Assessment of antibacterial activity and cytotoxic effects of in vitro and in vivo plant parts of a medicinal plant Gynura procumbens (Lour.) Merr.

The goal of this study was to evaluate the antibacterial and cytotoxic effects of both the in vitro and in vivo plant part extracts of the medicinal plant Gynura procumbens. An effective protocol for regeneration and callus formation was developed using nodal segments and regenerated leaf explants, respectively. The highest fresh and dry weight calli were produced after four weeks of culture on Murashige and Skoog (MS) medium containing 2.0 mg/L BAP and 2.0 mg/L NAA, while the most shoots were produced on MS medium containing 1.0 mg/L BAP and 0.5 mg/L IAA. The in vitro shoots developed roots on MS media with 0.1 mg/L IBA. The antibacterial activity of extracts against various bacteria was examined to determine their significance (p < 0.05). The least significant difference (LSD) test results showed that the regenerated leaf extract had the highest antibacterial activity while the callus extract had the lowest. The minimum bactericidal concentration (MBC) and the minimum inhibitory concentration (MIC) were also established. Regenerated leaf extract had the highest toxicity and the lowest lethal concentration (LC50) value (1.21 ± 0.03 µg/mL) in a brine shrimp lethality bioassay. In contrast, callus extract had the lowest toxicity and the highest LC50 (11.09 ± 0.4 µg/mL). In addition, the in vitro cytotoxicity test results revealed that the callus and field leaf extracts had anti-cell-proliferative properties. The regenerated leaf and stem extract, however, could induce cell growth.

In silico exploration of Serratia sp. BRL41 genome for detecting prodigiosin Biosynthetic Gene Cluster (BGC) and in vitro antimicrobial activity assessment of secreted prodigiosin.

The raising concern of drug resistance, having substantial impacts on public health, has instigated the search of new natural compounds with substantial medicinal activity. In order to find out a natural solution, the current study has utilized prodigiosin, a linear tripyrrole red pigment, as an active ingredient to control bacterial proliferation and prevent cellular oxidation caused by ROS (Reactive Oxygen Species). A prodigiosin-producing bacterium BRL41 was isolated from the ancient Barhind soil of BCSIR Rajshahi Laboratories, Bangladesh, and its morphological and biochemical characteristics were investigated. Whole genome sequencing data of the isolate revealed its identity as Serratia sp. and conferred the presence of prodigiosin gene cluster in the bacterial genome. "Prodigiosin NRPS", among the 10 analyzed gene clusters, showed 100% similarity with query sequences where pigC, pigH, pigI, and pigJ were identified as fundamental genes for prodigiosin biosynthesis. Some other prominent clusters for synthesis of ririwpeptides, yersinopine, trichrysobactin were also found in the chromosome of BRL41, whilst the rest displayed less similarity with query sequences. Except some first-generation beta-lactam resistance genes, no virulence and resistance genes were found in the genome of BRL41. Structural illumination of the extracted red pigment by spectrophotometric scanning, Thin-Layer Chromatography (TLC), Fourier Transform Infrared Spectroscopy (FTIR), and change of color at different pH solutions verified the identity of the isolated compound as prodigiosin. Serratia sp. BRL41 attained its maximum productivity 564.74 units/cell at temperature 30˚C and pH 7.5 in two-fold diluted nutrient broth medium. The compound exhibited promising antibacterial activity against Gram-positive and Gram-negative bacteria with MIC (Minimum Inhibitory Concentration) and MBC (Minimum Bactericidal Concentration) values ranged from 3.9 to15.62 µg/mL and 7.81 to 31.25 µg/mL respectively. At concentration 500 µg/mL, except in Salmonella enterica ATCC-10708, prodigiosin significantly diminished biofilm formed by Listeria monocytogens ATCC-3193, Pseudomonas aeruginosa ATCC-9027, Escherichia coli (environmental isolate), Staphylococcus aureus (environmental isolate). Cellular glutathione level (GSH) was elevated upon application of 250 and 500 µg/mL pigment where 125 µg/mL failed to show any free radical scavenging activity. Additionally, release of cellular components in growth media of both Gram-positive and Gram-negative bacteria were facilitated by the extract that might be associated with cell membrane destabilization. Therefore, the overall findings of antimicrobial, antibiofilm and antioxidant activities suggest that in time to come prodigiosin might be a potential natural source to treat various diseases and infections.

M gene targeted qRT-PCR approach for SARS-CoV-2 virus detection.

Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) is the gold standard method for SARS-CoV-2 detection, and several qRT-PCR kits have been established targeting different genes of the virus. Due to the high mutation rate of these genes, false negative results arise thus complicating the interpretation of the diagnosis and increasing the need of alternative targets. In this study, an alternative approach for the detection of SARS-CoV-2 viral RNA targeting the membrane (M) gene of the virus using qRT-PCR was described. Performance evaluation of this newly developed in-house assay against commercial qRT-PCR kits was done using clinical oropharyngeal specimens of COVID-19 positive patients. The limit of detection was determined using successive dilutions of known copies of SARS-CoV-2 pseudovirus. The M gene based assay was able to detect a minimum of 100 copies of virus/mL indicating its capacity to detect low viral load. The assay showed comparable accuracy, sensitivity and specificity with commercially available kits while detecting all the variants efficiently. The study concluded that the in-house M gene based assay might be an effective alternative for the currently available commercial qRT-PCR kits.

Computational design of medicinal compounds to inhibit RBD-hACE2 interaction in the Omicron variant: unveiling a vulnerable target site.

The COVID-19 pandemic, caused by SARS-CoV-2, has globally affected both human health and economy. Several variants with a high potential for reinfection and the ability to evade immunity were detected shortly after the initial reported case of COVID-19. A total of 30 mutations in the spike protein (S) have been reported in the SARS-CoV-2 (BA.2) variant in India and South Africa, while half of these mutations are in the receptor-binding domain and have spread rapidly throughout the world. Drug repurposing offers potential advantages over the discovery of novel drugs, and one is that it can be delivered quickly without lengthy assessments and time-consuming clinical trials. In this study, computational drug design, such as pharmacophore-based virtual screening and MD simulation has been concentrated, in order to find a novel small molecular inhibitor that prevents hACE2 from binding to the receptor binding domain (RBD). three medicinal compound databases: North-East African, North African, and East African were screened and carried out a multi-step screening approach that identified three compounds, which are thymoquinol 2-O-beta-glucopyranoside (C1), lanneaflavonol (C2), and naringenin-4'-methoxy-7-O-Alpha-L-rhamnoside (C3), with excellent anti-viral properties against the RBD of the omicron variant. Furthermore, PAIN assay interference, computation bioactivity prediction, binding free energy, and dissociation constant were used to validate the top hits, which indicated good antiviral activity. The three compounds that were found may be useful against COVID-19, though more research is required. These findings could aid the development of novel therapeutic drugs against the emerging Omicron variant of SARS-CoV-2.

A multicentre study reveals dysbiosis in the microbial co-infection and antimicrobial resistance gene profile in the nasopharynx of COVID-19 patients.

The impact of SARS-CoV-2 infection on the nasopharyngeal microbiome has not been well characterised. We sequenced genetic material extracted from nasopharyngeal swabs of SARS-CoV-2-positive individuals who were asymptomatic (n = 14), had mild (n = 64) or severe symptoms (n = 11), as well as from SARS-CoV-2-negative individuals who had never-been infected (n = 5) or had recovered from infection (n = 7). Using robust filters, we identified 1345 taxa with approximately 0.1% or greater read abundance. Overall, the severe cohort microbiome was least diverse. Bacterial pathogens were found in all cohorts, but fungal species identifications were rare. Few taxa were common between cohorts suggesting a limited human nasopharynx core microbiome. Genes encoding resistance mechanisms to 10 antimicrobial classes (> 25% sequence coverages, 315 genes, 63 non-redundant) were identified, with ß-lactam resistance genes near ubiquitous. Patients infected with SARS-CoV-2 (asymptomatic and mild) had a greater incidence of antibiotic resistance genes and a greater microbial burden than the SARS-CoV-2-negative individuals. This should be considered when deciding how to treat COVID-19 related bacterial infections.

Immunoinformatics approach to epitope-based vaccine design against the SARS-CoV-2 in Bangladeshi patients.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the ongoing coronavirus disease 2019 (COVID-19) pandemic which has brought a great challenge to public health. After the first emergence of novel coronavirus SARS-CoV-2 in the city of Wuhan, China, in December 2019. As of March 2020, SARS-CoV-2 was first reported in Bangladesh and since then the country has experienced a steady rise in infections, resulting in 13,355,191 cases and 29,024 deaths as of 27 February 2022. Bioinformatics techniques are used to predict B cell and T cell epitopes from the new SARS-CoV-2 spike glycoprotein in order to build a unique multiple epitope vaccine. The immunogenicity, antigenicity scores, and toxicity of these epitopes were evaluated and chosen based on their capacity to elicit an immune response. RESULT: The best multi-epitope of the possible immunogenic property was created by combining epitopes. EAAAK, AAY, and GPGPG linkers were used to connect the epitopes. In several computer-based immune response analyses, this vaccine design was found to be efficient, as well as having high population coverage. CONCLUSION: This research is entirely reliant on the development of epitope-based vaccines, and these in silico findings would represent a major step forward in the development of a vaccine that might eradicate SARS-CoV-2 in Bangladeshi patients.

Genomic characterization of the dominating Beta, V2 variant carrying vaccinated (Oxford-AstraZeneca) and nonvaccinated COVID-19 patient samples in Bangladesh: A metagenomics and whole-genome approach.

Bangladesh is experiencing a second wave of COVID-19 since March 2021, despite the nationwide vaccination drive with ChAdOx1 (Oxford-AstraZeneca) vaccine from early February 2021. Here, we characterized 19 nasopharyngeal swab (NPS) samples from COVID-19 suspect patients using genomic and metagenomic approaches. Screening for SARS-CoV-2 by reverse transcriptase polymerase chain reaction and metagenomic sequencing revealed 17 samples of COVID-19 positive (vaccinated = 10, nonvaccinated = 7) and 2 samples of COVID-19 negative. We did not find any significant correlation between associated factors including vaccination status, age or sex of the patients, diversity or abundance of the coinfected organisms/pathogens, and the abundance of SARS-CoV-2. Though the first wave of the pandemic was dominated by clade 20B, Beta, V2 (South African variant) dominated the second wave (January 2021 to May 2021), while the third wave (May 2021 to September 2021) was responsible for Delta variants of the epidemic in Bangladesh including both vaccinated and unvaccinated infections. Noteworthily, the receptor binding domain (RBD) region of S protein of all the isolates harbored similar substitutions including K417N, E484K, and N501Y that signify the Beta, while D614G, D215G, D80A, A67V, L18F, and A701V substitutions were commonly found in the non-RBD region of Spike proteins. ORF7b and ORF3a genes underwent a positive selection (dN/dS ratio 1.77 and 1.24, respectively), while the overall S protein of the Bangladeshi SARS-CoV-2 isolates underwent negative selection pressure (dN/dS = 0.621). Furthermore, we found different bacterial coinfections like Streptococcus agalactiae, Neisseria meningitidis, Elizabethkingia anophelis, Stenotrophomonas maltophilia, Klebsiella pneumoniae, and Pseudomonas plecoglossicida, expressing a number of antibiotic resistance genes such as tetA and tetM. Overall, this approach provides valuable insights on the SARS-CoV-2 genomes and microbiome composition from both vaccinated and nonvaccinated patients in Bangladesh.

SARS-CoV-2 infection reduces human nasopharyngeal commensal microbiome with inclusion of pathobionts.

The microbiota of the nasopharyngeal tract (NT) play a role in host immunity against respiratory infectious diseases. However, scant information is available on interactions of SARS-CoV-2 with the nasopharyngeal microbiome. This study characterizes the effects of SARS-CoV-2 infection on human nasopharyngeal microbiomes and their relevant metabolic functions. Twenty-two (n = 22) nasopharyngeal swab samples (including COVID-19 patients = 8, recovered humans = 7, and healthy people = 7) were collected, and underwent to RNAseq-based metagenomic investigation. Our RNAseq data mapped to 2281 bacterial species (including 1477, 919 and 676 in healthy, COVID-19 and recovered metagenomes, respectively) indicating a distinct microbiome dysbiosis. The COVID-19 and recovered samples included 67% and 77% opportunistic bacterial species, respectively compared to healthy controls. Notably, 79% commensal bacterial species found in healthy controls were not detected in COVID-19 and recovered people. Similar dysbiosis was also found in viral and archaeal fraction of the nasopharyngeal microbiomes. We also detected several altered metabolic pathways and functional genes in the progression and pathophysiology of COVID-19. The nasopharyngeal microbiome dysbiosis and their genomic features determined by our RNAseq analyses shed light on early interactions of SARS-CoV-2 with the nasopharyngeal resident microbiota that might be helpful for developing microbiome-based diagnostics and therapeutics for this novel pandemic disease.