Biosynthesis of silver nanoparticle using Bacillus licheniformis culture-supernatant for combating pathogenic biofilms.

Bacterial biofilms pose a significant threat to healthcare due to their recalcitrance to antibiotics and disinfectants. This study explores the anti-biofilm potential of Bacillus licheniformis cell-free culture supernatant (CFS) and its derived silver nanoparticles (bSNPs) against Staphylococcus aureus and Pseudomonas aeruginosa. The CFS exhibited potent anti-biofilm activity against both bacterial species, even at low concentrations, while devoid of significant bactericidal effects, mitigating resistance risks. Characterization studies revealed the non-proteinaceous nature and thermal stability of the CFS's anti-biofilm agent, suggesting a robust and heat-resistant structure. Green synthesis of bSNPs from CFS resulted in nanoparticles with significant anti-biofilm properties, particularly against P. aeruginosa, indicating differences in susceptibility between the bacterial species. Epifluorescence microscopy confirmed bSNPs' ability to inhibit and partially disrupt biofilm formation without inducing cellular lysis. The study highlights the potential of B. licheniformis CFS and bSNPs as promising biofilm control agents, offering insights into their mechanisms of action and broad-spectrum efficacy. Further research elucidating the underlying molecular mechanisms and identifying specific bioactive compounds is warranted for the translation of these findings into clinically relevant applications for combating biofilm-associated infections.

Combating bacterial biofilms and related drug resistance: Role of phyto-derived adjuvant and nanomaterials.

The emergence of antimicrobial resistance (AMR) in clinical microbes has led to a search for novel antibiotics for combating bacterial infections. The treatment of bacterial infections becomes more challenging with the onset of biofilm formation. AMR is further accelerated by biofilm physiology and differential gene expression in bacteria with an inherent resistance to conventional antibiotics. In the search for innovative strategies to control the spread of AMR in clinical isolates, plant-derived therapeutic metabolites can be repurposed to control biofilm-associated drug resistance. Unlike antibiotics, designed to act on a single cellular process, phytochemicals can simultaneously target multiple cellular components. Furthermore, they can disrupt biofilm formation and inhibit quorum sensing, offering a comprehensive approach to combat bacterial infections. In bacterial biofilms, the first line of AMR is due to biofilms associated with the extracellular matrix, diffusion barriers, quorum sensing, and persister cells. These extracellular barriers can be overcome using phytochemical-based antibiotic adjuvants to increase the efficacy of antibiotic treatment and restrict the spread of AMR. Furthermore, phytochemicals can be used to target bacterial intracellular machinery such as DNA replication, protein synthesis, efflux pumps, and degrading enzymes. In parallel with pristine phytochemicals, phyto-derived nanomaterials have emerged as an effective means of fighting bacterial biofilms. These nanomaterials can be formulated to cross the biofilm barriers and function on cellular targets. This review focuses on the synergistic effects of phytochemicals and phyto-derived nanomaterials in controlling the progression of biofilm-related AMR. IT provides comprehensive insights into recent advancements and the underlying mechanisms of the use of phyto-derived adjuvants and nanomaterials.

Starch wall of urea: Facile starch modification to residue-free stable urea coating for sustained release and crop productivity.

Quick leaching of urea fertilizer encourages different coatings, but achieving a stable coating without toxic linkers is still challenging. Here, the naturally abundant bio-polymer, i.e., starch, has been groomed to form a stable coating through phosphate modification and the support of eggshell nanoparticles (ESN) as a reinforcement agent. The ESN offers a calcium ion binding site for the phosphate to cause bio-mimetic folding. This coating retains hydrophilic ends in the core and gives an excellent hydrophobic surface (water contact angle 123°). Further, the phosphorylated starch+ESN led the coating to release only ∼30 % of the nutrient in the initial ten days and sustained for up to 60 days to show ∼90 % release. The stability of the coating has been attributed to its resistance to major soil factors viz., acidity and amylase degradation. The ESN also increases elasticity, cracking control, and self-repairing capacity by serving as buffer micro-bots. The coated urea enhanced the yield of rice grain by ∼10%.

Sequestration of cobalt and nickel by biofilm forming bacteria isolated from spent nuclear fuel pool water.

In the current study, six bacterial types, isolated from spent nuclear fuel (SNF) pool facility, were investigated for their ability to sequester heavy metals (cobalt and nickel). Biofilm formation by the six bacterial isolates, viz., Bacillus subtilis, Staphylococcus species, Staphylococcus arlettae, Staphylococcus epidermidis, Staphylococcus auricularis, and Chryseobacterium gleum, were assayed, and they were found to have significant biofilm forming property. Their biofilms were characterised using confocal scanning laser microscopy, and their potential to accumulate Co2+ and Ni2+ from bulk solutions was analysed with respect to time. A comparative assessment of bioaccumulation capacity was done using biofilms, planktonic cells, and live vs dead cells. The strains accumulated Co2+ and Ni2+ in the range of 4 × 10-4 to 1 × 10-5 g/mg of cell biomass. It is interesting to note that dead biomass also showed significant removal of the two metal ions, suggesting an alternative process for metal removal. This study suggests that hostile environments can be a repertoire of putative bacterial species with potential heavy metals and other contaminants remediation properties.

Targeting hydrophobicity in biofilm-associated protein (Bap) as a novel antibiofilm strategy against Staphylococcus aureus biofilm.

In this study, a comprehensive in silico characterization was performed on Bap-family proteins to develop novel approaches to deal with Staphylococcus biofilms with a better understanding of the functional, structural, and topological features of Bap proteins. This study showed that Bap-like proteins in staphylococci are highly acidic, large, and cell-wall anchored proteins with tandem repeats. Structurally, Bap-family proteins have two distinct parts. N terminal part, which contains at least 2-3 calcium-binding EF-hand motifs that play a regulatory role in Bap functioning. Whereas the C-terminal part which predominantly consists of tandem repeats (TR), plays a functional as well as structural role. Bioinformatic analysis of Bap proteins and other homologous proteins revealed the presence of an amyloidogenic heptapeptide (STVTVTF) in the hydrophobic core of TRs of protein, responsible for the protein-protein interactions. The synthetic heptapeptide was tested if the masking effect on surface proteins could inhibit the S. aureus biofilm development and act as an 'antibiofilm-peptide'. The results clearly showed that the heptapeptide was able to inhibit early adhesion as well as biofilm development in the S. aureus biofilms. This approach has a promising potential to treat persistent biofilm-based S. aureus infections where Bap-like proteins do play a significant role.

The role of S-layer protein (SlpA) in biofilm-formation of Deinococcus radiodurans.

AIMS: To investigate the molecular basis of biofilm formation in a recombinant lab strain of Deinococcus radiodurans with a plasmid harbouring gfp and kanR that acquired the biofilm-forming ability. METHODS AND RESULTS: Deinococcus radiodurans R1 is known as a nonbiofilm former bacterium and so far there are no reports on its biofilm-producing capabilities. In this study, we investigated the molecular basis of biofilm formation in a recombinant strain of D. radiodurans using classical biofilm assays, confocal laser scanning microscopy and real-time PCR. Biochemical analysis of D. radiodurans biofilm matrix revealed that it consisted predominantly of protein and carbohydrate complexes with a little amount of extracellular DNA (eDNA). Furthermore, studies showed that D. radiodurans biofilm formation was enhanced in the presence of 25 mM Ca2+ , which enhanced the exopolysaccharide and protein content in the biofilm matrix. Enzymatic treatments with proteinase K, alginate lyase and DNase I indicated the involvement of some proteinaceous components to be critical in the biofilm formation. RT-PCR studies showed that increased expression of a surface layer protein SlpA conferred the biofilm ability to D. radiodurans. CONCLUSION: Overexpression of SlpA in D. radiodurans conferred the biofilm formation ability to the bacterium, in which a partial role was also played by the recombinant plasmid pKG. It was also shown that the presence of Ca2+ in the growth medium enhanced SlpA production, thus improving biofilm stability and biofilm maturation of D. radiodurans. SIGNIFICANCE AND IMPACT: This study shows how biofilm formation can be augmented in D. radiodurans. The finding has implications for the development of D. radiodurans biofilm-based biotechnological applications.

Porous Silica Biofiber: A Reusable, Sustainable Fertilizer Reservoir.

Nitrogen fertilizers, namely urea, are prone to leaching that causes inefficiency in crop production and environmental pollution; hence porous particles were explored for slow release. Nevertheless, discrete particles add cost; therefore, jute cellulose has been tested as twine to tether silica together for reusability. On the other hand, silica serves as an exoskeleton to give pore memory property to cellulose, which otherwise is susceptible to loss of porosity during irrigation. The composite shows ∼70% more absorption capacity in the fifth cycle than the fiber without silica coating. The urea release kinetics shows only <1/3 and 3/4 of urea release from the jute-silica composite compared to naked porous silica and cellulose, respectively. The slow and sustained release of fertilizer from the composite results in a continuous increase in the chlorophyll content in rice crops.

Kinetic modelling of the uranium biosorption by Deinococcus radiodurans biofilm.

Increasing number of reports on uranium contamination in groundwater bodies is a growing concern. Deinococcus radiodurans biofilm-based U(VI) bioremediation has great potential to provide solution. This study focuses on the kinetic modelling of uranium biosorption by D. radiodurans biofilm biomass and identification of the functional groups involved in the sequestration process. The effect of temperature, pH and amount of biofilm dry mass were studied using two uranyl ion concentrations (100 and 1000 mg/L). D. radiodurans dry biomass showed good affinity for uranyl ion adsorption. The kinetic experiments revealed that the biosorption process was spontaneous and exothermic in nature. The modelling of kinetic adsorption data revealed that U(VI) sorption by D. radiodurans biofilm biomass follows a pseudo-second-order reaction. Mechanism of U(VI) sorption was suggested to follow an intra-particle diffusion model, which includes covalent bonding between U(VI) and functional groups present on the surface of biofilm biomass, and diffusional barrier acts as a rate limiting step. External mass transfer was the rate-limiting step as evident from Boyd and Elovich plot. Chemical modifications in surface functional groups of biofilm biomass, confirmed the involvement of carboxyl, phosphate, and hydroxyl groups in uranium binding as a significant loss in U(VI) sorption capacity was recorded in these chemically modified biomasses. XRD data indicated the formation of metal deposits, predominantly as uranyl phosphates.

Uranium bioremediation by acid phosphatase activity of Staphylococcus aureus biofilms: Can a foe turn a friend?

In this study, Staphylococcus aureus biofilms, which are considered a foe for being pathogenic, were tested for their uranium bioremediation capacity to find out if they can turn out to be a friend. Acid phosphatase activity, which is speculated to aid in bio-precipitation of U(VI) from uranyl nitrate solution, was assayed in biofilms of seven different S. aureus strains. The presence of acid phosphatase enzyme was detected in the biofilms of all S. aureus strains (in the range of 3.1 ± 0.21 to 26.90 ± 2.32 µi.u./g), and found to be higher when compared to that of their planktonic phenotypes. Among all, S. aureus V329 biofilm showed highest biofilm formation ability along with maximum phosphatase activity (26.9 ± 2.32 µi.u./g of biomass). Addition of phosphate enhanced the U(VI) remediation when treated with uranyl nitrate solution. S. aureus V329 biofilm showed significant U tolerance with only a 3-log reduction when exposed to 10 ppm U(VI) for 1 h. When treated in batch mode, V329 biofilm successfully remediated up to 47% of the 10 ppm U(VI). This new approach using the acid phosphatase from the S. aureus V329 biofilm presents an alternative method for the remediation of uranium contamination.

Microbiota of spent nuclear fuel pool water with emphasis on their biofilm forming ability on stainless steel (SS-304L).

Spent nuclear fuel (SNF) pool is an essential unit of a nuclear power plant infrastructure, where radioactive fuel rods are kept for cooling and shielding, before reprocessing. This study explored the presence of bacteria in SNF pool water with emphasis on their capability to form biofilms on pool wall cladding material stainless steel (SS-304L). Bacteria were isolated from SNF pool water and were characterized using 16S rRNA gene sequencing. The six bacterial isolates (Bacillus subtilis, Staphylococcus sps., S. arlettae, S. epidermidis, S. auricularis and Chryseobacterium gleum) can grow and form biofilms at very low nutrient condition as well as in chronic radioactivity. The bacterial isolates formed biofilm on SS-304L and glass. However, the biofilm parameters assessed by CLSM microscopy showed that the strains preferred SS-304L surface for biofilm formation. On SS-304L, the maximum biomass (0.45 l µm3/l µm2) was formed by S. arlettae when compared to maximum biomass (0.054 l µm3/l µm2) by Staphylococcus sp., on glass. Maximum biofilm thickness on SS- 304L was observed by Staphylococcus sp. (8.81 l µm) when compared to that of S. epidermidis (4.16 l µm) on the glass surface. The biofilm formation on SS-304L surface suggests the possible risk of microbial-induced corrosion of SNF pool cladding material. This study highlights the need for mandatory monitoring of microbial biofilm formation in an extreme environment such as SNF pool.

A new uranium bioremediation approach using radio-tolerant Deinococcus radiodurans biofilm.

Deinococcus radiodurans is the most radiation-tolerant organism ever known. It has gained importance in recent years as a potential candidate for bioremediation of heavy metals, especially the radioactive type. This study investigates the efficiency of a recombinant D. radiodurans (DR1-bf+) strain with an ability to form biofilm for uranium remediation. The modified Arsenazo III dye method was used to estimate the uranium concentration. Uranyl nitrate aqueous solution was generated during the operation of nuclear fuel reprocessing. The D. radiodurans biofilm (DR1-bf+) grown in the presence of 20 mM Ca2+ showed remarkable ability of uranyl ion removal. DR1-bf+ (Ca2+) biofilm removed ~75+/-2% of 1000 mg/L uranium within 30 min post-treatment from uranyl nitrate aqueous solution. Uranium removal rate was also found to be directly proportional to biofilm age. This study discusses the ability of D. radiodurans biofilm in uranium removal.

3'O-Methyltransferase, Ps3'OMT, from opium poppy: involvement in papaverine biosynthesis.

KEY MESSAGE: Using, in silico, in vitro and in planta functional assays, we demonstrate that Ps3'OMT, an 3'-O methyl transferase is linked to papaverine biosynthesis in opium poppy. Papaverine, one of the benzylisoquinoline alkaloids (BIA) synthesized in the medicinally important plant, Papaver somniferum, is known for the potent pharmacological properties. Papaverine biosynthesis has remained debatable as two different pathways, NH (involving N-desmethylated intermediates) and the NCH3 (involving N-methylated intermediates), have been proposed. In addition, there are several intermediate steps in both the proposed pathways that are not very well characterized in terms of specific enzymes. In this study, we report the identification and functional characterization of 3'O-methyltransferase (Ps3'OMT) which might participate in the 3'O-methylation of the intermediates in the papaverine biosynthesis. Comparison of transcript and metabolite profiles of high and low papaverine producing cultivar revealed the occurrence of a 3'O-methyltransferase, Ps3'OMT, which was abundant in aerial organs and shared 72% identity with the GfLOMT7 predicted to have 3'OMT activity. In silico studies based on homology modeling, docking and MD simulations predicted (S)-norlaudanine as the potential substrate forming a stable complex with Ps3'OMT. Suppression of Ps3'OMT through virus-induced gene silencing resulted in a remarkable decrease in the level of papaverine in comparison to control plants. The characterization of the functionally unique Ps3'OMT involved in BIA metabolism suggests an involvement of the NH pathway leading to papaverine biosynthesis.

Assessment of various genetic components through NCD-I and NCD-III designs of biparental mating in opium poppy.

Creation of genetic variability and development of varieties having higher yield potential depends on information about nature of gene action. The present investigation was undertaken to decipher the nature of gene action and allied genetic parameters involved in the inheritance of yield and yield-related component traits in opium poppy (Papaver somniferum L.). The biparental inbreeding progenies derived from four segregating base populations of crosses NB-1Kr40-3/3×NB-1Kr30+0.2-2/1, NB-5Kr40-7/2×58/1, NB-1Kr30+0.2-2/1×58/1 and NB-Kr40-3/3×NB-5Kr40-7/2 of opium poppy were analysed to study the gene actions involved in the inheritance of yield and component traits. Additive component of variance played a predominant role in North Carolina design (NCD)-I, while both additive and dominance genetic components were found important in NCD-III design. The presence of additive as well as nonadditive components of variance suggested that one or two generations of intermating in further generations followed by selection may lead to development of novel genotypes.

Celebratory firing leads to serious injuries - a case report.

Firearms are extensively used in shooting incidents, functions or for recreational purposes around the world. However, illegal modifications to a firearm can pose a major challenge to the forensic community, as their examination and subsequent individualisation are not always easy when compared to standard firearms due to the non-availability of standard comparison material and the damage caused to the firearm during test firing. We present a case report of unusual firing by an improvised firearm adapted to produce a loud noise during a marriage party. This led to high pressure inside the barrel, damaging the extractor assembly of the rifle and caused serious injuries to bystanders. An expert must be aware of all usual and unusual results of firings. In this case, an unusual method of firing two bullets shows the generation of high pressure inside the barrel causing the barrel and extractor assembly to break and act as secondary projectile. These cases need thorough investigation and careful observation to achieve positive outcomes.

Elicitation Enhanced the Yield of Glycyrrhizin and Antioxidant Activities in Hairy Root Cultures of Glycyrrhiza glabra L.

Glycyrrhiza glabra L. has become an endangered medicinal plant due to the unabated extraction of glycyrrhizin. Glycyrrhizin is a triterpenoid saponin that is a root centric secondary metabolite having numerous pharmacological properties, such as anti-inflammatory, immunomodulatory, antiallergic, antiulcer, and is found to be effective even against HIV. Harvesting of the roots for high value glycyrrhizin destroys the whole plant causing existential threat to the plant itself and consequent damage to biodiversity. The present study establishes that hairy root cultures of G. glabra, using an optimized elicitor, can dramatically enhance focused production of glycyrrhizin at a much faster pace year-round without causing destruction of the plant. Hairy root cultures of G. glabra were developed using the Agrobacterium rhizogenes A4 strain. The glycyrrhizin content was enhanced using different biotic and abiotic elicitors, for example, PEG (polyethylene glycol), CdCl2, cellulase, and mannan at different concentrations and durations. PEG at 1% concentration enhanced the yield of glycyrrhizin up to 5.4-fold after 24 h of exposure, whereas 200 µg mL-1 cellulase enhanced glycyrrhizin yield to 8.6-fold after 7 days of treatment. Mannan at 10 mg L-1 concentration enhanced the production of glycyrrhizin up to 7.8-fold after 10 days of stress. Among different antioxidant enzymes, SOD activity was significantly enhanced under drought, cellulase and mannan stress. This identification of elicitors can result in abundant supply of valuable glycyrrhizin to meet broad spectrum demand through commercial production without endangering G. glabra L.

Isolation and characterization of culturable bacteria present in the spent nuclear fuel pool water.

A spent nuclear fuel (SNF) pool is a key facility for safe management of nuclear waste, where spent nuclear fuel rods are stored in a water pool. The spent fuel rods carry a significant amount of radioactivity; they are either recycled or stored for further processing. Pool water acts as a heat sink as well as a shield against the radiation present in spent/burned fuel rods. The water used in these pools is filtered by an ultra-filtration process which makes certain the purity of water. As the life span of these pools is approximately 20 to 40 years, the maintenance of pure water is a big challenge. A number of researchers have shown the presence of bacterial communities in this ultrapure water. The bacterial types present in SNF pool water is of increasing interest for their potential bioremediation applications for radioactive waste. The present study showed the isolation of six bacterial species in the SNF pool water samples, which had significant radio-tolerance (D10 value 248 Gy to 2 kGy) and also biofilm-forming capabilities. These strains were also investigated for their heavy metal removal capacity. Maximum biofilm-mediated heavy metal (Co and Ni) removal (up to 3.8 µg/mg of biomass) was observed by three isolates (FPB1, FPB4, and FPB6). The ability of these bacterial isolates to survive in radioactive environments can be of great interest for remediation of radioactive contaminants.

Staphylococcus aureus biofilm removal by targeting biofilm-associated extracellular proteins.

BACKGROUND & OBJECTIVES: Among cell surface proteins, biofilm-associated protein (Bap) promotes biofilm development in Staphylococcus aureus strains. The aim of this study was to investigate proteinase-mediated biofilm dispersion in different isolates of S. aureus. METHODS: Biofilm assay was done in 96-well microtitre plate to evaluate the effect of proteinase K on biofilms of bovine mastitis S. Aureus isolates. Extracellular polymeric substances were extracted and evaluated for their composition (protein, polysaccharides and extracellular DNA), before and after the proteinase K treatment. RESULTS: Biofilm assay showed that 2 µg/ml proteinase K significantly inhibited biofilm development in bap-positive S. aureus V329 as well as other S. aureus isolates (SA7, SA10, SA33, SA352), but not in bap-mutant M556 and SA392 (a weak biofilm-producing strain). Proteinase K treatment on S. aureus planktonic cells showed that there was no inhibition of planktonic growth up to 32 µg/ml of proteinase K. Proteinase K treatment on 24 h old preformed biofilms showed an enhanced dispersion of bap-positive V329 and SA7, SA10, SA33 and SA352 biofilms; however, proteinase K did not affect the bap-mutant S. aureus M556 and SA392 biofilms. Biofilm compositions study before and after proteinase K treatment indicated that Bap might also be involved in eDNA retention in the biofilm matrix that aids in biofilm stability. When proteinase K was used in combination with antibiotics, a synergistic effect in antibiotic efficacy was observed against all biofilm-forming S. aureus isolates. INTERPRETATION & CONCLUSIONS: Proteinase K inhibited biofilms growth in S. aureus bovine mastitis isolates but did not affect their planktonic growth. An enhanced dispersion of preformed S. aureus biofilms was observed on proteinase K treatment. Proteinase K treatment with antibiotics showed a synergistic effect against S. aureus biofilms. The study suggests that dispersing S. aureus by protease can be of use while devising strategies againstS. aureus biofilms.

Ageratum enation virus Infection Induces Programmed Cell Death and Alters Metabolite Biosynthesis in Papaver somniferum.

A previously unknown disease which causes severe vein thickening and inward leaf curl was observed in a number of opium poppy (Papaver somniferum L.) plants. The sequence analysis of full-length viral genome and associated betasatellite reveals the occurrence of Ageratum enation virus (AEV) and Ageratum leaf curl betasatellite (ALCB), respectively. Co-infiltration of cloned agroinfectious DNAs of AEV and ALCB induces the leaf curl and vein thickening symptoms as were observed naturally. Infectivity assay confirmed this complex as the cause of disease and also satisfied the Koch's postulates. Comprehensive microscopic analysis of infiltrated plants reveals severe structural anomalies in leaf and stem tissues represented by unorganized cell architecture and vascular bundles. Moreover, the characteristic blebs and membranous vesicles formed due to the virus-induced disintegration of the plasma membrane and intracellular organelles were also present. An accelerated nuclear DNA fragmentation was observed by Comet assay and confirmed by TUNEL and Hoechst dye staining assays suggesting virus-induced programmed cell death. Virus-infection altered the biosynthesis of several important metabolites. The biosynthesis potential of morphine, thebaine, codeine, and papaverine alkaloids reduced significantly in infected plants except for noscapine whose biosynthesis was comparatively enhanced. The expression analysis of corresponding alkaloid pathway genes by real time-PCR corroborated well with the results of HPLC analysis for alkaloid perturbations. The changes in the metabolite and alkaloid contents affect the commercial value of the poppy plants.

Gene actions for yield and its attributes and their implications in the inheritance pattern over three generations in opium poppy (Papaver somniferum L.).

The gene actions for yield and its attributes and their inheritance pattern based on five parameter model have been explored in four single crosses (NBIHT-5 × NBIHT-6, NBIHT-5 × NBMHT-1, NBMHT-1 × NBIHT-6 and NBMHT-2 × NBMHT-1) obtained using thebaine rich pure lines of opium poppy (Papaver somniferum L.) for three consecutive generations. All the traits showed nonallelic mode of interaction, however, dominance effect (h) was more pronounced for all the traits except thebaine and papaverine. The dominance × dominance (l) effects were predominant over additive × additive (i) for all traits in all the four crosses except for papaverine. The seed and opium yield, and its contributing traits inherited quantitatively. The fixable gene effects (d) and (i) were lower in magnitude than nonfixable (h) and (l) gene effects. The estimates of heterosis were also higher in comparison to the respective parents which suggested preponderance of dominance gene action for controlling most of the traits. The phenotypic coefficient of variation was marginally higher than those of genotypic coefficient of variation for all the traits. The traits thebaine, narcotine, morphine and opium yield had high heritability coupled with high genetic advance. The leaf number, branches per plant and stem diameter showed positive correlation with opium and seed yields. The selection of plants having large number of leaves, branches and capsules with bigger size would be advantageous to enhance the yield potential.