Repurposing fusidic acid as an antimicrobial against enterococci with a low probability of resistance development.

Drug repurposing constitutes a strategy to combat antimicrobial resistance, by using agents with known safety, pharmacokinetics, and pharmacodynamics. Previous studies have implemented new fusidic acid (FA) front-loading-dose regimens, allowing higher serum levels than those achievable with ordinary doses. As susceptibility breakpoints are affected by serum level, we evaluated the repurposing of FA as an antimicrobial product against enterococci. FA minimum inhibitory concentrations (MICs) against standard enterococci strains; Enterococcus faecalis ATCC 29212 and Enterococcus faecium ATCC 27270 were 2 and 4 µg/mL, respectively. The MIC against 98 enterococcal clinical isolates was ≤ 8 µg/mL; all would be susceptible if categorized according to recalculated breakpoints (≥ 16 µg/mL), based on the serum level achieved using the front-loading regimen. FA administration in vivo, using the BALB/c mouse infection model, significantly reduced bacterial burden by two to three log10 units in the liver and spleen of mice infected with vancomycin-susceptible and -resistant strains. Exposure of the standard enterococcal strains to increasing, but not fixed, FA concentrations resulted in resistant strains (MIC = 128 µg/mL), with thicker cell walls and slower growth rates. Only one mutation (M651I) was detected in the fusA gene of the resistant strain derived from serial passage of E. faecium ATCC 27270, which was retained in the revertant strain after passage in the FA-free medium. In conclusion, FA can be repurposed as an antimicrobial drug against enterococci with a low probability of mutational resistance development, and can be employed for treatment of infections attributable to vancomycin-resistant enterococci.

Nationwide hepatitis C virus screening and treatment of adolescents in Egyptian schools.

Until 2018, Egypt had the highest prevalence of hepatitis C virus (HCV) infection globally, affecting approximately 7% of the population. Despite efforts in diagnosis and treatment since 2006, nearly 2 million individuals with chronic HCV infection had yet to be diagnosed as of early 2018. In December, 2018, a mass HCV screening campaign for adolescents aged 15-18 years was initiated. Among 3 024 325 adolescents screened, the HCV antibody seroprevalence was 11 477 (0·38%), of whom 8187 (78·7%) were HCV RNA-positive. Sustained virological response 12 weeks after completion of treatment (SVR12) was attained by 7327 (99·6%) adolescents with a fixed-dose combination of generic ledipasvir 90 mg plus sofosbuvir 400 mg. Although mass screening in this age group might not be regularly adopted by many health systems and its cost-effectiveness might be lower than the screening of adults and high-risk groups (eg, patients on haemodialysis, people who inject drugs), breaking the chain of transmission in younger populations should lead to a reduction in HCV incidence and complications, and hasten the elimination of the disease.

Biodegradation of aflatoxin by bacterial species isolated from poultry farms.

Aflatoxins are carcinogenic compounds produced by certain Aspergillus spp and naturally contaminate poultry rations. Exposure to low levels of Aflatoxin B1 (AFB1) in poultry feeds is the second most threatening issue facing the poultry industry in Egypt; it can cause a reduction in growth, egg production, and compromised immune functions, resulting in significant economic loss. Hence, a safe, effective and eco-friendly detoxification method is strongly required. Biological decontamination is a promising approach to reduce aflatoxin levels within threshold limits. This study explores the biodegradation capacity of bacteria isolated from the moldy feed, soil and poultry feces in various poultry farms against AFB1 (100 ppb), G1 (100 ppb), B2 (30 ppb), G2 (30 ppb). Sixty-five bacterial isolates were initially screened using coumarin media with a concentration of (0.01%-0.5%) coumarin. Only one soil isolate (SZ1) grew at the highest concentration (0.5%). Coumarin and Aflatoxin degradation rates of ten promising isolates were measured using spectrophotometry and HPLC. Six isolates reduced AFG1 by more than 90% in the liquid medium, five reduced AFB2 while only four did the same with AFB1& AFG2. Impressively, isolate SZ1 (identified as Pseudomonas fluorescens) exhibited the best degradation capacity to both coumarin and aflatoxin with 100% degradation of AFG1 and 99% degradation of AFB1, AFB2 and AFG2. Biochemical and molecular identification of the ten isolates revealed that they belong to four genera; Bacillus (6), Pseudomonas (2), Enterococcus (1) and Stenotrophomonas (1). Factors affecting Pseudomonas fluorescens SZ1 degradation activity was further investigated. Optimum temperature, time and pH for maximum aflatoxin degradation were at 37 °C, 72 h and 7, respectively. Treatment with proteinase K reduced the degradation activity of G1 (31% ± 1.438), B1 (42% ± 1.438), G2 (19% ± 1.097), and B2 (25% ± 1.732), suggesting that the effective component in aflatoxin degradation may be protein in nature. Our study suggests the biocontrol potential of several different species isolated from poultry farms; B. haynesii, B. licheniformis, B. tequilensis, B. subtilis, B. amyloliquefaciens, Pseudomonas fluorescens, Enterococcus casseliflavus, and Stenotrophomonas maltophilia. The results proposed Pseudomonas fluorescens SZ1 as an excellent candidate for bioremediation and decontamination of aflatoxin in feed matrices. To the best of our knowledge, this is the first report identifying B. haynesii, Enterococcus casseliflavus, B. tequilensis and B. amyloliquefaciens with aflatoxin degradation activity.

Purification and characterization of bacteriocins-like inhibitory substances from food isolated Enterococcus faecalis OS13 with activity against nosocomial enterococci.

Nosocomial infections caused by enterococci are an ongoing global threat. Thus, finding therapeutic agents for the treatment of such infections are crucial. Some Enterococcus faecalis strains are able to produce antimicrobial peptides called bacteriocins. We analyzed 65 E. faecalis isolates from 43 food samples and 22 clinical samples in Egypt for 17 common bacteriocin-encoding genes of Enterococcus spp. These genes were absent in 11 isolates that showed antimicrobial activity putatively due to bacteriocins (three from food, including isolate OS13, and eight from clinical isolates). The food-isolated E. faecalis OS13 produced bacteriocin-like inhibitory substances (BLIS) named enterocin OS13, which comprised two peptides (enterocin OS13α OS13ß) that inhibited the growth of antibiotic-resistant nosocomial E. faecalis and E. faecium isolates. The molecular weights of enterocin OS13α and OS13ß were determined as 8079 Da and 7859 Da, respectively, and both were heat-labile. Enterocin OS13α was sensitive to proteinase K, while enterocin OS13ß was resistant. Characterization of E. faecalis OS13 isolate revealed that it belonged to sequence type 116. It was non-hemolytic, bile salt hydrolase-negative, gelatinase-positive, and sensitive to ampicillin, penicillin, vancomycin, erythromycin, kanamycin, and gentamicin. In conclusion, BLIS as enterocin OS13α and OS13ß represent antimicrobial agents with activities against antibiotic-resistant enterococcal isolates.

Biomonitoring detoxification efficiency of an algal-bacterial microcosm system for treatment of coking wastewater: Harmonization between Chlorella vulgaris microalgae and wastewater microbiome.

Nowadays, due to worldwide water shortage, water utilities are forced to re-evaluate treated wastewater. Consequently, wastewater treatment plants need to conduct biomonitoring. Coking wastewater (CWW) has toxic, mutative and carcinogenic components with threatening effect on the environment. CWW was selected as a model for complex highly toxic industrial wastewater that should be treated. CWW from Egypt was treated in a nine-liter photobioreactor using an algal-bacterial system. The photobioreactor was operated for 154 days changing different parameters (toxic load and light duration) for optimization. Optimized conditions achieved significant reduction (45%) in the operation cost. The algal-bacterial system was monitored using chemical assays (chemical oxygen demand and phenol analysis), bioassays (phytotoxicity, Artemia-toxicity, cytotoxicity, algal-bacterial ratio and settleability) and Illumina-MiSeq sequencing of 16S rRNA gene. The algal-bacterial system detoxified (in terms of phytotoxicity, cytotoxicity and Artemia-toxicity) CWW introduced as influent through all phases. A significant difference was recorded in the microbial diversity between influent and effluent samples. Four phyla dominated influent samples; Proteobacteria (77%), Firmicutes (11%), Bacteroidetes (5%) and Deferribacteres (3%) compared to only two in effluent samples; Proteobacteria (66%) and Bacteroidetes (26%). The significant relative-abundance of versatile aromatic degraders (Comamonadaceae and Pseudomonadaceae families) in influent samples conformed to the nature of CWW. Microbial community shifted and promoted the activity of catabolically versatile and xenobiotics degrading families (Chitinophagaceae and Xanthomonadaceae). Co-culture of microalgae had a positive effect on the biodegrading bacteria that was reflected by enhanced treatment efficiency, significant increase in relative abundance of bacterial genera with cyanide-decomposing potential and negative effect on waterborne pathogens.

Comparative Structural Analysis of Different Mycobacteriophage-Derived Mycolylarabinogalactan Esterases (Lysin B).

Mycobacteriophage endolysins have emerged as a potential alternative to the current antimycobacterial agents. This study focuses on mycolylarabinogalactan hydrolase (LysB) enzymes of the α/ß-hydrolase family, which disrupt the unique mycolic acid layer of mycobacterium cell wall. Multiple sequence alignment and structural analysis studies showed LysB-D29, the only enzyme with a solved three-dimensional structure, to share several common features with esterases (lacking lid domain) and lipases (acting on long chain lipids). Sequence and structural comparisons of 30 LysB homology models showed great variation in domain organizations and total protein length with major differences in the loop-5 motif harboring the catalytic histidine residue. Docking of different p-nitrophenyl ligands (C4-C18) to LysB-3D models revealed that the differences in length and residues of loop-5 contributed towards wide diversity of active site conformations (long tunnels, deep and superficial funnels, shallow bowls, and a narrow buried cave) resembling that of lipases, cutinases, and esterases. A set of seven LysB enzymes were recombinantly produced; their activity against p-nitrophenyl esters could be related to their active site conformation and acyl binding site. LysB-D29 (long tunnel) showed the highest activity with long chain p-nitrophenyl palmitate followed by LysB-Omega (shallow bowl) and LysB-Saal (deep funnel).

Optimization and enhancement of textile reactive Remazol black B decolorization and detoxification by environmentally isolated pH tolerant Pseudomonas aeruginosa KY284155.

Azo dyes are complex derivatives of diazene used in food and textile manufacture. They are highly recalcitrant compounds, and account for severe environmental and health problems. Different strains of Pseudomonas species were isolated from textile wastewater effluents. The bioconversion of Remazol black B (a commonly used water soluble dye) by Pseudomonas aeruginosa was observed in static conditions. The bio-decolorization process was optimized by a multi factorial Plackett-Burman experimental design. Decolorization of 200 mg L-1 reached 100% in 32 h. Interestingly, the presence of yeast extract, magnesium and iron in the culture media, highly accelerated the rate of decolorization. Moreover, one of our isolates, P. aeruginosa KY284155, was kept high degradation rates at high pH (pH = 9), which represents the pH of most textile wastewater effluents, and was able to tolerate high concentration of dye up to 500 mg L-1. In bacteria, azo-dye degradation is often initiated by reductive azo compound cleavage catalyzed by azo-reductases. Three genes encoding azo-reductases, paazoR1, paazoR2 and paazoR3, could be identified in the genome of the isolated P. aeruginosa stain (B1). Bioinformatics analyses of the paazoR1, paazoR2 and paazoR3 genes reveal their prevalence and conservation in other P. aeruginosa strains. Chemical oxygen demand dramatically decreased and phyto-detoxification of the azo dye was accomplished by photocatalytic post treatment of the biodegradation products. We suggest applying combined biological photocatalytic post treatment for azo dyes on large scale, for effective, cheap decolorization and detoxification of azo-dyes, rendering them safe enough to be discharged in the environment.

Illumina sequencing and assessment of new cost-efficient protocol for metagenomic-DNA extraction from environmental water samples.

In this study, the development and assessment of a modified, efficient, and cost-efficient protocol for mDNA (metagenomic DNA) extraction from contaminated water samples was attempted. The efficiency of the developed protocol was investigated in comparison to a well-established commercial kit (Epicentre, Metagenomic DNA Isolation Kit for Water). The comparison was in terms of degree of shearing, yield, purity, duration, suitability for polymerase chain reaction and next-generation sequencing in addition to the quality of next-generation sequencing data. The DNA yield obtained from the developed protocol was 2.6 folds higher than that of the commercial kit. No significant difference in the alpha (Observed species, Chao1, Simpson and PD whole tree) and beta diversity was found between the DNA samples extracted by the commercial kit and the developed protocol. The number of high-quality sequences of the samples extracted by the developed method was 20% higher than those obtained by the samples processed by the kit. The developed economic protocol successfully yielded high-quality pure mDNA compatible with complex molecular applications. Thus we propose the developed protocol as a gold standard for future metagenomic studies investigating a large number of samples.

Illumina sequencing and assessment of new cost-efficient protocol for metagenomic-DNA extraction from environmental water samples

Abstract In this study, the development and assessment of a modified, efficient, and cost-efficient protocol for mDNA (metagenomic DNA) extraction from contaminated water samples was attempted. The efficiency of the developed protocol was investigated in comparison to a well-established commercial kit (Epicentre, Metagenomic DNA Isolation Kit for Water). The comparison was in terms of degree of shearing, yield, purity, duration, suitability for polymerase chain reaction and next-generation sequencing in addition to the quality of next-generation sequencing data. The DNA yield obtained from the developed protocol was 2.6 folds higher than that of the commercial kit. No significant difference in the alpha (Observed species, Chao1, Simpson and PD whole tree) and beta diversity was found between the DNA samples extracted by the commercial kit and the developed protocol. The number of high-quality sequences of the samples extracted by the developed method was 20% higher than those obtained by the samples processed by the kit. The developed economic protocol successfully yielded high-quality pure mDNA compatible with complex molecular applications. Thus we propose the developed protocol as a gold standard for future metagenomic studies investigating a large number of samples.

Illumina sequencing and assessment of new cost-efficient protocol for metagenomic-DNA extraction from environmental water samples

Abstract In this study, the development and assessment of a modified, efficient, and cost-efficient protocol for mDNA (metagenomic DNA) extraction from contaminated water samples was attempted. The efficiency of the developed protocol was investigated in comparison to a well-established commercial kit (Epicentre, Metagenomic DNA Isolation Kit for Water). The comparison was in terms of degree of shearing, yield, purity, duration, suitability for polymerase chain reaction and next-generation sequencing in addition to the quality of next-generation sequencing data. The DNA yield obtained from the developed protocol was 2.6 folds higher than that of the commercial kit. No significant difference in the alpha (Observed species, Chao1, Simpson and PD whole tree) and beta diversity was found between the DNA samples extracted by the commercial kit and the developed protocol. The number of high-quality sequences of the samples extracted by the developed method was 20% higher than those obtained by the samples processed by the kit. The developed economic protocol successfully yielded high-quality pure mDNA compatible with complex molecular applications. Thus we propose the developed protocol as a gold standard for future metagenomic studies investigating a large number of samples.

A Degradome-Based Polymerase Chain Reaction to Resolve the Potential of Environmental Samples for 2,4-Dichlorophenol Biodegradation.

A clean way to overcome environmental pollution is biodegradation. In this perspective, at the intersection of biodegradation and metagenomics, the degradome is defined as the totality of genes related to the biodegradation of a certain compound. It includes the genetic elements from both culturable and uncultured microorganisms. The possibility of assessing the biodegradation potential of an environmental samples, using a degradome-based polymerase chain reaction, was explored. 2,4-Dichlorophenol (2,4-DCP) was chosen as a model and the use of tfdB gene as a biodegradation marker was confirmed by bioinformatics study of TfdB protein. Five primer pairs were designed for the detection of different tfdB gene families. A total of 16 environmental samples were collected from Egyptian agricultural soils and wastewaters and tested for the presence of 2,4-DCP. The biodegradation capacity of 2,4-DCP was determined, for all isolated consortia, to reach up to 350 mg/l. Metagenomic DNA was extracted directly from the soil samples while successive 2,4-DCP-degrading microbial communities were enriched, with increasing concentrations of 2,4-DCP, then their DNA was extracted. The extracted DNA was tested for the distribution of the tfdB gene using a degradome-based polymerase chain reaction. tfdB-1 and tfdB-2 were detected in 5 and 9 samples, respectively. However, the co-existence of both genes was detected only in five samples. All tfdB positive samples were capable of 2,4-DCP degradation. The developed approach of assessing the potential of different environments for degrading 2,4-DCP was successfully measured in terms of accuracy (81.25%) and specificity (100%).

Biofilm formation in enterococci: genotype-phenotype correlations and inhibition by vancomycin.

Enterococci are nosocomial pathogens that can form biofilms, which contribute to their virulence and antibiotic resistance. Although many genes involved in biofilm formation have been defined, their distribution among enterococci has not been comprehensively studied on a genome scale, and their diagnostic ability to predict biofilm phenotypes is not fully established. Here, we assessed the biofilm-forming ability of 90 enterococcal clinical isolates. Major patterns of virulence gene distribution in enterococcal genomes were identified, and the differentiating virulence genes were screened by polymerase chain reaction (PCR) in 31 of the clinical isolates. We found that detection of gelE in Enterococcus faecalis is not sufficient to predict gelatinase activity unless fsrAB, or fsrB alone, is PCR-positive (P = 0.0026 and 0.0012, respectively). We also found that agg is significantly enriched in isolates with medium and strong biofilm formation ability (P = 0.0026). Additionally, vancomycin, applied at sub minimal inhibitory concentrations, inhibited biofilm in four out of five strong biofilm-forming isolates. In conclusion, we suggest using agg and fsrB genes, together with the previously established gelE, for better prediction of biofilm strength and gelatinase activity, respectively. Future studies should explore the mechanism of biofilm inhibition by vancomycin and its possible use for antivirulence therapy.

Remediation of a mixture of analgesics in a stirred-tank photobioreactor using microalgal-bacterial consortium coupled with attempt to valorise the harvested biomass.

An artificial microalgal-bacterial consortium was used to remediate a mixture of analgesics (ketoprofen, paracetamol and aspirin) in a stirred-tank photobioreactor. A hydraulic retention time (HRT) of 3days supported poor treatment because of the formation of p-aminophenol (paracetamol toxic metabolite). Increasing the HRT to 4days enhanced the bioremediation efficiency. After applying an acclimatization regime, 95% removal of the analgesics mixture, p-aminophenol and COD reduction were achieved. However, shortening the HRT again to 3days neither improved the COD reduction nor ketoprofen removal. Applying continuous illumination achieved the best analgesics removal results. The harvested biomass contained 50% protein, which included almost all essential amino acids. The detected fatty acid profile suggested the harvested biomass to be a good biodiesel-producing candidate. The water-extractable fraction possessed the highest phenolic content and antioxidant capacity. These findings suggest the whole process to be an integrated eco-friendly and cost-efficient strategy for remediating pharmaceutical wastewater.

Optimization of Cellulase Production by Halobacillus sp. QLS 31 Isolated from Lake Qarun, Egypt.

A halophilic cellulase-producing bacterium was isolated from a sediment sample collected from Lake Qarun (Fayoum Province, Egypt). Molecular identification based on 16S rDNA amplification and sequencing revealed 99% homology with Halobacillus sp. and hence was designated as Halobacillus sp. QLS 31. Medium composition and culture conditions were optimized for enhancing the production of cellulase enzyme using the Plackett-Burman statistical design. Ten variables were evaluated for their influence on cellulase production. Carboxymethyl cellulose (CMC), zinc sulfate (ZnSO4), and inoculum size were found to exert a significant effect on cellulase productivity by Halobacillus sp. QLS 31. The maximum specific activity of cellulase enzyme was 48.08 U/mg. Following the predicted conditions, a 7.5-fold increase in cellulase specific activity (175.47 U/mg) was achieved compared to the basal medium (23.19 U/mg) under the following optimized conditions: temperature (30 °C), fermentation time (2 days ), pH value (9), CMC concentration (1%), inoculum size (1%), yeast extract concentration (0.1%), ammonium sulfate ((NH3)2SO4) concentration (0.1%), sodium chloride (NaCl) concentration (20%), and metal inducers: ZnSO4 (0.1%) and Ca/Mg ratio (0.01%). Thus, the results of this study provide an important basis for more efficient, cheap industrial cellulase production from halophilic Halobacillus sp. QLS 31.

Studying the influence of formulation and process variables on Vancomycin-loaded polymeric nanoparticles as potential carrier for enhanced ophthalmic delivery.

Ocular topically applied Vancomycin (VCM) suffers poor bioavailability due to its high molecular weight and hydrophilicity. In the present investigation, VCM-loaded polymeric nanoparticles (PNPs) were developed aiming to enhance its ocular bioavailability through prolonging its release pattern and ophthalmic residence. PNPs were prepared utilizing double emulsion (W/O/O), solvent evaporation technique. 23×41 full factorial design was applied to evaluate individual and combined influences of polymer type, Eudragit® RS100, sonication time, and Span®80 concentration on PNPs particle size, encapsulation efficiency, and zeta potential. Further, the optimized formulae were incorporated in 1% Carbopol®-based gel. In-vivo evaluation of the optimized formulae was performed via Draize test followed by microbiological susceptibility testing on albino rabbits. Results revealed successful formulation of VCM-loaded PNPs was achieved with particle sizes reaching 155nm and up to 88% encapsulation. Draize test confirmed the optimized formulae as non-irritating and safe for ophthalmic administration. Microbiological susceptibility testing confirmed prolonged residence, higher Cmax. with more than two folds increment in the AUC(0.25-24) of VCM-PNPs over control groups. Thus, VCM-loaded PNPs represent promising carriers with superior achievements for enhanced Vancomycin ophthalmic delivery over the traditional use of commercially available VCM parenteral powder after constitution into a solution by the ophthalmologists.

Gut microbiome alterations in patients with stage 4 hepatitis C.

BACKGROUND: Hepatitis C virus (HCV) causes debilitating liver diseases, which may progress to cirrhosis and cancer, and claims 500,000 annual lives worldwide. While HCV epidemiology, pathophysiology, and therapy are being deeply studied, rare attention is given to reciprocal interactions between HCV infection , HCV-induced chronic liver diseases, and the human gut microbiome. As Egypt has the world's highest prevalence of HCV infections, we launched this study to monitor differences in the gut microbial community composition of Egyptian HCV patients that may affect, or result from, the patients' liver state. RESULTS: To this end, we analyzed stool samples from six stage 4-HCV patients and eight healthy individuals by high-throughput 16S rRNA gene sequencing using Illumina MiSeq. Overall, the alpha-diversity of the healthy persons' gut microbiomes was higher than those of the HCV patients. Whereas members of phylum Bacteroidetes were more abundant in HCV patients, healthy individuals had higher abundance of Firmicutes, Proteobacteria, and Actinobacteria. Genus-level analysis showed differential abundance of Prevotella and Faecalibacterium (higher in HCV patients) vs. Ruminococcus and Clostridium (healthy group), indicating that the higher abundance of Bacteroidetes in HCV patients is most likely due to Prevotella overabundance. The probiotic genus, Bifidobacterium, was only observed in the microbiotas of healthy individuals. CONCLUSIONS: To the best of our knowledge, this study provides a first overview of major phyla and genera differentiating stage 4-HCV patients from healthy individuals and suggests possible microbiome remodeling in chronic hepatitis C, possibly shaped by bacterial translocation as well as the liver's impaired role in digestion and protein synthesis. Future studies will investigate the microbiome composition and functional capabilities in more patients while tracing some potential biomarker taxa (e.g., Prevotella, Faecalibacterium vs. Bifidobacterium).

Biodegradation of ketoprofen using a microalgal-bacterial consortium.

OBJECTIVE: To test the toxicity of ketoprofen (a commonly-used NSAIDs) using two microalgal strains and Artemia sp. following the isolation of bacterial and microalgal strains and testing their ability to biodegrade and tolerate ketoprofen. RESULTS: Chlorella sp. was the most resistant to ketoprofen. A defined bacterial consortium (K2) degraded 5 mM ketoprofen as a sole carbon source both in the dark or continuous illumination. Ketoprofen did not undergo photodegradation. In the dark, biodegradation was faster with a lag phase of 10 h, 41% COD removal and 82 % reduction in toxicity. The consortium degraded up to 16 mM ketoprofen. The consortium was composed of four bacterial isolates that were identified. MS/MS analysis suggested a ketoprofen biodegradation pathway that has not been previously reported. Combining Chlorella sp. and the K2 consortium, ketoprofen was degraded within 7 days under a diurnal cycle of 12 h light/12 h dark. CONCLUSION: The feasibility of using a microalgal-bacterial system to treat pharmaceutical wastewater is promising for the reduction of the process cost and providing a safer technology for pharmaceutical wastewater treatment.

Nanoparticles as tool for enhanced ophthalmic delivery of vancomycin: a multidistrict-based microbiological study, solid lipid nanoparticles formulation and evaluation.

CONTEXT: A microbiological multidistrict-based survey from different Egyptian governorates was conducted to determine the most prevalent causative agents of ocular infections in the Egyptian population. Antibiotic sensitivity testing was then performed to identify the most potent antimicrobial agent. Vancomycin (VCM) proved the highest activity against gram-positive Staphylococcus bacteria, which are the most commonly isolated causative agents of ocular infection. However, topically applied VCM suffers from poor ocular bioavailability because of its high molecular weight and hydrophilicity. OBJECTIVE: The aim of the present study was to develop VCM-loaded solid lipid nanoparticles (SLNs) using water-in-oil-in-water (W/O/W) double emulsion, solvent evaporation technique to enhance ocular penetration and prolong ophthalmic residence of VCM. METHOD: Two consecutive full factorial designs (2(4) followed by 3(2)) were adopted to study the effect of different formulation and process parameters on SLN formulation. The lipid type and structure, polyvinyl alcohol (PVA) molecular weight and concentration, sonication time, as well as lipid:drug ratio were studied as independent variables. The formulated SLN formulae were evaluated for encapsulation efficiency (EE%), particle size (PS), and zeta potential as dependent variables. RESULTS: The statistically-optimized SLN formula (1:1 ratio of glyceryltripalmitate:VCM with 1% low molecular weight PVA and 1 min sonication time) had average PS of 277.25 nm, zeta potential of -20.45, and 19.99% drug encapsulation. Scanning and transmission electron micrographs showed well-defined, spherical, homogenously distributed particles. CONCLUSION: The present study suggests that VCM incorporation into SLNs is successfully achievable; however, further studies with different nanoencapsulation materials and techniques would be valuable for improving VCM encapsulation.

Direct Detection of Burkholderia cepacia in Susceptible Pharmaceutical Products Using Semi-Nested PCR.

Burkholderia cepaciahas recently received a considerable attention as one of the major risks in susceptible pharmaceutical products. This microorganism can easily propagate and cause vast and severe contamination, especially to the water supplies for pharmaceutical companies. Moreover, it proliferates within the products and can cause severe infections for humans. Therefore, fast and sensitive detection of these bacteria is of a great demand. The present study introduces improved application of a polymerase chain reaction assay with relatively high sensitivity and specificity for the direct detection ofB. cepaciafrom the aqueous pharmaceutical products. A semi-nested polymerase chain reaction approach using the primer set BCR1/BCR2 followed by BCR1/Mr yielding a 465 bp fragment of the recA gene was applied and tested using both crude lysate from isolated colonies and DNA directly extracted from artificially prepared and spiked reference syrup. The polymerase chain reaction assay showed no interference with other bacterial reference and environmental strains tested, includingStaphylococcus aureusATCC® 6538,Pseudomonas aeruginosaATCC® 9027,Escherichia coliATCC® 8739,Salmonella abonyNCTC® 6017,Bacillus subtilisATCC® 6633,Micrococcus luteus, Staphylococcus warneri, Pseudomonas fluorescens, Pseudomonas putida, andRalstonia pickettii Moreover, this semi-nested assay showed a detection limit of around 10 colony-forming units per sample and could detectB. cepaciastrains isolated from a municipal pre-treated potable water tank. Comparing the results for detection ofB. cepaciain 100 randomly collected commercial syrup preparations using both conventional standard method and polymerase chain reaction assay revealed thatB. cepaciawas detected in two samples using polymerase chain reaction assay while all samples showed negative results by conventional culturing and biochemical methods. These results highlight the advantage of using this polymerase chain reaction assay to detectB. cepaciain contaminated pharmaceutical products and even water for pharmaceutical purposes, without the need of culturing or pre-enrichment, where it may give false-negative results and may be misidentified when biochemically tested.

Optimization of rhamnolipid production by biodegrading bacterial isolates using Plackett-Burman design.

Biosurfactants are biological surfactants produced by microorganisms. Pseudomonas species are well known for the production of the rhamnolipid biosurfactant. In this work, the production of rhamnolipid biosurfactant by Pseudomonas spp. was investigated and further optimized. Two Plackett-Burman designs to study the effect of carbon source, nitrogen source, C/N ratio, iron concentration, magnesium concentration, phenol toxicity, pH, temperature, agitation and sampling time were tested. The first design revealed an optimization that increased biosurfactant productivity by almost two to fivefolds for the tested isolates. However, using the second design showed no remarkable increase in biosurfactant productivity. An additional validation run was adopted using the predicted optimal medium with predicted optimal conditions. The validation run showed remarkable increase in the productivity of the tested isolates. The use of microorganisms with biodegradation ability coupled with optimization of the parameters affecting productivity provides an efficient strategy for biosurfactant production.