Characterizations of biogenic selenium nanoparticles and their anti-biofilm potential against Streptococcus mutans ATCC 25175.

INTRODUCTION: S. mutans has been identified as the primary pathogenic bacterium in biofilm-mediated dental caries. The biogenic selenium nanoparticles (SeNPs) produced by L. plantarum KNF-5 were used in this study against S. mutans ATCC 25175. OBJECTIVES: The aims of this study were: (1) the biosynthesis of SeNPs by L. plantarum KNF-5, (2) the characterization of SeNPs, (3) the investigation of the inhibitory effect of biogenic SeNPs against S. mutans ATCC 25175, and (4) the determination of the anti-biofilm potential of SeNPS against S. mutans ATCC 25175. METHODOLOGY: 3 mL of the culture was added to 100 mL of MRS medium and incubated. After 4 h, Na2SeO3 solution (concentration 100 µg/mL) was added and incubated at 37 °C for 36 h. The color of the culture solution changed from brownish-yellow to reddish, indicating the formation of SeNPs. The characterization of SeNPs was confirmed by UV-Vis spectrophotometry, FTIR, SEM-EDS and a particle size analyzer. The antibacterial activity was determined by the disk diffusion method, the MIC by the micro-double dilution method, and the biofilm inhibitory potential by the crystal violet method and the MTT assay. The effect of SeNPs on S. mutans ATCC 25175 was determined using SEM and CLSM spectrometry techniques. The sulfate-anthrone method was used to analyze the effect of SeNPs on insoluble extracellular polysaccharides. The expression of genes in S. mutans ATCC 25175 was analyzed by real-time quantitative polymerase chain reaction (RT-qPCR). PREPARATION OF NANOPARTICLES: SeNPs produced by probiotic bacteria are considered a safe method. In this study, L. plantarum KNF-5 (probiotic strain) was used for the production of SeNPs. RESULTS: The biogenic SeNPs were spherical and coated with proteins and polysaccharides and had a diameter of about 270 nm. The MIC of the SeNPs against S. mutans ATCC 25175 was 3.125 mg/mL. Biofilm growth was also significantly suppressed at this concentration. The expression of genes responsible for biofilm formation (GtfB, GtfC, BrpA and GbpB,) was reduced when S. mutans ATCC 25175 was treated with SeNPs. CONCLUSION: It was concluded that the biogenic SeNPs produced by L. plantarum KNF-5 was highly effective to inhibit the growth of S. mutans ATCC 25175. NOVELTY STATEMENT: The application of biogenic SeNPs, a natural anti-biofilm agent against S. mutans ATCC 25175. In the future, this study will provide a new option for the prevention and treatment of dental caries.

Study of selenium enrichment metabolomics in Bacillus subtilis BSN313 via transcriptome analysis.

In this study, the transcriptome analysis was practiced to identify potential genes of probiotic Bacillus subtilis BSN313 involved in selenium (Se) enrichment metabolism. The transcriptomic variation of the strain was deliberated in presence of three different sodium selenite concentrations (0, 3, and 20 µg/mL). The samples were taken at 1 and 13 h subsequent to inoculation of selenite and gene expression profiles in Se metabolism were analyzed through RNA sequencing. The gene expression levels of the pre log phase were lower than the stationary phase. It is because, the bacteria has maximum grown with high concentration of Se (enriched with organic Se), at stationary phase. Bacterial culture containing 3 µg/mL concentration of inorganic Se (sodium selenite) has shown highest gene expression as compared to no or high concentration of Se. This concentration (3 µg/mL) of sodium selenite (as Se) in the medium promoted the upregulation of thioredoxin reductase expression, whereas its higher Se concentration inhibited the formation of selenomethionine (SeMet). The result of 5 L bioreactor fermentation showed that SeMet was also detected in the fermentation supernatant as the growth entered in the late stationary phase and reached up to 857.3 ng/mL. The overall intracellular SeMet enriched content in BSN313 was extended up to 23.4 µg/g dry cell weight. The other two selenoamino acids (Se-AAs), methyl-selenocysteine, and selenocysteine were hardly detected in medium supernatant. From this study, it was concluded that SeMet was the highest content of organic Se byproduct biosynthesized by B. subtilis BSN313 strain in Se-enriched medium during stationary phase. Thus, B. subtilis BSN313 can be considered a commercial probiotic strain that can be used in the food and pharmaceutical industries. This is because it can meet the commercial demand for Se-AAs (SeMet) in both industries.

Expression of BSN314 lysozyme genes in Escherichia coli BL21: a study to demonstrate microbicidal and disintegarting potential of the cloned lysozyme.

This study is an extension of our previous studies in which the lysozyme was isolated and purified from Bacillus subtilis BSN314 (Naveed et al., 2022; Naveed et al., 2023). In this study, the lysozyme genes were cloned into the E. coli BL21. For the expression of lysozyme in E. coli BL21, two target genes, Lyz-1 and Lyz-2, were ligated into the modified vector pET28a to generate pET28a-Lyz1 and pET28a-Lyz2, respectively. To increase the production rate of the enzyme, 0.5-mM concentration of IPTG was added to the culture media and incubated at 37 °C and 220 rpm for 24 h. Lyz1 was identified as N-acetylmuramoyl-L-alanine amidase and Lyz2 as D-alanyl-D-alanine carboxypeptidase. They were purified by multi-step methodology (ammonium sulfate, precipitation, dialysis, and ultrafiltration), and antimicrobial activity was determined. For Lyz1, the lowest MIC/MBC (0.25 µg/mL; with highest ZOI = 22 mm) were recorded against Micrococcus luteus, whereas the highest MIC/MBC with lowest ZOI were measured against Salmonella typhimurium (2.50 µg /mL; with ZOI = 10 mm). As compared with Aspergillus oryzae (MIC/MFC; 3.00 µg/mL), a higher concentration of lysozyme was required to control the growth of Saccharomyces cerevisiae (MIC/MFC; 50 µg/mL). Atomic force microscopy (AFM) was used to analyze the disintegrating effect of Lyz1 on the cells of selected Gram-positive bacteria, Gram-negative bacteria, and yeast. The AFM results showed that, as compared to Gram-negative bacteria, a lower concentration of lysozyme (Lyz1) was required to disintegrate the cell of Gram-positive bacteria.

Nutritional composition assessment and antimicrobial activity of Catostylus perezi, jellyfish blooms along the coast of Pakistan: an awareness to avoid food neophobia in Pakistan.

This study highlighted the nutritional importance of Catostylus perezi (an edible jellyfish) in Pakistan; a society where a large proportion of the population suffers from malnutrition, while C. perezi, a blessing of the sea, is wasted or exported. In the present study, the amino acid and fatty acid profiles of the oral arms and umbrella of C. perezi were determined by HPLC. The total amino acid concentration (ΣAA) in the oral arms was 151.19 mg/100g, while in the umbrella it was 100.17 mg/100g. The ratio of total essential amino acids to total non-essential amino acids (TEAA/TNEAA) was 0.72 in the oral arms, while it was 0.70 in the umbrella. Higher amount of ω-3 with lower ratio of ω-6/ω-3 in oral arms (0.52), rather umbrella (ω-6/ω-3 ratio; 0.62). The antimicrobial activity, MIC, MBC, and MFC of the whole body of the edible jellyfish were determined. On the basis of polarity, different solvents were used, e.g. water, methanol, dichloromethane, chloroform, and n-hexane. Among all the extracts, the water extract gave the highest ZOI against C. xerosis (29 mm), while the n-hexane extract gave the lowest ZOI against S. aureus (MRSA) ATCC 33591 (8.20 mm). The water extract of C. perezi had high potential against C. xerosis with the highest AMI and PAI (1.53 and 153, respectively), while the same extract had the highest TAI against E. coli (81.43 mL/g). For fungi/yeast, the methanolic extract had the highest ZOI (29.70 mm) against S. cerevisiae and the lowest MIC/MFC (2.40 µg/mL) against the same pathogen. The n-Hexane extract gave the lowest ZOI (11.10 mm) against P. variotii and the highest MIC/MFC (31.60 µg/mL) against Penicillium sp. Atomic force microscopy (AFM) was used to analyse the disintegrating effect of the extracts (with the highest antimicrobial effect) on the cells of selected Gram-positive, Gram-negative and yeast species. The amino acid and fatty acid profiles and antimicrobial assessment showed that C. perezi has great nutritional importance, so the use of C. perezi as food is highly recommended for the Pakistani community.

Purification, Characterization and Bactericidal Action of Lysozyme, Isolated from Bacillus subtillis BSN314: A Disintegrating Effect of Lysozyme on Gram-Positive and Gram-Negative Bacteria.

In the present study, lysozyme was purified by the following multi-step methodology: salt (ammonium sulfate) precipitation, dialysis, and ultrafiltration. The lysozyme potential was measured by enzymatic activity after each purification step. However, after ultrafiltration, the resulting material was considered extra purified. It was concentrated in an ultrafiltration centrifuge tube, and the resulting protein/lysozyme was used to determine its bactericidal potential against five bacterial strains, including three gram-positive (Bacillus subtilis 168, Micrococcus luteus, and Bacillus cereus) and two gram-negative (Salmonella typhimurium and Pseudomonas aeruginosa) strains. The results of ZOI and MIC/MBC showed that lysozyme had a higher antimicrobial activity against gram-positive than gram-negative bacterial strains. The results of the antibacterial activity of lysozyme were compared with those of ciprofloxacin (antibiotic). For this purpose, two indices were applied in the present study: antimicrobial index (AMI) and percent activity index (PAI). It was found that the purified lysozyme had a higher antibacterial activity against Bacillus cereus (AMI/PAI; 1.01/101) and Bacillus subtilis 168 (AMI/PAI; 1.03/103), compared to the antibiotic (ciprofloxacin) used in this study. Atomic force microscopy (AFM) was used to determine the bactericidal action of the lysozyme on the bacterial cell. The purified protein was further processed by gel column chromatography and the eluate was collected, its enzymatic activity was 21.93 U/mL, while the eluate was processed by native-PAGE. By this analysis, the un-denatured protein with enzymatic activity of 40.9 U/mL was obtained. This step shows that the protein (lysozyme) has an even higher enzymatic potential. To determine the specific peptides (in lysozyme) that may cause the bactericidal potential and cell lytic/enzymatic activity, the isolated protein (lysozyme) was further processed by the SDS-PAGE technique. SDS-PAGE analysis revealed different bands with sizes of 34 kDa, 24 kDa, and 10 kDa, respectively. To determine the chemical composition of the peptides, the bands (from SDS-PAGE) were cut, enzymatically digested, desalted, and analyzed by LC-MS (liquid chromatography-mass spectrometry). LC-MS analysis showed that the purified lysozyme had the following composition: the number of proteins in the sample was 56, the number of peptides was 124, and the number of PSMs (peptide spectrum matches) was 309. Among them, two peptides related to lysozyme and bactericidal activities were identified as: A0A1Q9G213 (N-acetylmuramoyl-L-alanine amidase) and A0A1Q9FRD3 (D-alanyl-D-alanine carboxypeptidase). The corresponding protein sequence and nucleic acid sequence were determined by comparison with the database.

Banned Sudan dyes in spices available at markets in Karachi, Pakistan.

Sudan dyes were investigated in branded and non-branded spices, commonly available in the markets of Karachi, Pakistan. High performance liquid chromatography (HPLC) with a variable wavelength detector (VWD) was applied to determine Sudan dyes I-IV. The non-branded samples had higher concentrations of Sudan dyes than the maximum limits of 0.1 mg/kg. The highest concentration of Sudan dye (I) was found in turmeric powder (8460 mg/kg) and the lowest concentration (1.50 mg/kg) of Sudan (IV) in Chaat Masala. This indicates that the use of non-branded spices is not safe, whereas no Sudan dye was found in the branded spice samples. Further studies regarding the higher carcinogenic risk posed by Sudan dye adulterated spices in Pakistan is strongly advised.

Biogenic Selenium Nanoparticles and Their Anticancer Effects Pertaining to Probiotic Bacteria-A Review.

Selenium nanoparticles (SeNPs) can be produced by biogenic, physical, and chemical processes. The physical and chemical processes have hazardous effects. However, biogenic synthesis (by microorganisms) is an eco-friendly and economical technique that is non-toxic to human and animal health. The mechanism for biogenic SeNPs from microorganisms is still not well understood. Over the past two decades, extensive research has been conducted on the nutritional and therapeutic applications of biogenic SeNPs. The research revealed that biogenic SeNPs are considered novel competitors in the pharmaceutical and food industries, as they have been shown to be virtually non-toxic when used in medical practice and as dietary supplements and release only trace amounts of Se ions when ingested. Various pathogenic and probiotic/nonpathogenic bacteria are used for the biogenic synthesis of SeNPs. However, in the case of biosynthesis by pathogenic bacteria, extraction and purification techniques are required for further useful applications of these biogenic SeNPs. This review focuses on the applications of SeNPs (derived from probiotic/nonpathogenic organisms) as promising anticancer agents. This review describes that SeNPs derived from probiotic/nonpathogenic organisms are considered safe for human consumption. These biogenic SeNPs reduce oxidative stress in the human body and have also been shown to be effective against breast, prostate, lung, liver, and colon cancers. This review provides helpful information on the safe use of biogenic SeNPs and their economic importance for dietary and therapeutic purposes, especially as anticancer agents.

Biosynthesis of Selenium Nanoparticles (via Bacillus subtilis BSN313), and Their Isolation, Characterization, and Bioactivities.

Among the trace elements, selenium (Se) has great demand as a health supplement. Compared to its other forms, selenium nanoparticles have minor toxicity, superior reactivity, and excellent bioavailability. The present study was conducted to produce selenium nanoparticles (SeNPs) via a biosynthetic approach using probiotic Bacillus subtilis BSN313 in an economical and easy manner. The BSN313 exhibited a gradual increase in Se reduction and production of SeNPs up to 5-200 µg/mL of its environmental Se. However, the capability was decreased beyond that concentration. The capacity for extracellular SeNP production was evidenced by the emergence of red color, then confirmed by a microscopic approach. Produced SeNPs were purified, freeze-dried, and subsequently characterized systematically using UV-Vis spectroscopy, FTIR, Zetasizer, SEM-EDS, and TEM techniques. SEM-EDS analysis proved the presence of selenium as the foremost constituent of SeNPs. With an average particle size of 530 nm, SeNPs were shown to have a -26.9 (mV) zeta potential and -2.11 µm cm/Vs electrophoretic mobility in water. SeNPs produced during both the 24 and 48 h incubation periods showed good antioxidant activity in terms of DPPH and ABST scavenging action at a concentration of 150 µg/mL with no significant differences (p > 0.05). Moreover, 200 µg/mL of SeNPs showed antibacterial reactivity against Escherichia coli ATCC 8739, Staphylococcus aureus ATCC 9027, and Pseudomonas aeruginosa ATCC 25923. In the future, this work will be helpful to produce biogenic SeNPs using probiotic Bacillus subtilis BSN313 as biofactories, with the potential for safe use in biomedical and nutritional applications.

A Size-dependent Bioaccumulation of Metal Pollutants, Antibacterial and Antifungal Activities of Telescopium telescopium, Nerita albicilla and Lunella coronata.

We assessed metal/metalloid pollutants (through multi-indices) in seawater, sediments, tissues and shells of gastropods using various indices such as contamination degree (modified/unmodified; Cd/mCd; 1875/187.5). From sediment indices; e.g. the potential ecological risk index/enrichment factor (Eri/EF; 3396.8/105.5) indicated the area to be highly contaminated with metal/metalloid pollutants. Indeed, bioaccumulation with these materials was gastropod size dependent. Antimicrobial and percentage activity indices (AMI/PAI) for; T. telescopium was (AMI/PAI; 1.59/159), N. albicilla (1.14/114) and L. coronata (0.95/95) against E. coli. Similarly T. telescopium (1.33/133), N. albicilla (1.19/119) and L. coronata (1.14/114) have AMI/PAI against A. terreus. The total activity index (TAI), for T. telescopium was the highest, while L. coronata has lowest for all pathogens. This study indicates, T. telescopium, N. albicilla and L. coronata, surviving under metal/metalloid stress exhibited altered natural defense to pathogens which was related to the degree of toxin bioaccumulation.

Evaluation of heavy metal pollutants in salt and seawater under the influence of the Lyari River and potential health risk assessment.

Heavy metals intoxication through edible salt poses serious health hazards. The conducted research assessed the levels of heavy metals, health risks of salt samples, and the pollution index of seawater obtained from saltpans located at Sandpit, Karachi, Pakistan, which receive untreated effluents through the Lyari River. Seawater (n = 27) and salt samples (n = 27) were prepared for the detection of Al, Cd, Pb, Cr, Fe, Cu, Hg, Ni, As and Zn through atomic absorption spectroscopy, mean concentrations ± S.D. (mg/mL) were compared with the National Environmental Quality Standards, Pakistan. The levels of Cr (40.06 ± 0.21) were the highest followed by Fe (39.77 ± 0.08) > As (25.12 ± 0.21) > Ni > Pb > Al > Hg > Zn > Cd > Cu. In sea salt (Mean ± S·D mg/Kg), the Cr (47.79 ± 0.19), Fe (47.5 ± 0.15), As (30.62 ± 0.22), and Ni were redundant elements followed by Al > Pb > Hg > Zn > Cu > Cd. The water comprehensive pollution index was 1000 times greater than the critical values. The single factor pollution index was highest for Hg (1321), followed by Cr (40), Ni (36), Pb (32), Al (31.4), Cd (31.3), and As (25). Health risk assessment indices (from salt samples), including target hazard quotient (THQ) for As, Pb, Al, Hg, and Cr were two to six times higher than the noncarcinogenic THQ reference range. Similarly, the hazardous index indicated that salt was 20 times hazardous (HI = 20.29), and the carcinogenic rate index for the heavy metals i.e., Cd, As, Cr, and Ni was above the reference CR value i.e., 1 × 10-4. In conclusion, the experimental and theoretical approaches recommend that the use of contaminated salt may impart health hazards.

A comprehensive assessment of environmental pollution by means of heavy metal analysis for oysters' reefs at Hab River Delta, Balochistan, Pakistan.

The heavy metal pollution status of oyster reefs has been assessed with respect to ten metals pollutants in seawater, sediments, and tissues of above two oysters (soft tissues and shells) for assessing the pollution status in a short food chain in Hab River Delta. The results showed that heavy metals accumulated in M. bilineata were higher than those in M. cuttackensis. Simultaneously, the population of M. bilineata species has been ironically decreasing as a results of high pollution. The determined concentrations revealed a significant differences in their profiles among sediments, seawater and bioaccumulation in tissues and shells of two native oysters. The present study also compared these metal concentrations with national and international database by applying different pollution indices. Heavy metals in all samples were above the national environmental quality standards (NEQS-Pakistan). High level of pollution with an alarming condition of Hab River Delta need more attention for coastal management.