because it exhibits a reduced macrophage response owing to inadequate expression of CCL2 proinflammatory cytokine. What causes a reduction in CCL2 expression in diabetic wounds early after injury remains unknown. In this study, we report that in contrast to prolonged exposure to high glucose, which makes monocytes proinflammatory, short-term exposure to high glucose causes a rapid monocyte reprogramming, manifested by increased expression and secretion of IL-10, which in an autocrine/paracrine fashion reduces glucose uptake and transforms monocytes into an anti-inflammatory phenotype by dampening signaling through toll-like receptors. We show that IL-10 expression is significantly increased in diabetic wounds during the acute phase of healing, causing significant reductions in toll-like receptor signaling and proinflammatory cytokine production, delaying macrophage and leukocyte responses, and underlying healing impairment in diabetic wounds. Importantly, blocking IL-10 signaling during the acute phase of healing improves toll-like receptor signaling, increases proinflammatory cytokine production, enhances macrophage and leukocyte responses, and stimulates healing in diabetic wounds. We posit that anti-IL-10 strategies have therapeutic potential if added topically after surgical debridement, which resets chronic wounds into acute fresh wounds.

Therapeutic evaluation of immunomodulators in reducing surgical wound infection.

Despite many advances in infection control practices, including prophylactic antibiotics, surgical site infections (SSIs) remain a significant cause of morbidity, prolonged hospitalization, and death worldwide. Our innate immune system possesses a multitude of powerful antimicrobial strategies which make it highly effective in combating bacterial, fungal, and viral infections. However, pathogens use various stealth mechanisms to avoid the innate immune system, which in turn buy them time to colonize wounds and damage tissues at surgical sites. We hypothesized that immunomodulators that can jumpstart and activate innate immune responses at surgical sites, would likely reduce infection at surgical sites. We used three immunomodulators; fMLP (formyl-Methionine-Lysine-Proline), CCL3 (MIP-1α), and LPS (Lipopolysaccharide), based on their documented ability to elicit strong inflammatory responses; in a surgical wound infection model with Pseudomonas aeruginosa to evaluate our hypothesis. Our data indicate that one-time topical treatment with these immunomodulators at low doses significantly increased proinflammatory responses in infected and uninfected surgical wounds and were as effective, (or even better), than a potent prophylactic antibiotic (Tobramycin) in reducing P. aeruginosa infection in wounds. Our data further show that immunomodulators did not have adverse effects on tissue repair and wound healing processes. Rather, they enhanced healing in both infected and uninfected wounds. Collectively, our data demonstrate that harnessing the power of the innate immune system by immunomodulators can significantly boost infection control and potentially stimulate healing. We propose that topical treatment with these immunomodulators at the time of surgery may have therapeutic potential in combating SSI, alone or in combination with prophylactic antibiotics.

Effect of Low Versus High-Heeled Footwear on Spinopelvic Alignment at Different Phases of Menstrual Cycle in Young Adult Women: A Biopsychosocial Perspective.

High-heeled shoes adversely affect spinal curvature, increase the risk of low back pain, and disturb the normal gait pattern. The purpose of this study was to examine, from a biopsychosocial point of view, the combined effect of wearing two different heel heights and of hormonal oscillation throughout different phases of the menstrual cycle on spinopelvic alignment. Notably, 70 females with an average age of 20.42 ± 1.51 years participated in this study, wearing each female two different heel heights as follows: low (2.5 cm) and high (7 cm). Spinopelvic alignment was evaluated by rasterstereography formetric 3D analysis during early follicular, ovulatory, and mid-luteal phases of the menstrual cycle. Statistical analysis showed that there was no significant difference (p > 0.05) on spinopelvic alignment [kyphotic angle (KA), trunk inclination (TI), and pelvic inclination] between wearing low- or high-heeled shoes during early follicular, ovulatory, and mid-luteal phases of the menstrual cycle. Considering that high-heeled shoes are traditionally associated with femininity, body image, beauty, and charm, this research has important biopsychosocial implications that should be explored in detail in future studies.

Pseudomonas aeruginosa ExoT induces G1 cell cycle arrest in melanoma cells.

Recently, we demonstrated that Pseudomonas aeruginosa Exotoxin T (ExoT) employs two distinct mechanisms to induce potent apoptotic cytotoxicity in a variety of cancer cell lines. We further demonstrated that it can significantly reduce tumour growth in an animal model for melanoma. During these studies, we observed that melanoma cells that were transfected with ExoT failed to undergo mitosis, regardless of whether they eventually succumbed to ExoT-induced apoptosis or survived in ExoT's presence. In this report, we sought to investigate ExoT's antiproliferative activity in melanoma. We delivered ExoT into B16 melanoma cells by bacteria (to show necessity) and by transfection (to show sufficiency). Our data indicate that ExoT exerts a potent antiproliferative function in melanoma cells. We show that ExoT causes cell cycle arrest in G1 interphase in melanoma cells by dampening the G1/S checkpoint proteins. Our data demonstrate that both domains of ExoT; (the ADP-ribosyltransferase (ADPRT) domain and the GTPase activating protein (GAP) domain); contribute to ExoT-induced G1 cell cycle arrest in melanoma. Finally, we show that the ADPRT-induced G1 cell cycle arrest in melanoma cells likely involves the Crk adaptor protein. Our data reveal a novel virulence function for ExoT and further highlight the therapeutic potential of ExoT against cancer.

A Library Approach to Cationic Amphiphilic Polyproline Helices that Target Intracellular Pathogenic Bacteria.

A number of pathogenic bacteria reproduce inside mammalian cells and are thus inaccessible to many antimicrobial drugs. Herein, we present a facile method to a focused library of antibacterial agents known as cationic amphiphilic polyproline helices (CAPHs). We identified three CAPHs from the library with superior cell penetration within macrophages and excellent antibacterial action against both Gram-positive and Gram-negative bacteria. These cell-penetrating antibacterial CAPHs have specific subcellular localizations that allow for targeting of pathogenic bacteria at their intracellular niches, a unique feature that promotes the successful clearance of intracellular pathogens ( Salmonella, Shigella, and Listeria) residing within macrophages. Furthermore, the selected CAPHs also significantly reduced bacterial infections in an in vivo model of Caenorhabditis elegans, with minimal in vivo toxicity.

Efficacy of Magnesium Sulfate Added to Local Anesthetic in a Transversus Abdominis Plane Block for Analgesia Following Total Abdominal Hysterectomy: A Randomized Trial.

BACKGROUND: Major abdominal surgeries are associated with severe pain, which can affect respiratory and cardiac functions if insufficiently treated; this increases postoperative morbidity. OBJECTIVE: We aim at evaluating the efficacy of magnesium sulfate as an adjuvant to local anesthetic in an ultrasound-guided transversus abdominis plane (TAP) block for postoperative analgesia in total abdominal hysterectomy. STUDY DESIGN: A prospective, randomized, double-blinded clinical trial. SETTING: An academic medical center. METHODS: This study is registered at https://clinicaltrials.gov (no.: NCT02930707). This randomized, double-blinded clinical trial included 60 women undergoing total abdominal hysterectomy that were divided into 2 groups (30 patients per group). Group I received a TAP block with 20 mL per side of 0.25% bupivacaine plus 2 mL magnesium sulphate 10% (200 mg). Group II received a TAP block with 20 mL per side of 0.25% bupivacaine. Visual analog scale (VAS) scores, the time of the first analgesic request, total morphine consumption, and any side effects were assessed and recorded. RESULTS: The mean postoperative VAS score was significantly reduced in group I compared to group II in all of the time-points except after 10 hours. The mean time of the first request for rescue analgesia was significantly prolonged in group I (15.67 hrs.) compared to group II (7.33 hrs.) (P < 0.001), and the mean total morphine consumption, over the first 24 hours postoperatively, was significantly lower in group I (7.63 ± 2.93 mg) than in group II (16.20 ± 3.24 mg) (P < 0.001). No significant difference in side effects was observed. LIMITATIONS: Sample size. CONCLUSION: The addition of 200 mg of magnesium sulfate to bupivacaine in an ultrasound-guided TAP block significantly reduced postoperative opioid requirements, prolonged the duration of analgesia, and reduced the VAS score in patients who underwent abdominal hysterectomy, without significant side effects. KEY WORDS: Magnesium sulfate, TAP block, postoperative pain, total abdominal hysterectomy.

Particle engineering for intracellular delivery of vancomycin to methicillin-resistant Staphylococcus aureus (MRSA)-infected macrophages.

Methicillin-resistant Staphylococcus aureus (MRSA) infection is a serious threat to the public health. MRSA is particularly difficult to treat when it invades host cells and survive inside the cells. Although vancomycin is active against MRSA, it does not effectively kill intracellular MRSA due to the molecular size and polarity that limit its cellular uptake. To overcome poor intracellular delivery of vancomycin, we developed a particle formulation (PpZEV) based on a blend of polymers with distinct functions: (i) poly(lactic-co-glycolic acid) (PLGA, P) serving as the main delivery platform, (ii) polyethylene glycol-PLGA conjugate (PEG-PLGA, p) to help maintain an appropriate level of polarity for timely release of vancomycin, (iii) Eudragit E100 (a copolymer based on dimethylaminoethyl methacrylate, butyl methacrylate and methyl methacrylate, E) to enhance vancomycin encapsulation, and (iv) a chitosan derivative called ZWC (Z) to trigger pH-sensitive drug release. PpZEV NPs were preferentially taken up by the macrophages due to its size (500-1000nm) and facilitated vancomycin delivery to the intracellular pathogens. Accordingly, PpZEV NPs showed better antimicrobial activity than free vancomycin against intracellular MRSA and other intracellular pathogens. When administered intravenously, PpZEV NPs rapidly accumulated in the liver and spleen, the target organs of intracellular infection. Therefore, PpZEV NPs is a promising carrier of vancomycin for the treatment of intracellular MRSA infection.

A short D-enantiomeric antimicrobial peptide with potent immunomodulatory and antibiofilm activity against multidrug-resistant Pseudomonas aeruginosa and Acinetobacter baumannii.

Antimicrobial peptides (AMPs) represent a promising therapeutic alternative for the treatment of antibiotic-resistant bacterial infections. The present study investigates the antimicrobial activity of new, rationally-designed derivatives of a short α-helical peptide, RR. From the peptides designed, RR4 and its D-enantiomer, D-RR4, emerged as the most potent analogues with a more than 32-fold improvement in antimicrobial activity observed against multidrug-resistant strains of Pseudomonas aeruginosa and Acinetobacter baumannii. Remarkably, D-RR4 demonstrated potent activity against colistin-resistant strains of P. aeruginosa (isolated from cystic fibrosis patients) indicating a potential therapeutic advantage of this peptide over several AMPs. In contrast to many natural AMPs, D-RR4 retained its activity under challenging physiological conditions (high salts, serum, and acidic pH). Furthermore, D-RR4 was more capable of disrupting P. aeruginosa and A. baumannii biofilms when compared to conventional antibiotics. Of note, D-RR4 was able to bind to lipopolysaccharide to reduce the endotoxin-induced proinflammatory cytokine response in macrophages. Finally, D-RR4 protected Caenorhabditis elegans from lethal infections of P. aeruginosa and A. baumannii and enhanced the activity of colistin in vivo against colistin-resistant P. aeruginosa.

Targeting biofilms and persisters of ESKAPE pathogens with P14KanS, a kanamycin peptide conjugate.

BACKGROUND: The worldwide emergence of antibiotic resistance represents a serious medical threat. The ability of these resistant pathogens to form biofilms that are highly tolerant to antibiotics further aggravates the situation and leads to recurring infections. Thus, new therapeutic approaches that adopt novel mechanisms of action are urgently needed. To address this significant problem, we conjugated the antibiotic kanamycin with a novel antimicrobial peptide (P14LRR) to develop a kanamycin peptide conjugate (P14KanS). METHODS: Antibacterial activities were evaluated in vitro and in vivo using a Caenorhabditis elegans model. Additionally, the mechanism of action, antibiofilm activity and anti-inflammatory effect of P14KanS were investigated. RESULTS: P14KanS exhibited potent antimicrobial activity against ESKAPE pathogens. P14KanS demonstrated a ≥128-fold improvement in MIC relative to kanamycin against kanamycin-resistant strains. Mechanistic studies confirmed that P14KanS exerts its antibacterial effect by selectively disrupting the bacterial cell membrane. Unlike many antibiotics, P14KanS demonstrated rapid bactericidal activity against stationary phases of both Gram-positive and Gram-negative pathogens. Moreover, P14KanS was superior in disrupting adherent bacterial biofilms and in killing intracellular pathogens as compared to conventional antibiotics. Furthermore, P14KanS demonstrated potent anti-inflammatory activity via the suppression of LPS-induced proinflammatory cytokines. Finally, P14KanS protected C. elegans from lethal infections of both Gram-positive and Gram-negative pathogens. CONCLUSIONS: The potent in vitro and in vivo activity of P14KanS warrants further investigation as a potential therapeutic agent for bacterial infections. GENERAL SIGNIFICANCE: This study demonstrates that equipping kanamycin with an antimicrobial peptide is a promising method to tackle bacterial biofilms and address bacterial resistance to aminoglycosides.

Dual Targeting of Intracellular Pathogenic Bacteria with a Cleavable Conjugate of Kanamycin and an Antibacterial Cell-Penetrating Peptide.

Bacterial infection caused by intracellular pathogens, such as Mycobacterium, Salmonella, and Brucella, is a burgeoning global health epidemic that necessitates urgent action. However, the therapeutic value of a number of antibiotics, including aminoglycosides, against intracellular pathogenic bacteria is compromised due to their inability to traverse eukaryotic membranes. For this significant problem to be addressed, a cleavable conjugate of the antibiotic kanamycin and a nonmembrane lytic, broad-spectrum antimicrobial peptide with efficient mammalian cell penetration, P14LRR, was prepared. This approach allows kanamycin to enter mammalian cells as a conjugate linked via a tether that breaks down in the reducing environment within cells. Potent antimicrobial activity of the P14KanS conjugate was demonstrated in vitro, and this reducible conjugate effectively cleared intracellular pathogenic bacteria within macrophages more potently than that of a conjugate lacking the disulfide moiety. Notably, successful clearance of Mycobacterium tuberculosis within macrophages was observed with the dual antibiotic conjugate, and Salmonella levels were significantly reduced in an in vivo Caenorhabditis elegans model.

Evaluation of short synthetic antimicrobial peptides for treatment of drug-resistant and intracellular Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) infections present a serious challenge because of the emergence of resistance to numerous conventional antibiotics. Due to their unique mode of action, antimicrobial peptides are novel alternatives to traditional antibiotics for tackling the issue of bacterial multidrug resistance. Herein, we investigated the antibacterial activity of two short novel peptides (WR12, a 12 residue peptide composed exclusively of arginine and tryptophan, and D-IK8, an eight residue ß-sheet peptide) against multidrug resistant staphylococci. In vitro, both peptides exhibited good antibacterial activity against MRSA, vancomycin-resistant S. aureus, linezolid-resistant S. aureus, and methicillin-resistant S. epidermidis. WR12 and D-IK8 were able to eradicate persisters, MRSA in stationary growth phase, and showed significant clearance of intracellular MRSA in comparison to both vancomycin and linezolid. In vivo, topical WR12 and D-IK8 significantly reduced both the bacterial load and the levels of the pro-inflammatory cytokines including tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in MRSA-infected skin lesions. Moreover, both peptides disrupted established in vitro biofilms of S. aureus and S. epidermidis significantly more so than traditional antimicrobials tested. Taken together, these results support the potential of WR12 and D-IK8 to be used as a topical antimicrobial agent for the treatment of staphylococcal skin infections.

Superior hypogastric plexus combined with ganglion impar neurolytic blocks for pelvic and/or perineal cancer pain relief.

BACKGROUND: The superior hypogastric plexus (SHGP) carries afferents from the viscera of the lower abdomen and pelvis. Neurolytic block of this plexus is used for reducing pain resulting from malignancy in these organs. The ganglion impar (GI) innervats the perineum, distal rectum, anus, distal urethra, vulva, and distal third of the vagina. Different approaches to the ganglion impar neurolysis have been described in the literature. OBJECTIVES: To assess the feasibility, safety, and efficacy of combining the block of the SHGP through the postero-median transdiscal approach with the GI block by the trans-sacro-coccygeal approach for relief of pelvic and/or perineal pain caused by pelvic and/or perineal malignancies or any cancer related causes. METHODS: Fifteen patients who had cancer-related pelvic pain, perineal pain, or both received a combined SHGP neurolytic block through the postero-median transdiscal approach using a 20-gauge Chiba needle and injection of 10 mL of 10% phenol in saline plus a GI neurolytic block by the trans-sacro-coccygeal approach using a 22-gauge 5 cm needle and injection of 4 - 6 mL of 8% phenol in saline. Pain intensity (measured using a visual analogue scale) and oral morphine consumption pre- and post-procedure were measured. RESULTS: All patients presented with cancer-related pelvic, perineal, or pelviperineal pain. Pain scores were reduced from a mean (± SD) of 7.87 ± 1.19 pre-procedurally to 2.40 ± 2.10 one week post-procedurally (P < 0.05). In addition, the mean consumption of morphine (delivered via 30 mg sustained-release morphine tablets) was reduced from 98.00 ± 34.89 mg to 32.00 ± 28.48 mg after one week (P < 0.05). No complications or serious side effects were encountered during or after the block. LIMITATIONS: This study is limited by its small sample size and non-randomized study. CONCLUSION: A combined neurolytic SHGP block with GI block is an effective and safe technique for reducing pain in cancer patients presented with pelvic and/or perineal pain. Also, a combined SHGP block through a posteromedian transdiscal approach with a GI block through a trans-sacrococcygeal approach may be considered more effective and easier to perform than the recently invented bilateral inferior hypogastric plexus neurolysis through a transsacral approach.

Efficacy of short novel antimicrobial and anti-inflammatory peptides in a mouse model of methicillin-resistant Staphylococcus aureus (MRSA) skin infection.

The therapeutic efficacy of two novel short antimicrobial and anti-inflammatory peptides (RR and RRIKA) was evaluated in a mouse model of staphylococcal skin infection. RR (2%) and RRIKA (2%) significantly reduced the bacterial counts and the levels of proinflammatory cytokines, tumor necrosis factor (TNF)-α, and interleukin (IL)-6, in methicillin-resistant Staphylococcus aureus USA 300-0114 skin lesions. Furthermore, the combined therapy of RRIKA (1%) and lysostaphin (0.5%) had significantly higher antistaphylococcal and anti-inflammatory activity compared to monotherapy. This study supports the potential use of these peptides for topical treatment of methicillin-resistant Staphylococcus aureus skin infections.

Targeting methicillin-resistant Staphylococcus aureus with short salt-resistant synthetic peptides.

The seriousness of microbial resistance combined with the lack of new antimicrobials has increased interest in the development of antimicrobial peptides (AMPs) as novel therapeutics. In this study, we evaluated the antimicrobial activities of two short synthetic peptides, namely, RRIKA and RR. These peptides exhibited potent antimicrobial activity against Staphylococcus aureus, and their antimicrobial effects were significantly enhanced by addition of three amino acids in the C terminus, which consequently increased the amphipathicity, hydrophobicity, and net charge. Moreover, RRIKA and RR demonstrated a significant and rapid bactericidal effect against clinical and drug-resistant Staphylococcus isolates, including methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-intermediate S. aureus (VISA), vancomycin-resistant S. aureus (VRSA), linezolid-resistant S. aureus, and methicillin-resistant Staphylococcus epidermidis. In contrast to many natural AMPs, RRIKA and RR retained their activity in the presence of physiological concentrations of NaCl and MgCl2. Both RRIKA and RR enhanced the killing of lysostaphin more than 1,000-fold and eradicated MRSA and VRSA isolates within 20 min. Furthermore, the peptides presented were superior in reducing adherent biofilms of S. aureus and S. epidermidis compared to results with conventional antibiotics. Our findings indicate that the staphylocidal effects of our peptides were through permeabilization of the bacterial membrane, leading to leakage of cytoplasmic contents and cell death. Furthermore, peptides were not toxic to HeLa cells at 4- to 8-fold their antimicrobial concentrations. The potent and salt-insensitive antimicrobial activities of these peptides present an attractive therapeutic candidate for treatment of multidrug-resistant S. aureus infections.

Antibacterial activity of novel cationic peptides against clinical isolates of multi-drug resistant Staphylococcus pseudintermedius from infected dogs.

Staphylococcus pseudintermedius is a major cause of skin and soft tissue infections in companion animals and has zoonotic potential. Additionally, methicillin-resistant S. pseudintermedius (MRSP) has emerged with resistance to virtually all classes of antimicrobials. Thus, novel treatment options with new modes of action are required. Here, we investigated the antimicrobial activity of six synthetic short peptides against clinical isolates of methicillin-susceptible and MRSP isolated from infected dogs. All six peptides demonstrated potent anti-staphylococcal activity regardless of existing resistance phenotype. The most effective peptides were RRIKA (with modified C terminus to increase amphipathicity and hydrophobicity) and WR-12 (α-helical peptide consisting exclusively of arginine and tryptophan) with minimum inhibitory concentration50 (MIC50) of 1 µM and MIC90 of 2 µM. RR (short anti-inflammatory peptide) and IK8 "D isoform" demonstrated good antimicrobial activity with MIC50 of 4 µM and MIC90 of 8 µM. Penetratin and (KFF)3K (two cell penetrating peptides) were the least effective with MIC50 of 8 µM and MIC90 of 16 µM. Killing kinetics revealed a major advantage of peptides over conventional antibiotics, demonstrating potent bactericidal activity within minutes. Studies with propidium iodide and transmission electron microscopy revealed that peptides damaged the bacterial membrane leading to leakage of cytoplasmic contents and consequently, cell death. A potent synergistic increase in the antibacterial effect of the cell penetrating peptide (KFF)3K was noticed when combined with other peptides and with antibiotics. In addition, all peptides displayed synergistic interactions when combined together. Furthermore, peptides demonstrated good therapeutic indices with minimal toxicity toward mammalian cells. Resistance to peptides did not evolve after 10 passages of S. pseudintermedius at sub-inhibitory concentration. However, the MICs of amikacin and ciprofloxacin increased 32 and 8 fold, respectively; under similar conditions. Taken together, these results support designing of peptide-based therapeutics for combating MRSP infections, particularly for topical application.

Diabetic heel ulcer in the Sudan: determinants of outcome.

Heel ulceration, on average, costs 1.5 times more than metatarsal ulceration. The aim of this study was to analyze the determinant factors of healing in diabetic patients with heel ulcers and the late outcomes at Jabir Abu Eliz Diabetic Centre Khartoum, Khartoum, Sudan. Data were collected prospectively for 96 of 100 diabetic patients presenting with heel ulcers at the Jabir Abu Eliz Diabetic Centre Khartoum from May 2003 to January 2005. Late outcome was assessed 3 years later (February 2008). Heeling was achieved in one half of the patients (n = 48). In the remaining 48 patients, 22 ended with major lower extremity amputation and 22 were still receiving wound care. A total of 8 patients died, 4 in each group, the healed and unhealed. The most significant determinants of healing using a logistic multivariate regression model, 95% confidence intervals, and odds ratios included a shorter duration of diabetes (p < .009), adequate lower limb perfusion (p < .043), and a superficial foot ulcer (p < .012). Three years later, of the 88 patients who could be traced, 78 were alive and 59 had healed ulcers (7 had died of unrelated causes and 3 of diabetic-related complications), and no additional lower extremity amputation was recorded. Mortality in the series was 18 patients, of whom 14 had undergone a previous lower extremity amputation. Superficial heel ulcers in diabetic patients with a short history of diabetes and with good limb circulation are more likely to heal within an average duration of 25 weeks. At 3 years of follow-up, 75% showed a favorable outcome for ulcer healing, and 22 patients underwent lower extremity amputation (25%), of whom 14 were dead within 3 years.