Molecular detection of different virulence factors genes harbor pslA, pelA, exoS, toxA and algD among biofilm-forming clinical isolates of Pseudomonas aeruginosa.

Pseudomonas aeruginosa (P. aeruginosa) is considered as the foremost cause of hospital-acquired infections due to its innate and plasmid-mediated resistance to multiple antibiotics making it a multi-drug resistant (MDR) pathogen. This study aimed to determine the biofilm formation ability and the presence of different virulence factors genes (pslA, pelA, exoS, toxA and algD) among biofilm-forming strains of P. aeruginosa clinical isolates from burn units in Ismailia Hospitals, Egypt. In our cross-sectional study, one hundred and twenty-six (126) non-duplicate clinical P. aeruginosa isolates were recovered from 450 clinical specimens from burn units in Ismailia Hospitals. The antibiotic sensitivity of strong and moderate biofilm producer isolates was investigated using the disc diffusion method. The isolated bacteria were tested for their ability to form biofilm using a microtiter plate assay. The expression of (pslA, pelA, exoS, toxA and algD) genes in biofilm producers isolates was detected using PCR. The MPA detected 80% (95 /126) isolates as biofilm producers, 18% (22/126) were strong biofilm producers, 34% (43/126) were moderate biofilm producers, 28% (35/126) were weak biofilm producers and 20% (31/126) non-biofilm producers. Susceptibility pattern analysis of biofilm-forming P. aeruginosa isolates (95) detected that 60% (68/ 95) were multi-drug resistant isolates (MDR). Resistance to all used antibiotics and multidrug resistance was higher among biofilm-producing than non-biofilm-producing strains, but the difference was statistically non-significant. Investigation of virulence factors associated genes revealed that 96%, 94%, 86.4%, 80.0% and 74% of the biofilm producers isolates were harboring algD, pslA, pel A, toxA and exoS gene, respectively. The present study confirmed that antimicrobial resistance and virulence genes were more prominent in biofilm-producing P. aeruginosa than in non-biofilm-producers.

Antiviral and protective effect of small interfering RNAs against rift valley fever virus in vitro.

BACKGROUND: Rift Valley Fever Virus (RVFV) is an arbovirus, a zoonotic disease that resurfaces as a potential hazard beyond geographic boundaries. Fever that can proceed to encephalitis, retinitis, hemorrhagic fever, and death is the main manifestation observed in human infections. RVFV has no authorized medication. The RNA interference (RNAi) gene silencing pathway is extremely well conserved. By targeting specific genes, small interfering RNA (siRNA) can be used to suppress viral replication. The aim of this study was to design specific siRNAs against RVFV and evaluate their prophylactic and antiviral effects on the Vero cells. METHODS AND RESULTS: Various siRNAs were designed using different bioinformatics tools. Three unique candidates were tested against an Egyptian sheep cell culture-adapted strain BSL-2 that suppressed RVFV N mRNA expression. SiRNAs were transfected a day before RVFV infection (pre-transfection), and 1 h after the viral infection (post-transfection), and were evaluated to detect the silencing activity and gene expression decrease using real-time PCR and a TCID50 endpoint test. The degree of N protein expression was determined by western blot 48 h after viral infection. D2 which targets the (488-506 nucleotides), the middle region of RVFV N mRNA was the most effective siRNA at 30 nM concentration, it almost eliminates N mRNA expression when utilized as antiviral or preventive therapy. siRNAs had a stronger antiviral silencing impact when they were post-transfected into Vero cells. CONCLUSION: Pre and post-transfection of siRNAs significantly reduced RVFV titer in cell lines, offering novel and potentially effective anti-RVFV epidemics and epizootics therapy.

Metagenomic and metatranscriptomic exploration of the Egyptian Red Sea sponge Theonella sp. associated microbial community.

Marine sponges associated microorganisms are considered to be prolific source of bioactive natural products. Omics-based techniques such as metagenomics and metatranscriptomics have been used as effective tools to discover natural products. In this study, we used integrated metagenomic and metatranscriptomic analysis of three samples of the Egyptian Red Sea sponge Theonella sp. microbiome to obtain a complete picture of its biosynthetic activity to produce bioactive compounds. Our data revealed high biodiversity of the Egyptian sponge microbiota represented by 38 bacterial phyla with Candidate Phylum Poribacteria as the most abundant phyla with an average of 27.5% of the microbial community. The analysis also revealed high biosynthetic activity of the sponge microbiome through detecting different types of biosynthetic gene clusters (BGCs) with predicted antibacterial, cytotoxic and inhibitory bioactivity and the majority of these clusters were found to be actively transcribed. The complete BGCs of the cytotoxic theonellamide and misakinolide were detected and found to be actively transcribed. The majority of the detected BGCs were predicted to be novel as they did not show any similarity with any known cluster in the MIBiG database.

Designing and fabrication of electrochemical nano-biosensor for the fast detection of SARS-CoV-2-RNA.

SARS-CoV-2 caused a global panic among populations. Rapid diagnostic procedures for the virus are crucial for disease control. Thus, the designed signature probe from a highly conserved region of the virus was chemically immobilized onto the nanostructured-AuNPs/WO3-screen printed electrodes. Different concentrations of the matched oligonucleotides were spiked to test the specificity of the hybridization affinity whereas the electrochemical impedance spectroscopy was used for tracking the electrochemical performance. After a full assay optimization, limits of detection and quantification were calculated based on linear regression and were valued at 298 and 994 fM, respectively. Further, the high performance of the fabricated RNA-sensor chips was confirmed after testing the interference status in the presence of the mismatched oligos in one nucleotide and completely one. Worthy to mention that the single-stranded matched oligos can be hybridized to the immobilized probe in 5 min at room temperature. The designed disposable sensor chips are capable of detecting the virus genome directly. Therefore, the chips are a rapid tool for SARS-CoV-2 detection.

Bioactivity of bacteria associated with Red Sea nudibranchs and whole genome sequence of Nocardiopsis dassonvillei RACA-4.

Microorganisms associated with marine invertebrates consider an important source of bioactive products. This study aimed to screen for antimicrobial and anticancer activity of crude extracts of bacteria associated with Red sea nudibranchs and molecular identification of the bioactive isolates using 16Sr RNA sequencing, in addition to whole-genome sequencing of one of the most bioactive bacteria. This study showed that bacteria associated with Red sea nudibranchs are highly bioactive and 16Sr RNA sequencing results showed that two isolates belonged to Firmicutes, and two isolates belonged to Proteobacteria, and Actinobacteria. The whole genome sequence data of the isolated Nocardiopsis RACA4 isolate has an estimated genome length of 6,721,839 bp and the taxonomy showed it belongs to the bacteria Nocardiopsis dassonvillei. The De novo assembly of RACA-4 paired reads using Unicycler v0.4.8 initially yielded 97 contigs with an N50 value of 214,568 bp and L50 value of 10, The resulting assembly was further mapped to the reference genome Nocardiopsis dassonvillei strain NCTC10488 using RagTag software v.2.1.0 and a final genome assembly resulted in 39 contigs and N50 value of 6,726,007 and L50 of 1. Genome mining using anti-smash showed around 9.1% of the genome occupied with genes related to secondary metabolites biosynthesis. A wide variety of secondary metabolites belonging to Polyketides, Terpenes, and nonribosomal peptides were predicted with high degree of similarity to known compounds. Non-characterized clusters were also found which suggest new natural compounds discovered by further studies.

Metataxanomic, bioactivity and microbiome analysis of Red Sea marine sponges from Egypt.

Red Sea marine sponges (phylum Porifera) and associated microorganisms harbor a wide range of microorganisms, which are considered an essential source of bioactive products. In this study, we screened both the crude extracts of Red Sea marine sponges and their associated bacterial extract for antimicrobial activity and antiviral activity. Molecular characterization of bioactive producers was performed using16S rRNA sequencing, in addition to metagenomic analysis of three representative sponges utilizing the 16S rRNA gene V3-V4 region sequencing in two different seasons. Twelve samples were collected from five different sponge species by scuba diving, and all the crude extracts of sponges showed antimicrobial activity except Negombata corticata. Moreover, 84 out of 110 bacterial isolates extracts demonstrated antimicrobial activity against at least one tested microorganism. These results revealed the bioactivity and biodiversity of the Red Sea marine invertebrates-associated bacteria. It was found that the bioactive isolates belong to several bacterial groups. The bacterial communities of Negombata magnifica, Negombata corticata, and Siphonochalina siphonella were shown with great diversity and differences in the bacterial percentage, diversity, and unique community composition at different seasons in each sponge species. Unique microenvironment for each sponge species may be linked to the production of specific bioactive product.

Design of Staphylococcus aureus New Vaccine Candidates with B and T Cell Epitope Mapping, Reverse Vaccinology, and Immunoinformatics.

An effective vaccine against Staphylococcus aureus infection is a major planetary heath priority, particularly with increasing antibiotic resistance worldwide. Previous efforts for a highly effective S. aureus vaccine were largely unsuccessful, in part, because the vaccine designs have tended to target mainly the B cell immunity and development of opsonic antibodies. In contrast, recent observations suggest that cell mediated immunity may be critical for protection against S. aureus. In addition, the S. aureus surface proteins are among the key immunodominant antigens because they are the first molecules to interact with the host organism cells and tissues. We report here an original vaccinomics study in which we used a reverse vaccinology and immunoinformatics in silico strategy integrated with genomics. After analyzing 2767 proteins, we defined 16 proteins of S. aureus as promising subunit vaccine candidates. Phosphatidylinositol phosphodiesterase (Plc) is secreted by extracellular pathogens such as S. aureus. We mapped the B and T cell epitopes for the Plc protein, tested the reactivity of the synthesized epitopes by Western blotting, and verified our findings in a pilot study of 10 patients with S. aureus infection. The peptides were then tested for their protective effect in groups of mice challenged with pathogenic S. aureus strain, which showed high protection level. These findings warrant further translational research for development of novel vaccines against S. aureus infection. Reverse vaccinology is an advanced approach that can be applied to identify new vaccine candidates against a host of microorganisms, including S. aureus.

Antimicrobial activity of bacteria isolated from Red Sea marine invertebrates.

Marine invertebrates-associated microorganisms were considered to be important sources of marine bioactive products. This study aims to isolate marine invertebrates associated bacteria with antimicrobial activity from the Red Sea and test their biosynthetic potential through the detection of PKS and NRPS gene clusters involved with the production of bioactive secondary metabolites. In this respect, fifty bacterial strains were isolated from eight different Red Sea marine invertebrates and screened for their antimicrobial activity against standard pathogenic bacteria (Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, Bacillus subtilis ATCC 6633) and yeast (Candida albicans ATCC 10231) using the standard well diffusion assay. Five isolates showed antifungal activity against Candida albicans with no activity recorded against other pathogenic bacterial strains. On the other hand when these isolates were screened for the presence of biosynthetic gene clusters (PKS and NRPS) by PCR using five sets of degenerative primers, 60% of the isolates were shown to contain at least one type of PKS and NRPS gene clusters, which indicates the biosynthetic potential of these isolates even if the isolates didn't express any biological activity in vitro. Moreover the 16S rRNA molecular identification of the isolates reveal the biodiversity of the red sea marine invertebrates associated bacteria as they were found to belong to several bacterial groups present in Alphaproteobacteria, Gammaproteobacteria, Actinobacteria and Firmicutes.

Antiviral activity of Ribavirin nano-particles against measles virus.

Measles virus considers an important cause of child morbidity and mortality in some areas as Africa. Ribavirin's activity as a nucleoside analog can disclose the surprisingly broad spectrum action against several RNA viruses under laboratory cell culture conditions. The Current study aimed to investigate the antiviral activity of ribavirin Nano gold particles (AuNPs) against measles virus on vero cell line. Ribavirin- AuNPs was prepared, characterization and the cytotoxicity of ribavirin, AuNPs and ribavirin -AuNPs were tested on vero cells using MTT assay. Antiviral activiry of ribavirin, AuNPs and ribavirin- AuNPswere determined on vero cells using simultaneous, pre-infection and post-infection protocols. Results indicated safety of ribavirin and ribavirin-AuNPs on vero cells, there was a reduction by 78.1% when vero cells treated with ribavirin -AuNPs at 99.5µg/ml while, the viral reduction was 25.4% when ribavirin 500 µg /ml was used for the same viral concentration. Our results concluded that ribavirin - AuNPs had a higher antiviral activity with lower dose than ribavirin alone and the maximal activity showed when it used after the virus infection.

Clonal Complexes and Antimicrobial Susceptibility Profiles of Staphylococcus pseudintermedius Isolates from Dogs in the United States.

Staphylococcus pseudintermedius is the primary cause of canine pyoderma and has been associated with diseases in other animals, including human beings. A high prevalence of methicillin and multidrug resistance has been reported in this bacterium in some geographic regions of the United States. Multilocus sequence type (MLST) 68 was implicated, initially, as the major clonal genotype based on a limited number of samples. The objectives of this study were to determine the population genetics of S. pseudintermedius isolated from a cross-section of the United States using a seven-locus multilocus sequence typing method, to identify clonal complexes (CCs), and to correlate sequence types with antimicrobial susceptibility profiles. A total of 190 S. pseudintermedius with 86 different MLSTs were detected and the constituents of three major CCs of methicillin-resistant S. pseudintermedius (MRSP), CC68, CC71, and CC84, were identified. Different patterns of resistance were associated with each CC. CC71 from the United States had notable differences with CC71 studied on other continents with chloramphenicol, tetracycline, and trimethoprim/sulfamethoxazole resistance. Some isolates with resistance to the broadest range of drugs tested, including that to chloramphenicol, had STs unrelated to the major CCs, suggesting the potential for the emergence of new clonal populations of MRSP that are resistant to most therapeutically useful antimicrobials.