Bacteriophage therapy as an alternative technique for treatment of multidrug-resistant bacteria causing diabetic foot infection / La terapia con bacteriófagos como técnica alternativa para el tratamiento de bacterias multirresistentes causantes de la infección del pie diabético

Diabetic foot ulcer (DFU) represented the most feared diabetic complication that caused the hospitalization of the diabetic patient. DFU was usually characterized with delayed healing as the diabetic neuropathy, angiopathy, and ulcer concomitant infections, among them, are multidrug-resistant (MDR) bacteria that emphasized the clinical importance for developing new therapeutic strategy with safe and effective alternatives for the antibiotics to overcome DFU-MDR bacterial infection. Bacteriophage therapy was considered a novel approach to eradicate the MDR, but its role in the polymicrobial infection of the DFU remains elusive. Thus, the current work was designed to investigate the effect of the topical application of the phage cocktail on the healing of the diabetic wound infected with clinical isolates of Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella variicola, Escherichia coli, and Proteus mirabilis. Bacterial isolation was performed from clinical hospitalized and non-hospitalized cases of DFU, identified morphologically, biochemically, molecularly via 16 s rRNA sequencing, and typed for the antibiotic resistance pattern. Moreover, phages were isolated from the aforementioned clinical isolates and identified with electron microscope. Forty-five adult male Sprague–Dawley rats were assigned in 3 groups (15 rats each), namely, the diabetic infected wound group, diabetic infected wound ceftriaxone-treated group, and the diabetic infected wound phage cocktail-treated group. The results revealed that phage cocktail had a superior effect over the ceftriaxone in wound healing parameters (wound size, wound index, wound bacterial load, and mRNA expression); wound healing markers (Cola1a, Fn1, MMP9, PCNA, and TGF-β); inflammatory markers (TNF-α, NF-κβ, IL-1β, IL-8, and MCP-1); anti-inflammatory markers (IL-10 and IL-4); and diabetic wound collagen deposition; and also the histomorphic picture of the diabetic infected wound. Based on the current findings...(AU)

Bacteriophage therapy as an alternative technique for treatment of multidrug-resistant bacteria causing diabetic foot infection.

Diabetic foot ulcer (DFU) represented the most feared diabetic complication that caused the hospitalization of the diabetic patient. DFU was usually characterized with delayed healing as the diabetic neuropathy, angiopathy, and ulcer concomitant infections, among them, are multidrug-resistant (MDR) bacteria that emphasized the clinical importance for developing new therapeutic strategy with safe and effective alternatives for the antibiotics to overcome DFU-MDR bacterial infection. Bacteriophage therapy was considered a novel approach to eradicate the MDR, but its role in the polymicrobial infection of the DFU remains elusive. Thus, the current work was designed to investigate the effect of the topical application of the phage cocktail on the healing of the diabetic wound infected with clinical isolates of Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella variicola, Escherichia coli, and Proteus mirabilis. Bacterial isolation was performed from clinical hospitalized and non-hospitalized cases of DFU, identified morphologically, biochemically, molecularly via 16 s rRNA sequencing, and typed for the antibiotic resistance pattern. Moreover, phages were isolated from the aforementioned clinical isolates and identified with electron microscope. Forty-five adult male Sprague-Dawley rats were assigned in 3 groups (15 rats each), namely, the diabetic infected wound group, diabetic infected wound ceftriaxone-treated group, and the diabetic infected wound phage cocktail-treated group. The results revealed that phage cocktail had a superior effect over the ceftriaxone in wound healing parameters (wound size, wound index, wound bacterial load, and mRNA expression); wound healing markers (Cola1a, Fn1, MMP9, PCNA, and TGF-ß); inflammatory markers (TNF-α, NF-κß, IL-1ß, IL-8, and MCP-1); anti-inflammatory markers (IL-10 and IL-4); and diabetic wound collagen deposition; and also the histomorphic picture of the diabetic infected wound. Based on the current findings, it could be speculated that phage therapy could be considered a novel antibiotic substitute in the DFU with MDR-polymicrobial infection therapeutic strategies.

Microbial chitinases: properties, enhancement and potential applications.

Chitinases are a category of hydrolytic enzymes that catalyze chitin and are formed by a wide variety of microorganisms. In nature, microbial chitinases are primarily responsible for chitin decomposition and play a vital role in the balance of carbon and nitrogen ratio in the ecosystem. The physicochemical attributes and the source of chitinase are the main bases that determine their functional characteristics and hydrolyzed products. Several chitinases have been reported and characterized, and they obtain a wider consideration for their utilization in a large number of uses such as in agriculture, food, environment, medicine and pharmaceutical companies. The antifungal and insecticidal impacts of several chitinases have been extensively studied, aiming to protect crops from phytopathogenic fungi and insects. Chitooligosaccharides synthesized by chitin degradation have been shown to improve human health through their antimicrobial, antioxidant, anti-inflammatory and antitumor properties. This review aims at investigating chitinase production, properties and their potential applications in various biotechnological fields.

Human gut microbiota/microbiome in health and diseases: a review.

The human gut microbiota has received considerable interest in the recent years and our knowledge of the inhabitant species and their potential applications is increased particularly after the development of metagenomic studies. Gut microbiota is highly diverse and harboring trillions of microorganisms in human digestive system. The shaping and multiplication of gut microbiome starts at birth, while the modification of their composition depends mainly on various genetic, nutritional and environmental factors. The modification in the composition and function of the gut microbiota can change intestinal permeability, digestion and metabolism as well as immune responses. The pro inflammatory state caused by alternation of gut microbiota balance lead to the onset of many diseases ranging from gastrointestinal and metabolic conditions to immunological and neuropsychiatric diseases. In this context, the present review clarifies the role of gut microbiota in maintaining host health and investigates how nutritional and environmental factors affect the gut microbial structure and function. In addition, many therapeutic strategies of gut microbiota aimed at modulating and restoring of the intestinal ecosystem balance have been surveyed.

Detoxification of Aflatoxin B1 by Antifungal Compounds from Lactobacillus brevis and Lactobacillus paracasei, Isolated from Dairy Products.

Aflatoxins are a large group of highly toxic, mutagenic, and carcinogenic mycotoxins produced by specific species of fungi. Potential contamination of food commodities by these compounds causes extensive damage that lead to great economic losses. This study explored the potential use of antifungal compounds, produced by Lactobacillus brevis and Lactobacillus paracasei, for growth inhibition and subsequent aflatoxin B1 production from select strains of Aspergillus flavus and Aspergillus parasiticus. Lactobacilli strains were isolated from traditional Egyptian dairy products, whereas fungal strains were isolated from infected cereal seeds. There were noticeable decreases in mycelium biomass and aflatoxin production as well. L. brevis exhibited the highest reduction of aflatoxin B1 production by A. flavus and A. parasiticus, 96.31 and 90.43%, respectively. The concentrations of amino acids of the antifungal compound produced by L. brevis were significantly higher than that produced by L. paracasei. Asparagine, glutamine, glycine, alanine, and leucine were the most concentrated amino acids for both strains. The antifungal compounds produced by L. brevis and L. paracasei were active in a wide range of pH, heat stable and inactivated by proteolytic enzymes (protease K and trypsin A). The expression of Omt-A gene that involved in the later step of aflatoxin production was evaluated by real-time PCR. There was a vigorous reduction at transcriptional level of Omt-A gene observed in A. flavus that is treated by L. brevis and L. paracasei (80 and 70%, respectively). However, the reduction of Omt-A gene observed in A. parasiticus that is treated by L. brevis and L. paracasei was 64.5 and 52%, respectively. Treating maize seeds with antifungal compounds exhibited great efficiency in controlling fungal infection and increasing seed germination. The results confirmed that lactic acid bacteria are a promising strategy to control food contamination of fermented food and dairy products.

Silver nanoparticles as an antimicrobial agent: A case study on Staphylococcus aureus and Escherichia coli as models for Gram-positive and Gram-negative bacteria.

The current research was focused on the characterization and antimicrobial activity of silver nanoparticles (AgNPs) produced by Bacillus licheniformis NM120-17. The synthesis was initially observed by a colour change from pale yellow to brown which was further confirmed by UV-Vis spectroscopy. The AgNPs were characterized using TEM, EDAX and FTIR. The synthesized nanoparticles were found to be spherical and uniformly distributed with a size in the range of 9-27.5 nm. The antibacterial activities and acting mechanism of AgNPs were studied with respect to Staphylococcus aureus and Escherichia coli by measuring the growth curves, protein and reducing sugar leakage, respiratory chain dehydrogenase activity, as well as the formation of bactericidal reactive oxygen species (ROS). The experimental results indicated that 50 mg/ml AgNPs could completely inhibit the growth of bacterial cells and destroy the permeability of bacterial membranes and depress the activity of some membranous enzymes, which cause bacteria to die eventually. These nontoxic nanomaterials, which can be prepared in a simple and cost-effective manner, may be suitable for the formulation of new types of bactericidal materials.

Antimicrobial, antioxidant and antitumor activities of silver nanoparticles synthesized by Allium cepa extract: A green approach.

An economic and efficient method for the synthesis of silver nanoparticles (AgNPs) was performed using onion (Allium cepa) extract as reducing and capping agent. UV-vis spectroscopy confirmed the formation of silver nanoparticles by observing the typical surface plasmon resonance peak at 420 nm. Transmission electron microscopy studies revealed that AgNPs were spherical in shape with a size range of 10-23 nm. AgNPs were further demonstrated by the characteristic peaks observed in the XRD image. The possible functional groups of AgNPs were identified by FTIR analysis. AgNPs exhibited potential antimicrobial activity against all the microbial strains tested. Antioxidant activity of AgNPs revealed that they can be used as potential radical scavenger against deleterious damages caused by the free radicals. Additionally, AgNPs had antitumor activities against human breast (MCF-7), hepatocellular (HepG-2) and colon (HCT-116) carcinoma cell lines in a dose-dependent manner with IC50 of 1.6, 2.3 and 2.2 µg/ml, respectively. The overall results indicate promising baseline information for the potential uses of AgNPs in the treatment of infectious diseases and tumors.

Statistical Modelling and Optimization of Fermentation Medium for Lincomycin Production by Streptomyces lincolnensis Immobilized Cells

ABSTRACT Response surface methodology was used to optimize lincomycin production by Streptomyces lincolnensis NRRL ISP-5355 in submerged fermentation. Screening of fermentation medium components to find their relative effect on lincomycin production was done using Plackett-Burman design. Malt extract, dextrin, soluble starch and (NH4)2SO4 were the most significant nutrient influenced on lincomycin production. Central composite design was applied to determine optimal concentrations of these factors and the effect of their mutual interactions. The interaction between soluble starch and (NH4)2SO4 was found to enhance the production, whereas malt extract and dextrin exhibited an influence independent from the other two factors. Using this statistical optimization method, maximum lincomycin concentration of 1345 μg/ml was obtained which represented a 40.5 % increase in titer than that acquired from the non-optimized medium. This statistically optimized medium was employed for lincomycin production through immobilization of Streptomyces lincolnensis by adsorption on synthetic cotton fibers. Immobilization technique improved the concentration to 1350 μg/ml higher than that produced from free cells cultures and could be maintained for longer than 17 days in a repeated batch system.

Cryoprotection of probiotic bacteria with poly-γ-glutamic acid produced by Bacillus subtilis and Bacillus licheniformis.

Poly-γ-glutamic acid (γ-PGA) is a naturally occurring biopolymer made up of repeating units of glutamic acid and can be potentially used for multiple applications. This study compared the production of γ-PGA by Bacillus subtilis and Bacillus licheniformis in GS and E media. The highest γ-PGA production was achieved using initial glycerol concentration of 40 and 80 g/l, ammonium chloride as the nitrogen source, 20 g/l glutamic acid at pH 6.5 for 72 h using E medium. On characterization, it was observed that glutamic acid was the sole component of the purified material. It contained a mixture of Na-γ-PGA and H+-γ-PGA. The survival of probiotics during freeze drying was improved by combining them with γ-PGA polymer. For Lactobacilli, 10% γ-PGA protected the cells significantly than 10% sucrose during freeze drying. γ-PGA protection was shown to improve the viability of probiotic bacteria in orange juice for 40 days. No considerable change was observed in the concentrations of citric acid, malic acid and ascorbic acid when probiotic bacteria and γ-PGA were introduced into orange juice and hence, it could be used as a non-dairy delivery platform for these bacteria.

Production of polyhydroxyalkanoates (PHAs) by Bacillus subtilis and Escherichia coli grown on cane molasses fortified with ethanol

The aim of this work was to study the production of polyhydroxyalkanoates (PHAs) by Bacillus subtilis and Escherichia coli isolated from the industrial contaminated soil samples using cane molasses as an inexpensive substrate. The amount of PHA accumulated followed a similar pattern to its growth for each of treatment indicating a growth-related production, yielding maximum PHA production of 54.1 and 47.16% for B. subtilis and E. coli, respectively after 96 h cultivation in the medium containing 6 and 8 % molasses, respectively and decreased thereafter. The growth and PHA yields were improved by introducing 1% ethanol into the molasses medium. Ammonium sulphate and ammonium nitrate at a concentration of 1 g/L served as the best nitrogen sources for bacterial growth, allowing B. subtilis and E. coli cells to accumulate PHA up to 62.21 and 58.7%, respectively. The optimum environmental conditions that influenced the PHA production by the two strains were inoculum concentration of 8%, pH 7.0 and a temperature of 35°C. The functional groups of the extracted PHA granules were identified by Fourier transform infrared (FTIR) spectroscopy analysis.

Optimization and characterization of alkaline protease and carboxymethyl-cellulase produced by Bacillus pumillus grown on Ficus nitida wastes.

The potentiality of 23 bacterial isolates to produce alkaline protease and carboxymethyl-cellulase (CMCase) on Ficus nitida wastes was investigated. Bacillus pumillus ATCC7061 was selected as the most potent bacterial strain for the production of both enzymes. It was found that the optimum production of protease and CMCase were recorded at 30 °C, 5% Ficus nitida leaves and incubation period of 72 h. The best nitrogen sources for protease and CMCase production were yeast extract and casein, respectively. Also maximum protease and CMCase production were reported at pH 9 and pH 10, respectively. The enzymes possessed a good stability over a pH range of 8-10, expressed their maximum activities at pH10 and temperature range of 30-50 °C, expressed their maximum activities at 50 °C. Ions of Hg(2+), Fe2+ and Ag(+) showed a stimulatory effect on protease activity and ions of Fe(2+), Mg(2+), Ca(2+), Cu(2+) and Ag(+) caused enhancement of CMCase activity. The enzymes were stable not only towards the nonionic surfactants like Triton X-100 and Tween 80 but also the strong anionic surfactant, SDS. Moreover, the enzymes were not significantly inhibited by EDTA or cystein. Concerning biotechnological applications, the enzymes retained (51-97%) of their initial activities upon incubation in the presence of commercials detergents for 1 h. The potential use of the produced enzymes in the degradation of human hair and cotton fabric samples were also assessed.

Antimicrobial activity of a biosurfactant produced by Bacillus licheniformis strain M104 grown on whey

The aim of the present study was to investigate the antimicrobial effect of the lipopeptide biosurfactants produced by Bacillus licheniformis strain M104 grown on whey. The biosurfactant was investigated for potential antimicrobial activity by using the disc-diffusion method against different Gram-positive bacteria {B subtilis, B. thuringiensis (two strains), B. cereus, Staphylococcus aureus (two strains) and Listeria monocytogenes)}, Gram-negative bacteria {(Pseudomonas aeruginosa, Escherichia coli (two strains), Salmonella typhimurium, Proteous vulgaris and Klebsiella pneumoniae) and a yeast (Candida albicans)}. The biosurfactant showed profoundly distinct antibacterial activity toward tested bacteria and displayed an antifungal activity against the tested yeast. Maximum antimicrobial activity of the biosurfactant was shown against S. aureus ATCC 25928. The biosurfactant had a broad inhibition effect on intracellular components of S. aureus ATCC 25928. The antimicrobial effect of lipopeptide biosurfactant produced by B. licheniformis strain M104 was time and concentration dependent. When biosurfactant was added to S. aureus medium in a concentration of (48 μg / ml), the maximum reduction of acid soluble phosphorous (53.06 %), total lipid (90.47 %) total proteins (53.43%), RNA (83.29 %) and DNA (48.50%) were recorded after 12 h of incubation period. From the preliminary characterization results, it could be concluded that biosurfactants were a suitable alternative in potential applications of medical fields.

Optimization and characterization of alkaline protease and carboxymethyl-cellulase produced by Bacillus pumillus grown on Ficus nitida wastes

The potentiality of 23 bacterial isolates to produce alkaline protease and carboxymethyl-cellulase (CMCase) on Ficus nitida wastes was investigated. Bacillus pumillus ATCC7061 was selected as the most potent bacterial strain for the production of both enzymes. It was found that the optimum production of protease and CMCase were recorded at 30 °C, 5% Ficus nitida leaves and incubation period of 72 h. The best nitrogen sources for protease and CMCase production were yeast extract and casein, respectively. Also maximum protease and CMCase production were reported at pH 9 and pH 10, respectively. The enzymes possessed a good stability over a pH range of 8-10, expressed their maximum activities at pH10 and temperature range of 30-50 °C, expressed their maximum activities at 50 °C. Ions of Hg2+, Fe2+ and Ag+ showed a stimulatory effect on protease activity and ions of Fe2+, Mg2+, Ca2+, Cu2+ and Ag+ caused enhancement of CMCase activity. The enzymes were stable not only towards the nonionic surfactants like Triton X-100 and Tween 80 but also the strong anionic surfactant, SDS. Moreover, the enzymes were not significantly inhibited by EDTA or cystein. Concerning biotechnological applications, the enzymes retained (51-97%) of their initial activities upon incubation in the presence of commercials detergents for 1 h. The potential use of the produced enzymes in the degradation of human hair and cotton fabric samples were also assessed.

Chitinase production by Bacillus thuringiensis and Bacillus licheniformis: their potential in antifungal biocontrol.

Thirty bacterial strains were isolated from the rhizosphere of plants collected from Egypt and screened for production of chitinase enzymes. Bacillus thuringiensis NM101-19 and Bacillus licheniformis NM120-17 had the highest chitinolytic activities amongst those investigated. The production of chitinase by B. thuringiensis and B. licheniformis was optimized using colloidal chitin medium amended with 1.5% colloidal chitin, with casein as a nitrogen source, at 30°C after five days of incubation. An enhancement of chitinase production by the two species was observed by addition of sugar substances and dried fungal mats to the colloidal chitin media. The optimal conditions for chitinase activity by B. thuringiensis and B. licheniformis were at 40°C, pH 7.0 and pH 8.0, respectively. Na(+), Mg(2+), Cu(2+), and Ca(2+) caused enhancement of enzyme activities whereas they were markedly inhibited by Zn(2+), Hg(2+), and Ag(+). In vitro, B. thuringiensis and B. licheniformis chitinases had potential for cell wall lysis of many phytopathogenic fungi tested. The addition of B. thuringiensis chitinase was more effective than that of B. licheniformis in increasing the germination of soybean seeds infected with various phytopathogenic fungi.