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.

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.

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.

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.

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.

Detection, characterization, and molecular typing of human Mycoplasma spp. from major hospitals in Cairo, Egypt.

Mycoplasmas are fastidious slow growing organisms lacking a cell wall and mostly isolated from the mucosal surfaces of the respiratory and genitourinary tracts. There is a dearth of information regarding clinical Mycoplasma spp. isolates among Egyptian patients. A total of 170 samples were collected from patients and apparently healthy personnel in local public hospitals in Cairo, Egypt. Isolation of Mycoplasma spp. was carried out using appropriate culture media and further identification was carried out by biochemical tests followed by serotyping using specific antisera. Confirmation was done by PCR for detection of different Mycoplasma spp. using genus-specific primers targeting 16S ribosomal RNA gene. Characterization of the antibiotic resistance and sensitivity pattern against different antimicrobials was carried out using disc diffusion test. The results indicated the presence of six Mycoplasma spp. in 22.94% of the samples. Mycoplasmas were detected more frequently in throat swabs than sputum. Mycoplasma pneumoniae was highly sensitive to macrolides and quinolones but less sensitive to aminoglycosides and tetracyclines. Molecular techniques were found to be of more rapid, highly sensitive, able to detect nonviable organisms, and cost effective. These results shed light on difficulties of Mycoplasma detection and the superiority of molecular techniques over culture.