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.

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.

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.

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.

Microbial reduction of [Co(III)-EDTA]⁻ by Bacillus licheniformis SPB-2 strain isolated from a solar salt pan.

Naturally stressed habitats are known to be repositories for novel microorganisms with potential bioremediation applications. In this study, we isolated a [Co(III)-EDTA](-) reducing bacterium Bacillus licheniformis SPB-2 from a solar salt pan that is exposed to constant cycles of hydration and desiccation in nature. [Co(III)-EDTA](-) generated during nuclear waste management process is difficult to remove from the waste due to its high stability and solubility. It is reduced form i.e. [Co(II)-EDTA](2-) is less stable though it is toxic. This study showed that B. licheniformis SPB-2 reduced 1mM [Co(III)-EDTA](-) in 14 days when grown in a batch mode. However, subsequent cycles showed an increase in the reduction activity, which was observed up to four cycles. Interestingly, the present study also showed that [Co(III)-EDTA](-) acted as an inducer for B. licheniformis SPB-2 spore germination. Vegetative cells germinated from the spores were found to be involved in [Co(III)-EDTA](-) reduction. More detailed investigations showed that after [Co(III)-EDTA](-) reduction, i.e. [Co(II)-EDTA](2-) complex was removed by B. licheniformis SPB-2 from the bulk liquid by adsorption phenomenon. The bacterium showed a D10 value (radiation dose required to kill 90% cells) of ∼250 Gray (Gy), which signifies the potential use of B. licheniformis SPB-2 for bioremediation of moderately active nuclear waste.

Phenotypic switching in biofilm-forming marine bacterium Paenibacillus lautus NE3B01.

Biofilm-forming marine bacterium Paenibacillus lautus NE3B01 was isolated from a mangrove ecosystem, Odisha, India. This isolate formed a swarming type of colony pattern on the solid culture medium with 0.5-2 % agar. Phase contrast microscopy study of a growing colony of P. lautus on solid media and swarming pattern revealed the existence of two phenotypically distinct cells (i.e. cocci and rods) across the colonies. However, in actively growing planktonic culture, only rod-shaped cells were observed. Biofilm growth studies (crystal violet assay) with the isolate showed significant biofilm formation by 6 h, and the detachment phase was observed after 18 h. Biofilm parameters (such as total biomass, roughness coefficient, biofilm thickness, etc.) of 24-h-old P. lautus biofilm were studied by confocal scanning laser microscopy (CSLM). The CSLM study showed that P. lautus formed a biofilm with an average thickness of 14.8 ± 2.6 µm, a high roughness coefficient (0.379 ± 0.103) and surface to bio-volume ratio (4.59 ± 1.12 µm(2)/µm(3)), indicating a highly uneven topography of the biofilm. This also indicates that the 24-h-old biofilm is in dispersal phase. Scanning electron microphotographs of P. lautus also supported the existence of two distinct phenotypes of P. lautus. The current findings suggest that P. lautus has two vegetative phenotypes and to decongest the overcrowded biofilm the bacterium can switch over to motile rods from nonmotile cocci and vice versa.

Differential radio-tolerance of nutrition-induced morphotypes of Deinococcus radiodurans R1.

Deinococcus radiodurans R1 is a highly radio-tolerant bacterium. Depending on the nutrient availability D. radiodurans R1 exists in three morphologies viz. monococcal, diplococcal and tetracoccal. In this study, we examined whether nutrition-induced morphotypes of D. radiodurans showed similar DNA damage upon gamma radiation exposure. Total DNA damage after radiation exposure was estimated by comparing percent double-strand breaks (DSBs) in genomic DNA. It was found that all three morphotypes exhibited different radiation tolerances which were also dependent on the radiation dose given. Monococcal forms were found to be most radio-tolerant at most of the tested radiation doses. Results showed that these nutrient-starved-condition induced morphotypes show lesser DNA DSBs upon irradiation, hence show higher radio-tolerance.

Removal of toxic Co-EDTA complex by a halophilic solar-salt-pan isolate Pseudomonas aeruginosa SPB-1.

In this study, a promising bioremediation approach was developed to remove [Co(III)-EDTA](-) complex that is generated during the waste management process. Though several studies have been reported on bioremediation of cobalt, the removal of [Co(III)-EDTA](-) complex has not been tested. A [Co(III)-EDTA](-) resistant bacterium, Pseudomonas aeruginosa SPB-1 was isolated from the solar-salt-pan and physical parameters were optimized for its growth. The various studies showed that the removal of [Co(III)-EDTA](-) from the bulk liquid was due to the adsorption of the complex by the biomass. Using absorption/desorption isotherm over a range of pH (1-8), the maximum adsorption of [Co(III)-EDTA](-) was found to be at pH 7.0 and maximum desorption from the biomass occurred at pH 1.0, thus rendering an ion exchange property to P. aeruginosa SPB-1 biomass. P. aeruginosa SPB-1 biomass could be used as bio-resin that showed 80.4±3.27% adsorption capacity up to fourth cycle and the biomass was viable till the ninth cycle with 10.5±7.3% adsorption. Radiation tolerance potential i.e. D10 value for the strain was found to be ~300 Gy, which suggests the potential use of the bacterium in bioremediation of moderately active nuclear waste.

Calcium-mediated modulation of Pseudomonas mendocina NR802 biofilm influences the phenanthrene degradation.

A potential biofilm forming and phenanthrene utilizing marine bacterium Pseudomonas mendocina NR802 was isolated from Rushukulya, Odisha, East Coast of India. The effect of Ca(2+) and Mg(2+) on biofilm growth and phenanthrene degradation was evaluated. Among the various tested concentrations, 20 mM of Ca(2+) and Mg(2+) showed a significant enhancement in biofilm production by the bacterium. The SEM-EDAX study showed that the elemental composition of the biofilm varied significantly when grown in the presence of Ca(2+) and Mg(2+). The CSLM analysis of biofilms grown in the presence of 20 mM Ca(2+) and Mg(2+) reveal the critical role of these ions on biofilm architectural parameters such as total biomass, biofilm thickness, roughness coefficient and surface to biovolume ratio. Ca(2+) was found to enhance the extracellular polymeric substances (EPS) production and phenanthrene degradation. Ca(2+) enhanced the biofilm growth in a dose dependent manner, whereas Mg(2+) significantly increased the cell growth in biofilm. More than 15% increase in phenanthrene degradation was observed when biofilm was grown in the presence of an additional 20 mM Ca(2+). This study also supports the fundamental role of Ca(2+) in biofilm growth, architecture as well as biofilm-mediated pollutant degradation.

Determination of cyclamate in urine by derivatized gas chromatography-mass spectrometry.

AIM: It is important in toxicological/drug screening work to rule out the possible interfering analytes, to eliminate the false positive or negative results. In this paper, we describe a simple, selective, and sensitive derivatized GC-MS method for the determination of cyclohexylsulfamic acid (cyclamate) in urine. MATERIALS AND METHODS: Elite- 5MS capillary column was used for the separation of analytes and detection using GC-MS. The analysis was carried out in selected ion monitoring mode (SIM) in the range of 26 to 200 using m/z values of 57, 30, 55, 41, 44, 67, 82, 98, and 39. RESULTS AND DISCUSSION: The method is based on the conversion of cyclamate into nitroso derivative of cyclamate followed by its gas chromatography-mass spectrometry determination. The limit of detection, limit of quantitation, and linearity range of the proposed method were found to be 0.2 µg/ ml, 0.7 µg/ml, and 1-15 µg/ml, respectively. The recovery of the present method is in the range of 88-94%. CONCLUSION: The proposed method can be applied for detection and quantification of cyclamate in urine.

Effect of calcium on Staphylococcus aureus biofilm architecture: a confocal laser scanning microscopic study.

Bacterial adhesion is a threshold event in the formation of biofilms. Several studies on molecular and biochemical aspects have highlighted that the protein matrix of the biofilm is of interest in developing strategies to combat biofouling. The prevalent role of biofilm associated protein (Bap) of Staphylococcus aureus in early adhesion and the putative presence of Ca(2+) binding EF hand motif in Bap was the motivation for this study. Biofilm assays (S. aureus strains V329 and M556) were done in micro-titer plates and confocal laser scanning microscopy (CLSM) was used to study the biofilm architecture. The results showed that Ca(2+) did not influence planktonic growth of the cultures; however, it modulated the biofilm architecture of S. aureus V329 in a dose dependent manner. Strain M556 was found to be a weak biofilm former and showed no significant change in the presence of Ca(2+). When tested with increasing NaCl concentration, there was no reversal of the Bap-dependent Ca(2+) inhibition of S. aureus V329 biofilm. This indicates that the interaction of Bap and Ca(2+) is not mere electrostatic. CLSM images of V329 biofilm showed reduction in biofilm thickness as well as altered biofilm topography with varying Ca(2+) concentrations. The inhibition effect of Ca(2+) on strain V329 biofilm disappeared in the presence of chelating agent EDTA at a non-inhibiting concentration (0.15 mM). The paper elaborates the role of Ca(2+) in biofilm architecture of S. aureus.

Radiation sterilization of fluoroquinolones in solid state: investigation of effect of gamma radiation and electron beam.

The effect of gamma radiation from (60)Co source and 2MeV electron beam was studied on two fluoroquinolone antibiotics viz norfloxacin and gatifloxacin in the solid state. The changes in reflectance spectrum, yellowness index, vibrational characteristics, thermal behavior, UV spectrum, chemical potency (HPLC) and microbiological potency were investigated. ESR measurement gave the number of free radical species formed and their population. The nature of final radiolytic impurities was assessed by studying the HPLC impurity profile. Both norfloxacin and gatifloxacin were observed to be radiation resistant, and did not show significant changes in their physico-chemical properties. They could be radiation sterilized at a dose of 25kGy.