Pseudomonas aeruginosa uses T3SS to inhibit diabetic wound healing

Diabetic foot ulcers are responsible for more hospitalizations than any other complication of diabetes. Bacterial infection is recognized as an important factor associated with impaired healing in diabetic ulcers. Pseudomonas aeruginosa is the most frequently detected Gram‐negative pathogen in diabetic ulcers. P. aeruginosa infection has been shown to impair healing in diabetic wounds in a manner that correlates with its ability to form biofilm. While the majority of infections in diabetic ulcers are biofilm associated, 33% of infections are nonbiofilm in nature. P. aeruginosa is the most prevalent Gram‐negative pathogen in all diabetic wound types, which suggests that the deleterious impact of P. aeruginosa on healing in diabetic wounds goes beyond its ability to form biofilm and likely involves other factors. The Type III Secretion System (T3SS) virulence structure is required for the pathogenesis of all P. aeruginosa clinical isolates, suggesting that it may also play a role in the inhibition of wound repair in diabetic skin ulcers. We evaluated the role of T3SS in mediating P. aeruginosa–induced tissue damage in the wounds of diabetic mice. Our data demonstrate that P. aeruginosa establishes a robust and persistent infection in diabetic wounds independent of its ability to form biofilm and causes severe wound damage in a manner that primarily depends on its T3SS.     

Evaluation of clinical isolates of Pseudomonas aeruginosa for ribosomal resistance to tobramycin

The suggested use of tobramycin in selective decontamination of the digestive tract led to the investigation of the part played clinically by ribosomal resistance in Pseudomonas aeruginosa. Examination of 32 tobramycin-resistant isolates, both enzyme and non-enzyme producing, suggests that ribosomal resistance is clinically important.     

Endovascular photodynamic therapy using mono-L-aspartyl-chlorin e6 to inhibit Intimal hyperplasia in balloon-injured rabbit arteries

BACKGROUND AND OBJECTIVE: Intimal hyperplasia (IH) leading to restenosis is a major complication of arterial revascularization. The purpose of this study was to investigate the effect of photodynamic therapy (PDT) using mono-L-aspartyl chlorin e6 (NPe6) as a photosensitizer and intraluminal radial irradiation for inhibition of IH experimentally. STUDY DESIGN/MATERIALS AND METHODS: Study of laser transmission through the blood indicated that exclusion of blood is a prerequisite for intraluminal PDT. For homogeneous radial laser irradiation to the vessel wall, we used a newly developed cylindrical diffusing balloon laser fiber. Injuries were induced by pulling a balloon catheter through the right iliac artery of rabbits. One and 6 hours after the NPe6 injection (5mg/kg i.v.), drug distribution was examined by fluorescence microscopy. Nineteen rabbits received NPe6 at the time of injuries and PDT was performed with 664-nm laser at 30 and 10 J/cm(2) (20, 30, 40 mW/cm(2)) 1 hour after the injuries. The arteries were harvested at 2 days. In a second group of rabbits, PDT was given at 30 mW/cm(2) (30 J/cm(2)). Two weeks after treatment, the arteries were removed and examined histologically. RESULTS: NPe6 was found to be distributed selectively in the injured media. Endovascular NPe6-PDT showed complete depletion of smooth muscle cells even with 10 J/cm(2) at 2 days. IH was significantly inhibited at 14 days after PDT. CONCLUSIONS: Endovascular PDT of injured artery using NPe6 can prevent IH in this model of arterial wall injury and may become clinically useful for the prophylaxis of IH.     

Characterization of antibiotic resistance profiles in Pseudomonas aeruginosa isolates from burn patients

ObjectiveTo investigate the prevalence of multidrug-resistant (MDR) Pseudomonas aeruginosa (PA) producing extended-spectrum beta-lactamases (ESBLs) and metallo-beta-lactamases (MBLs) in burn patients in Algeria.MethodsBetween April 2016 and October 2019, 47 non-redundant isolates of PA were collected from 47 burn patients admitted to the Department of Burns at the Military Hospital of Algiers in Algeria. Antibiotic susceptibility testing was performed by agar diffusion and the Phoenix automated method. Resistance genes were identified by PCR, and molecular typing of isolates was carried out by enterobacterial repetitive intergenic consensus (ERIC) sequences-polymerase chain reaction (PCR).ResultsAmong the 47 non-redundant MDR PA strains isolated, 59.57% were phenotypically ESBLs-positive, and 100% were phenotypically MBL-positive. The ESBL-positive isolates were subsequently screened for six groups of bla genes encoding ESBL-type enzymes, namely blaCTX-M2, blaPER, blaTEM, blaSHV, blaVEB, and blaGES. Out of the 28 ESBL-producing strains, 23 (82.14%) were blaCTX-M2 positive; 18 (38.29%) were blaPER positive, and 16 (34.04%) were blaTEM positive, while 5 (17.9%) were co-harboring blaCTX-M2, blaTEM, and blaPER genes. The blaSHV, blaVEB, and blaGES genes were not detected in any of the ESBL positive isolates. Since all isolates were MBL-positive, all 47 strains were screened for the blaNDM-1, blaIMP, blaVIM genes that produce MBLs; however, none of these genes were detected. Additional screening for the oprD gene demonstrated that 45 (95.74%) of the isolates were positive for this gene. Finally, ERIC PCR revealed 11 distinct PA clones among the blaCTX-M2 positive strains.ConclusionThis is the first study to report the presence of CTX-M2-producing PA in the North Africa region and the first to detect blaCTX-M2-positive and blaPER-positive PA clinical isolates in Algeria, therefore demonstrating the spread of such MDR strains to this part of the world. Identification of bacterial genotypic alterations that confer antibiotic resistance is critical in determining the most effective antimicrobial strategies to be employed. Therefore, our findings could potentially facilitate clinical decision making regarding the antibiotics of choice for the treatment of burn patients that suffer from PA infections in Algeria.     

Effect of methylene blue-mediated antimicrobial photodynamic therapy on dentin caries microcosms

Antimicrobial photodynamic therapy (aPDT) has been proposed as an adjuvant treatment of dental caries, although there are no well-defined protocols to its clinical application. This study aimed to evaluate the influence of aPDT on the viability of microorganisms, vitality of biofilms, and lactic acid production of dentin caries microcosms. Biofilms were grown on bovine dentin discs in anaerobic conditions at 37 °C for 5 days, inoculating infected carious dentin in modified McBain medium plus 1% sucrose. The biofilms were treated by the combination of deionized water or 100 mg L^?1 methylene blue (MB) with 0, 37.5, or 75 J cm^?2 LED at 630 nm. The counts of total microorganisms, total streptococci, mutans streptococci, and total lactobacilli were determined by colony-forming units (CFU). The vitality of microbial cells in intact biofilms was measured by confocal laser scanning microscope (CLSM). The lactic acid production was analyzed by enzymatic spectrophotometry at 340 nm. Statistical analysis was conducted by Kruskal-Wallis and post hoc Dunn’s tests (P < 0.05). MB and 37.5 J cm^?2 LED alone did not interfere in the viability of microorganisms, unlike 75 J cm^?2 LED alone that decreased the total microorganism and lactobacillus counts. The combination of MB and 75 J cm^?2 LED reduced the viability of all microorganisms and the vitality of intact biofilms. The production of lactic acid was statistically lower in all treatment groups in comparison with that of the control (no treatment), except for MB alone. Therefore, the MB-mediated aPDT was effective in controlling the viability, vitality and the acidogenicity of dentin caries microcosms.     

The impact of antimicrobial photodynamic therapy in an artificial biofilm model

The susceptibility of bacterial cultures in biofilm formations is important for a variety of clinical treatment procedures. Therefore, the aim of the study was to assess the impact of laser-induced antimicrobial photodynamic therapy on the viability of Streptococcus mutans cells employing an artificial biofilm model. Using sterile chambered coverglasses, a salivary pellicle layer was formed in 40 chambers. Streptococcus mutans cells were inoculated in a sterile culture medium. Employing a live/dead bacterial viability kit, bacteria with intact cell membranes stained fluorescent green. Each pellicle-coated test chamber was filled with 0.7 ml of the bacterial suspension and analysed using a confocal laser scanning microscope within a layer of 10 μm at intervals of 1 μm from the pellicle layer. Phenothiazine chloride was used as a photosensitizer in all 40 test chambers. A diode laser (wavelength 660 nm, output power 100 mW) was used to irradiated 20 chambers for 2 min. Fluorescence values in the test chambers after laser irradiation (median 2.1 U, range 0.4–3.4 U) were significantly lower than baseline values after adding the photosensitizer (median 3.6 U, range 1.1–9.0; p?p?>?0.05). The present study indicated that laser irradiation is an essential part of antimicrobial photodynamic therapy to reduce bacteria within a layer of 10 μm. Further studies are needed to evaluate the maximum biofilm thickness that still allows a toxic effect on microorganisms.     

Comparison of the efficacy of Rose Bengal and erythrosin in photodynamic therapy against Enterobacteriaceae

The aim of this study was to evaluate the effects of the photosensitizers Rose Bengal and erythrosin combined with a light-emitting diode (LED) on Enterobacteriaceae. Twelve Enterobacteriaceae strains isolated from the oral cavities of patients undergoing prolonged antibiotic therapy, including three Escherichia coli, three Enterobacter cloacae, three Klebsiella oxytoca and three Klebsiella pneumoniae, were studied. An Enterobacteriaceae suspension (10⁶ cells/ml) was prepared from each clinical strain isolated from the human oral cavity and subjected to the following treatments: LED and Rose Bengal, LED and erythrosin, LED and physiological solution, and physiological solution only as control. A blue LED unit (460 nm), and Rose Bengal and erythrosin at a concentration of 50 μmol/l were used. After incubation at 37°C for 48 h, the number of colony-forming units (CFU) was calculated and subjected to analysis of variance (ANOVA). The Enterobacterial strains were sensitive to photodynamic therapy with Rose Bengal. There was a reduction of approximately 7.14 log10 for Enterobacter cloacae, 7.73 log10 for Escherichia coli, 6.76 log10 for Klebsiella pneumoniae and 7.21 log10 for Klebsiella oxytoca. However, photodynamic therapy using erythrosin did not reduce the numbers of CFUs per milliliter compared to the control group. The use of the LED alone had no toxic effect on the strain tested. The Enterobacteriaceae strains studied were sensitive to photodynamic therapy with Rose Bengal.     

Influence of pre-irradiation time employed in antimicrobial photodynamic therapy with diode laser

The aim of the present study was to evaluate, in vitro, the effect of different pre-irradiation times of the photosensitizer in photodynamic therapy in biofilms formed by Streptococcus mutans and Candida albicans, through the evaluation of the microbial load. The factors under study were as follows: times of pre-irradiation of the photosensitizer in three levels (1, 2, or 5 min). For the control of the cariogenic dental biofilm with antimicrobial photodynamic therapy (aPDT), methylene blue (0.01%) was used in association with the diode laser (InGaAlP) with a wavelength of 660 nm. Chlorhexidine digluconate (0.12% CHX) and saline were used as positive and negative controls, respectively. The study design was carried out in complete and randomized blocks. The sample consisted of 15 S. mutans biofilms cultures, randomly divided into five groups and 15 C. albicans cultures, also divided into five groups. The experiment was performed in triplicate (n = 3) and the response variables were obtained through quantitative analysis of bacterial viability, expressed in colony-forming units (CFU) per square millimeter of the specimen area. The data were analyzed with the aid of the ANOVA one-way test and Tukey’s post-test. All analyses were performed using the Graph Pad Prism 4.0 program, with a significance level of 5%. For the S. mutans group, only the saline solution presented a statistically significant difference when compared to the other treatments (p < 0.05), that is, the treatment with aPDT, irrespective of the irradiation time applied, was similar to the treatment with CHX and both were more effective in reducing cariogenic biofilm compared to saline. For the group of C. albicans, there was no statistical difference between the groups (p > 0.05). Therefore, it can be concluded that the treatment with aPDT reduced the number of CFUs of S. mutans in a similar way to CHX, independently of the pre-irradiation time applied. No effect of this therapy or of the different pre-irradiation times on the C. albicans biofilm could be observed. In this way, the pre-irradiation time of 1 min can be used to reduce the microbial load of S. mutans.     

Fosfomycin therapy for multiresistant Pseudomonas aeruginosa in cystic fibrosis.

BACKGROUND: Increasing resistance to standard antibiotics has been demonstrated in CF patients colonised by Pseudomonas aeruginosa. The antibiotic Fosfomycin has a unique mode of action against this organism, and may protect against aminoglycoside mediated renal and ototoxic effects. However, there is little published experience of this drug in IV form, and it is not licensed for use in the UK. METHODS: In combination with other antibiotics, we used Fosfomycin to treat 30 pulmonary exacerbations in 15 adult CF patients colonised by P. aeruginosa, mainly multiresistant strains. All patients gave informed consent. We cultured sputum prior to treatment and measured spirometry, renal function, and symptoms before and after treatment, and recorded any side effects. RESULTS: One patient developed nausea and Fosfomycin treatment was withdrawn. The remaining patients showed clinical resolution of their chest exacerbations (mean FEV1% predicted: pre 41.1 vs. post 49.4, P<0.001). Although there was a statistical increase in plasma urea (pre 3.9 mmol/l vs. post 4.3, P<0.03), this was still within the normal range. Plasma creatinine was unchanged. CONCLUSIONS: This study shows that IV Fosfomycin is well tolerated by adult patients with CF and can be useful in the treatment of those colonised with multiresistant P. aeruginosa.     

Treatment of experimental periodontitis in rats using repeated adjunctive antimicrobial photodynamic therapy

The aim of this study was to histologically and histometrically evaluate the influence of repeated adjunctive antimicrobial photodynamic therapy (aPDT) on bone loss (BL) in furcation areas in rats. Periodontitis was induced by placing a ligature around the mandibular molar in 75 rats. The animals were divided into five groups: the SS group was treated with saline solution (SS); the SRP group received scaling and root planing (SRP); the aPDT1 group received SRP as well as toluidine blue (TBO) and low-level laser therapy (LLLT; InGaAlP, 660 nm; 4.94 J/cm²/point) postoperatively at 0 h; the aPDT2 group received SRP as well as TBO and LLLT postoperatively at 0, 24, 28, and 72 h; and the aPDT3 group received SRP, TBO, and LLLT postoperatively at 0, 48, 96, and 144 h. The area of BL in the furcation region of the molar was histometrically analyzed. Data were analyzed statistically (P?<?0.05). Animals treated with a single episode of aPDT showed less BL at days 7 and 30 than those who received only SRP treatment. No significant differences were found among the aPDT groups (P?>?0.05). Repeated aPDT did not improve BL reduction when compared to a single episode of aPDT.     

Effect of antimicrobial photodynamic therapy on periodontally infected tooth sockets in rats

Lasers in Medical Science - The aim of the present study was to evaluate the antimicrobial effect of antimicrobial photodynamic therapy (aPDT) in alveolar treatment of areas with induced...     

Cell-mediated immune responses to Pseudomonas aeruginosa.

Of six healthy laboratory workers with no history of illness caused by Pseudomonas aeruginosa, four exhibited positive skin responses at 24 hours, strong inhibition of leukocyte migration, and marked stimulation of blastogenesis of peripheral lymphocytes when exposed to a "cocktail" of three phenol-killed strains of P aeruginosa. There was no correlation between the extent of these responses with serum hemagglutinin titers. It is postulated that cell-mediated immunity against P aeruginosa may have a biologic role in host defense.     

Hyperbaric oxygen treatment increases killing of aggregating Pseudomonas aeruginosa isolates from cystic fibrosis patients

Background: Pseudomonas aeruginosa is a major pathogen of the chronic lung infections in cystic fibrosis (CF) patients. These persistent bacterial infections are characterized by bacterial aggregates with biofilm-like properties and are treated with nebulized or intravenous tobramycin in combination with other antibiotics. However, the chronic infections are close to impossible to eradicate due to reasons that are far from fully understood. Recent work has shown that re‑oxygenation of hypoxic aggregates by hyperbaric oxygen (O₂) treatment (HBOT: 100% O₂ at 2.8 bar) will increase killing of aggregating bacteria by antibiotics. This is relevant for treatment of infected CF patients where bacterial aggregates are found in the endobronchial secretions that are depleted of O₂ by the metabolism of polymorphonuclear leukocytes (PMNs). The main objective of this study was to investigate the effect of HBOT as an adjuvant to tobramycin treatment of aggregates formed by P. aeruginosa isolates from CF patients.Methods: The effect was tested using a model with bacterial aggregates embedded in agarose. O₂ profiling was used to confirm re‑oxygenation of aggregates.Results: We found that HBOT was able to significantly enhance the effect of tobramycin against aggregates of all the P. aeruginosa isolates in vitro. The effect was attributed to increased O₂ levels leading to increased growth and thus increased uptake of and killing by tobramycin.Conclusions: Re‑oxygenation may in the future be a clinical possibility as adjuvant to enhance killing by antibiotics in cystic fibrosis lung infections.