Halotolerant bacterial biofilms for desalination and water treatment: a pilot study.

Salinity has a significant impact on the water quality and crop yield. Physical desalination techniques were once thought to be expensive and time-consuming. Among biological techniques, halotolerant bacteria were thought to be the fastest and most effective way to reduce the salt content in brackish saltwater water. In the current study, halotolerant bacterial biofilms were used to desalinate saline water on abiotic substrates (such as sand, pebbles, glass beads, and plastic beads), and studied subsequently for the effects on Zea mays germination. Briefly, salt samples (SLT7 and SLT8) from the Khewra site in Punjab, Pakistan, as well as seawater and sea sand samples (USW1, USW3, USW6, DSW1, DSW4, SS1, and SS3) from Karachi, Sindh, Pakistan's Arabian Sea, were collected. Halotolerant bacteria were isolated and characterized. Crystal violet ring assays and capsule staining were used to estimate extracellular polymeric substance (EPS) and biofilm development, respectively. All halotolerant bacterial strains were spore formers and produced EPS and formed biofilms well. 16S rRNA gene sequencing of the best halotolerant bacteria, USW6, showed the closest (100%) similarity to Bacillus aerius strain G-07 (a novel species) (accession number ON202984). A pilot-scale experiment for desalinating the artificial water (supplemented with 1 M NaCl) using biofilm adhered abiotic beads showed declined level of NaCl from 1 M to 0.00003 M after 15 days in treated water. Also, Zea mays germination was observed in the plants using treated water compared to no growth in the non-treated saline water. Estimations of chlorophyll, total soluble sugar, and protein revealed that plants cultivated using elute collected from a desalinated pilot scale setup contained less chlorophyll (i.e., 5.994 and 116.76). Likewise, plants grown with elute had a total soluble protein and sugar content of 1.45 mg/ml and 1.3 mg/ml, respectively. Overall, in treated water plants, a minor drop in chlorophyll content, a slight increase in total soluble sugar content, and a slight increase in protein content were noted. The study concluded that biofilm-treated desalt water has the potential to significantly reduce the effects of droughts, soil salinization, and economic and environmental issues associated with agricultural drainage. The results specified the application of halotolerant bacteria biofilms (Bacillus aerius, a novel species, USW6) for water desalination to overcome the problem of water scarcity caused by global warming and the increased salinity.

Efficacy of rice husk and halophilic bacterial biofilms in the treatment of saline water.

Water salinity causes less production of agricultural productivity, low economic returns, soil destructions, less sustainability, and reduction in the germination rate. The current study was aimed to understand the combined potential of halophilic bacteria and rice husk in treating water salinity. In total, 10 halophilic bacterial isolates were isolated from Khewra Mines, Pakistan. Bacterial isolates were characterized by biochemical tests. 16S rRNA gene sequencing identified the isolate SO 1 as Bacillus safensis (accession number ON203008) being the promising halophilic bacteria tolerating upto 3 M NaCl concentration. Next, rice husk was used as carbon source for bacterial biofilm formation, growth and propagation. For saline water treatment, the experimental setting comprising glass wool, rice husk and artificial sea water (3 M) was set. B. safensis biofilm was developed in test samples to desaline the saline water containing 3 M NaCl concentration. Following NaCl decline, flame photometric analysis was used to check the desalination extent of treated saline water. Results showed decreased sodium level in sea water in the presence of rice husk and glass wool. The eluted water used for the germination of Zea mays seeds showed improved growth performance. Also, decreased photosynthetic pigments (chlorophyll "a" = 18.99, and chlorophyll "b" = 10.65), sugar contents (0.7593), and increased carotenoid (1526.91), protein contents (0.4521) were noted compared to control. This eco-friendly approach for bioremediation of salt-affected soils to optimize crop yields under stress through halophilic bacteria and rice husk may overcome the problem of the reduced yield of cash crops/agriculture and water shortage by salinity.

Evaluation of Safety of Stewart's Wood Fern (Dryopteris stewartii) and Its Anti-Hyperglycemic Potential in Alloxan-Induced Diabetic Mice.

Diabetes has become a critical challenge to the global health concerns. Cytotoxicity and development of resistance against available drugs for management of diabetes have shifted the focus of global scientific researchers from synthetic to herbal medications. Therefore, the current study was conducted to investigate the possible anti-hyperglycemic potential of Dryopteris stewartii using Swiss albino mice. To evaluate any possible toxic effect of the plant, acute oral toxicity test was performed while the anti-diabetic effects of aqueous and ethanol extracts at 500 mg/kg, positive, negative and normal control were assessed simultaneously. The anti-diabetic study revealed that aqueous extract has higher anti-diabetic potential than ethanol extract while lowered blood glucose level at second week reaching 150 mg/dL, exerting stronger anti-diabetic effects, compared to ethanol extract (190 mg/dL). Oral glucose tolerance findings revealed that aqueous extract decreased blood glucose level by -0.41-fold, compared to ethanol extract showing a decrease by only -0.29-folds. The histopathological evaluation of liver and pancreas of all groups revealed normal cell architecture with no morphological abnormalities. These results suggested the possible use of D. stewartii as anti-diabetic herbal drug in near future. However, these recommendations are conditioned by deep mechanistic studies.

Application of green silver nanoparticles synthesized from the red seaweeds Halymenia porphyriformis and Solieria robusta against oral pathogenic bacteria by using microscopic technique.

Aqueous extracts of two red seaweeds Halymenia porphyriformis and Solieria robusta were used to synthesize green silver nanoparticles. These biogenic nanoparticles were tested against four strains of oral pathogenic bacteria which cause tooth decay or cavities. Staphylococcus aureus (MT416445), Streptococcus viridans (MT416448), Lactobacillus acidophilus (MT416447) and Lactobacillus brevis (MT416446) were used. Characterization of AgNPs was done by UV-Visible spectroscopy, SEM, XRD and FTIR. XRD analysis revealed the crystalline nature of the particles. The size analysis by XRD of the green AgNPs by H. porphyriformis indicated it had smaller particles, 15.23 nm, when compared to AgNPs by S. robusta (17 nm). Both green synthesized silver nanoparticles showed moderate antibacterial activity against all strains of bacteria, except L. acidophilus. Both particles showed their maximum zone of inhibition against L. acidophilus at a lower concentration of 50 and 100 µg. However, it was concluded that silver nanoparticles of H. porphyriformis are more effective than that of S. robusta due to their smaller size.

A Comparative Analysis of Antimicrobial, Antibiofilm and Antioxidant Activity of Silver Nanoparticles Synthesized from Erythrina Suberosa Roxb. and Ceiba Pentandra.

Biofilm forming bacteria can cause serious health problems that are difficult to combat. Silver nanoparticles (Ag-NPs) synthesized from plant extracts have potential to fight and eradicate biofilmforming bacteria. In the present research, AgNPs were synthesized using leaf and bark extract of Erythrina suberosa Roxb. and Ceiba pentandra L. and their antibiofilm, antioxidant and antibacterial activity was checked. Phytochemical analysis of the plant extracts showed important bioactive compounds such as tannins, saponins, steroids, phenolics, alkaloids, flavonoids and glycosides. The AgNPs were synthesized and confirmed by visual color observation and UV-Vis spectrophotometer. Visual color observation showed that the color of the leaf and bark extracts of E. suberosa and C. pentandra turned into brown. UV-Vis spectra analysis showed absorbance peak range between 430-450 nm. The antioxidant activity of the AgNPs was determined by FRAC (Ferrous reducing antioxidant capacity) assay. Synthesized AgNPs from all sources showed significant antioxidant activity. However, antioxidant activity of E. suberosa AgNPs was significant compared to other sources. Antibacterial activity and biofilm forming assay was analyzed against Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus. The synthesized AgNPs silver nanoparticles showed significant (p ≤ 0.05) antibacterial activity against all the bacteria. The maximum zone of inhibition was found in case of E. suberosa AgNPs bark extract against P. aeruginosa was 20±1.154 mm. The results of biofilm forming assay showed that the AgNPs from all sources significantly (p ≤ 0.05) inhibited the activity of biofilms by all the tested bacteria. From results, it can be concluded that AgNPs synthesized from both plants can be used in developing antimicrobial compounds.

Black coffee mitigates diethyl phthalate disrupted folliculogenesis, reduced gonadotropins, and ovarian lesions in female albino mice.

Phthalates are multifunctional compounds with extensive applications and emerging environmental pollutants. Due to their ubiquity in the environment and unavoidable exposure to humans, concerns have been voiced about public health dangers. This study was aimed to explore the diethyl phthalate (DEP) toxicity and the potential protective effect of black coffee in female Swiss albino mice. Four-week-old mice, weighing 12 ± 1 g were segregated into five groups (n = 10), designated as G-I (without any treatment), G-II (treated with corn oil), G-III (exposed to 1.5 mg/g body wt. (B.W.) DEP), G-IV (received 2 µg/g B.W coffee), and G-V (co-administrated with 1.5 mg/g DEP and 2 µg/g B.W coffee). Before dose administration, the coffee extract was assessed for its antioxidant potential through FRAP, TPC, and GC-MS analyses. Respective phthalates/coffee doses were administrated orally, once a day for 8 weeks consecutively starting from the prepubescent stage. After 56 days, mice were acclimated for 4 days then dissected. Morphological assessments showed an irregular shape of the ovaries in DEP-treated mice as compared to the control. The average bodyweight of DEP-intoxicated mice (p ≤ 0.05) increased notably against control, while DEP plus coffee group showed a regular gain in the average weight of mice. The gonado-somatic index showed non-significant variations among all groups. Micrometric studies showed that the diameter of secondary follicles (115 µm) in the ovaries of DEP-exposed mice (p ≤ 0.001) decreased significantly as compared to control (204 µm); conversely, follicular diameter in the coffee control group (248) increased significantly. Serum FSH and LH levels were significantly increased in DEP-exposed mice with a noteworthy decrease in estrogen level while hormonal levels of all other groups were comparable to control. Histological sections of DEP-exposed mice ovaries showed anatomical disruptions contrary to other groups, which were comparable with control. Antioxidant potential was checked in ovaries homogenates; FRAP values showed a notable decrease in DEP group in comparison with the control group, in contrast to G-V, when DEP was co-administrated with coffee. This study concluded that black coffee has protective effect, against DEP-instigated reproductive toxicity in Swiss albino female mice.

Biogenic Synthesis, Characterization and Antibacterial Properties of Silver Nanoparticles against Human Pathogens.

Biogenic synthesis of silver nanoparticles (AgNPs) is more eco-friendly and cost-effective approach as compared to the conventional chemical synthesis. Biologically synthesized AgNPs have been proved as therapeutically effective and valuable compounds. In this study, the four bacterial strains Escherichia coli (MT448673), Pseudomonas aeruginosa (MN900691), Bacillus subtilis (MN900684) and Bacillus licheniformis (MN900686) were used for the biogenic synthesis of AgNPs. Agar well diffusion assay revealed to determine the antibacterial activity of all biogenically synthesized AGNPs showed that P. aeruginosa AgNPs possessed significantly high (p < 0.05) antibacterial potential against all tested isolates. The one-way ANOVA test showed that that P. aeruginosa AgNPs showed significantly (p < 0.05) larger zones of inhibition (ZOI: 19 to 22 mm) compared to the positive control (rifampicin: 50 µg/mL) while no ZOI was observed against negative control (Dimethyl sulfoxide: DMSO). Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) concentration against four test strains also showed that among all biogenically synthesized NPs, P. aeruginosa AgNPs showed effective MIC (3.3-3.6 µg/mL) and MBC (4.3-4.6 µg/mL). Hence, P. aeruginosa AGNPs were characterized using visual UV vis-spectroscopy, X-ray diffractometer (XRD), fourier transform infrared (FTIR) and scanning electron microscopy (SEM). The formation of peak around 430 nm indicated the formation of AgNPs while the FTIR confirmed the involvement of biological molecules in the formation of nanoparticles (NPs). SEM revealed that the NPs were of approximately 40 nm. Overall, this study suggested that the biogenically synthesized nanoparticles could be utilized as effective antimicrobial agents for effective disease control.

Toxicological effects of the chemical and green ZnO NPs on Cyprinus carpio L. observed under light and scanning electron microscopy.

Nanoparticles in aquatic bodies cause serious harm to the aquatic organisms when accumulated in high amounts. However, green nanoparticles synthesized using plants can be less toxic as compared to chemical nanoparticles. Hence, we designed our study to investigate the toxicological effects of chemical and green zinc oxide nanoparticles (ZnO NPs) on the biological activity of juvenile Cyprinus carpio. The green ZnO NPs were synthesized from Solieria robusta, and chemical ZnO NPs were synthesized using zinc chloride solution and ammonium hydroxide. Characterization was done by using light microscopy, scanning electron microscope (SEM), Fourier transmission infrared radiation, and X-ray diffraction (XRD) techniques. The highest absorbance of nanoparticles was observed at 360 which confirmed the synthesis of ZnO. The SEM analysis showed that green nanoparticles were hexagonal while the chemical nanoparticles were spherical to cubic in shape. Definite peaks were observed in XRD of green and chemical NPs at 2θ angles 45.84° and 32.18°, respectively. Oxidative stress was determined by chemical analysis of catalase, glutathione S-transferase (GST), glutathione (GSH), and lipid peroxidation (LPO) activities. The toxicological effects of chemical ZnO NPs on the catalase, LPO, GST, and GSH activities were more than green ZnO NPs. The histopathological investigation proved that the effect of chemical nanoparticles was worse than green ZnO NPs. More tissue damage was found in chemical nanoparticles than green synthesized nanoparticles. It was concluded that chemical nanoparticles can be replaced by green nanoparticles, as green nanoparticles are eco-friendly with less toxicological effects. This replacement can limit the toxic effect of nanoparticles when they get accumulated in high amounts in water bodies.

Antioxidant potential and chemical characterization of bioactive compounds from a medicinal plant Colebrokea oppositifolia Sm.

Colebrookea oppositifolia is a highly used medicinal plant and an enriched source of essential oils. Therefore, the present study was designed with the aim to extract the chemical constituents and to evaluate its antioxidant potential. Fresh plant parts were subjected to the extraction of volatile chemical constituents by maceration using n-hexane as the menstruum. The resulting n-hexane fractions were purified and then subjected to GC-MS and FTIR analysis. In-vitro antioxidant abilities were evaluated by, DPPH, total phenolic content (TPC), total flavonoid content (TFC) method against the standard solutions of (Gallic acid, Quercetin) as a positive control. The GC-MS analysis of leaves, stem and inflorescence showed a total of 100, 98 and 48 components out of which 47, 16 and 17 peaks were identified representing the 67.64 %, 73.16 % and 61.93 % of the total oily fractions, respectively. The FTIR spectrum indicated the presence of various functional groups. In-vitro antioxidant results exhibited that leaves showed the highest antioxidant potential by DPPH (3.365 ± 0.002), and the highest total phenolic content by FC method (203.00 ± 0.091). Foliar micromorphological features were found significant in the authentication of C. oppositifolia. Further pharmacognostic studies of this plant are recommended to evaluate its therapeutic potential.

Toxicological Evaluation of Essential Oils from Some Plants of Rutaceae Family.

Essential oils are produced as secondary metabolites by aromatic plants, predominantly belonging to families Apiaceae, Lamiaceae, Myrtaceae, and Rutaceae. The family Rutaceae has great economic importance for its numerous edible fruits and essential oils. In the present study, essential oils of seven plants of family Rutaceae, Aegle marmelos, Murraya koenigii, Citrus reticulata Blanco, Zanthoxylum armatum, Skimmia laureola, Murraya paniculata, and Boenninghausenia albiflora, were used for their toxicological assessment. Seven groups of selected essential oils-treated Wistar rats were established against control group (n = 5) that received water for 14 days; animals were offered feed and water ad libitum and treated with essential oils at 400 mg/kg body weight. Hematological studies revealed significant elevation in TEC in animals treated with essential oils of M. koenigii, S. laureola, and B. albiflora, while an elevation in PCV and depletion in MCV were observed in animals treated with M. paniculata and B. albiflora, respectively. Serological investigations demonstrated significant depletion in triglycerides and elevation in blood sodium level in animals treated with essential oils of A. marmelos and C. reticulata Blanco. Boenninghausenia albiflora affected many markers including RBC, MCV, triglycerides, HDL, LDL, urea, and sodium. In conclusion, all oils except B. albiflora can be considered safe for internal use.

In vivo screening and evaluation of four herbs against MRSA infections.

BACKGROUND: Recently, we reported high in vitro antibacterial efficacy of Althaea officinalis, Ziziphus jujuba, Cordia latifolia and Thymus vulgaris out of a total 21 plants against wide range of bacteria including MRSA. This study was therefore, designed to confirm efficacy of these four herbs against MRSA in an animal model. METHODS: A pilot study was conducted to establish the dose of S. aureus (KY698020) required to induce clinical infection. Afterword, in main trial, efficacy of aforementioned plant extracts on the course of sore throat was checked by evaluating general health, gross lesion score, bacterial load and hematology in mice. RESULTS: Pilot study revealed that 40 µl dose of 107 CFU/ml could induce infection which persist upto 08 days post infection. Mice treated with T. vulgaris and Z. jujuba showed reduction in gross lesion score of both heart and lungs. Treatment with only some plants could significantly decrease bacterial load of throat (T. vulgaris) heart, blood and joint (C. latifolia, and T. vulagris). Hematological indicators confirmed in vivo control of MRSA infection in all treatment groups except A. officinalis. CONCLUSION: This is first report confirming in vivo anti-MRSA potential of C. latifolia and T. vulgaris and highlight the need to explore bioactive constituents of these plants. Moreover, previously reported in vitro antibacterial efficiency of A. officinalis could not be validated in current study.

Effect of medicinal plants, Heavy metals and antibiotics against pathogenic bacteria isolated from raw, Boiled and pasteurized milk.

Present study has been undertaken to isolate and identify the bacterial flora in raw, boiled and pasteurized milk. Agar disc diffusion method was used to determine their sensitivity using medicinal plants, antibiotics and heavy metals. Methylene blue reduction test was used to test the quality of milk samples. Total 10 pathogenic strains were isolated, five strains were isolated from raw milk, three from boiled milk and 2 two from pasteurized milk. To determine optimum conditions for growth, these pathogenic microorganisms were incubated at various temperatures and pH. Gram's staining and biochemical tests revealed that these pathogenic bacteria include Lactobacillus sp., E. coli, Salmonella sp., Pseudomonas sp., Streptococcus sp. and Staphylococcus. Ribotyping revealed S2 as Pseudomonas fluorescens, S5 as Lactococcus lactis and S9 as Lactobacillus acidophilus. Prevalence of pathogenic organisms provided the evidence that contamination of milk arises during milking, transportation and storage of milk. Raw milk is more contaminated than other two types of milk because it contains highest percentage of pathogenic organisms and pasteurized milk was found to be of best quality among three types. So it is recommended to drink milk after proper boiling or pasteurization. Proper pasteurization and hygienic packing of milk is essential to minimize contamination in milk which can save human beings from many milk borne diseases. Our study suggests that antimicrobial use in animal husbandry should be minimized to reduce the hazard of antibiotic resistance. Plant extracts are better alternative against pathogenic bacteria in milk.

Phytochemical, Antimicrobial, and Toxicological Evaluation of Traditional Herbs Used to Treat Sore Throat.

The in vitro antibacterial activities of 29 traditional medicinal plants used in respiratory ailments were assessed on multidrug resistant Gram-positive and Gram-negative bacteria isolated from the sore throat patients and two reference strains. The methanolic, n-hexane, and aqueous extracts were screened by the agar well diffusion assay. Bioactive fractions of effective extracts were identified on TLC coupled with bioautography, while their toxicity was determined using haemolytic assay against human erythrocytes. Qualitative and quantitative phytochemical analysis of effective extracts was also performed. Methanolic extract of 18 plants showed antimicrobial activity against test strains. Adhatoda vasica (ZI = 17-21 mm, MIC: 7.12-62.5 µg/mL), Althaea officinalis (ZI = 16-20 mm, MIC: 15.62-31.25 µg/mL), Cordia latifolia (ZI = 16-20 mm, MIC: 12.62-62.5 µg/mL), Origanum vulgare (ZI = 20-22 mm, MIC: 3-15.62 µg/mL), Thymus vulgaris (ZI = 21-25 mm, MIC: 7.81-31.25 µg/mL), and Ziziphus jujuba (ZI = 14-20 mm, MIC: 7.81-31.25 µg/mL) showed significant antibacterial activity. Alkaloid fractions of Adhatoda vasica, Cordia latifolia, and Origanum vulgare and flavonoid fraction of the Althaea officinalis, Origanum vulgare, Thymus Vulgaris, and Ziziphus jujuba exhibited antimicrobial activity. Effective plant extracts show 0.93-0.7% erythrocyte haemolysis. The results obtained from this study provide a scientific rationale for the traditional use of these herbs and laid the basis for future studies to explore novel antimicrobial compounds.

Isolation and characterization of biocides resistant bacteria from dental unit water line biofilms.

Six biocides resistant isolates were isolated from dental unit water lines (DUWL) in Pakistan. All isolates could tolerate 150 microg ml(-1 )of biocides (5.25% sodium hypocholrite, 35% H2O2, 4% tween 20, 1% povidine iodine, 0.2% chlorohexidine gluconate, 1% ethylene di-amino tetra acetic acid and 1% phenol) on l-agar and 100 microg ml(-1 )in l-broth. The growth rate of all isolates was determined by generating growth curves at 37 degrees C for 48 h. The isolates were found to differ in growth rates with lag phase varying from (4-6 h) in biocides supplemented media compared to 2-4 h in biocides free medium. They have wide temperatures (24-42 degrees C) and pH (5-9) ranges. Traditional ways of identification of bacteria by phenotypic characteristics were accomplished by phenotypic and biochemical characterization. Heavy metals and antimicrobial susceptibility tests indicated that all isolates examined were resistant to trimethoprim, chloramphenicol while sensitive to HgCl2 and Pb (NO3)2. Almost all isolates were opportunistic pathogens. The 16S rRNA-encoding genes from these six isolates were sequenced to confirm the identity of these isolates. 5 different genera (Bacillus, Achromobacter, Pseudomonas and Klebsiella) of bacteria were identified by 16S rDNA genes amplified from genomic DNA of biocides resistant DUWL biofilm isolates. Analysis of 16S rDNA genes revealed a much more clear identification of microorganisms than culture methods. However, different species of the same genera can have the same 16S rRNA gene sequence but are different due to phenotypic differences or different clinical manifestations.

Analysis of cell wall constituents of biocide-resistant isolates from dental-unit water line biofilms.

In past years, the significance of microbial resistance to biocides has increased. Twenty biocide-resistant bacterial strains were isolated from dental-unit water line biofilm. All strains resisted high biocide concentrations (up to 100 microg ml(-1)): sodium dodecyl sulphate, hydrogen peroxide, sodium hypochlorite, phenol, Tween 20, ethylenediaminetetraacetic acid, chlorohexidine gluconate, and povidine iodine. Among bacteria, biocide sensitivity is based on permeability of biocides through the cell wall. Gram-positive bacteria are more permeable and susceptible to biocides, whereas gram-negative bacteria have a more complex cell wall and are the least sensitive bacteria. The present study was designed to study the effect of biocides on the cell wall of biocide-resistant bacteria. Peptidoglycan (PG), diaminopimelic acid (DAP), and teichoic acid contents of the cell wall were determined in L-broth and L-broth supplemented with biocides at different temperatures (37 degrees C and 45 degrees C) and pH levels (7 and 9). In general and Gram staining-specific comparison, a significant increase (p < 0.05) in the DAP content of biocide-resistant bacteria was observed at pH 7 and at both temperatures. In tubing-specific comparison, a significant increase in the amount of teichoic acid in air water tubing (37 degrees C at pH 9) and DAP in patient tubing (pH 7 at both temperatures) was observed. In main water pipe, a significant decrease (p > 0.05) in PG content was noticed at 45 degrees C and pH 9. Overall, a significant increase in DAP content may be an important constituent in the manifestation of isolate resistance against various biocides.