Development of Data-driven Machine Learning Models and their Potential Role in Predicting Dengue outbreak.

ABSTRACT: Dengue fever is one of the most widespread vector-borne viral infections in the world, resulting in increased socio-economic burdens. The WHO has reported that 2.5 billion people are infected with dengue fever across the world, resulting in high mortalities in tropical and subtropical regions. The current article endeavors to present an overview of predicting dengue outbreaks through data-based machine-learning models. This artificial intelligence model uses real-world data such as dengue surveillance, climatic variables, and epidemiological data and combines big data with machine learning algorithms to forecast dengue. Monitoring and predicting dengue incidences have been significantly enhanced through innovative approaches. This involves gathering data on various climatic factors, including temperature, rainfall, relative humidity, and wind speed, along with monthly records of dengue cases. The study functions as an efficient warning system, enabling the anticipation of dengue outbreaks. This early warning system not only alerts communities but also aids relevant authorities in implementing crucial preventive measures.

Evaluating the Effect of pH, Temperature and Concentration on Antioxidant and Antibacterial Potential of Spectroscopically, Spectrophotometrically and Microscopically Characterized Mentha Spicata Capped Silver Nanoparticles.

The use of traditional plants has been tremendously increased due to their higher biological impact, minimal side effects, and comparatively low cost. Moreover, the emergence of antibacterial resistance is also shifting the scientific community to reconsider herbal remedies which provide relatively safer, cheap and biologically tolerable solutions. The present research was designed to fabricate the Mentha spicata conjugated silver nanoparticles (Me-AgNPs). Furthermore, the assessment of the bactericidal potential of Me-AgNPs against various bacterial strains was another motive behind this study. Fabricated NPs were characterized with the help of the UV-Visible spectrophotometric analysis, Fourier transform infrared spectroscopy (FTIR) and Scanning electron microscopy (SEM). Me-AgNPs showed a significant zone of inhibition (23 ± 0.2 mm) at 8 mg/mL against Staphylococcus aureus and a 4.0 ± 0.2 mm zone of growth inhibition at 2 mg/mL against Aeromonas veronii. The stability of Me-AgNPs was assessed at various pH (4, 7 and 11) and temperatures (25 °C, 4 °C, 37 °C, 75 °C). The significant zones of inhibition (11.3 ± 0.3 mm, 8.3 ± 0.3mm, 14.3 ± 0.3 mm, and 7.6 ± 0.2 mm) were observed at pH 11 against Escherichia coli, Staphylococcus aureus, Bacillus subtilis, and Klebsiella pneumoniae, respectively. Growth inhibition zones (14.0 ± 0.5 mm and 13.0 ± 0.5 mm) were also determined against B. subtilis and S. aureus at 25 °C. DPPH bioassay was conducted to find the antioxidant properties of Me-AgNPs. The highest (38.66 ± 0.2%) free radical scavenging activity was shown by Me-AgNPs at 4 mg/mL. Present study results concluded that biogenic Me-AgNPs have bactericidal as well as anti-oxidative potential. Moreover, these green synthesized Me-AgNPs could maintain their potency and stability at a wide range of pH and temperature.

Therapeutic Values of Earthworm Species Extract from Azad Kashmir as Anticoagulant, Antibacterial, and Antioxidant Agents.

AIMS: Current research aimed to explore the therapeutic values of different earthworms as antibacterial, anticoagulant, and antioxidant agents. METHODS: Ten different earthworms, i.e., Amynthas corticis, Amynthas gracilis, Pheretima posthuma, Eisenia fetida, Aporrectodea rosea, Allolobophora chlorotica, Aporrectodea trapezoides, Polypheretima elongata, Aporrectodea caliginosa, and Pheretima hawayana, were collected and screened for biological activities. Antibacterial effect analysis of earthworm species was done against fourteen bacterial pathogens, i.e., Escherichia coli, Serratia marcescens, Streptococcus pyogenes, Staphylococcus epidermidis, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa (1), Salmonella typhimurium, Shigella flexneri, Enterobacter amnigenus, Serratia odorifera, Pseudomonas aeruginosa (2), Staphylococcus warneri, and Lactobacillus curvatus, via agar well diffusion, crystal violet, MTT, agar disc diffusion, and direct bioautography assays. Antioxidant potential was evaluated through ABTS and DPPH assays. Lipolytic, proteolytic, and amylolytic assays were done for lipase, protease, and amylase enzymes confirmation. In vitro anticoagulant effects were examined in the blood samples by measuring prothrombin time. RESULTS: Results revealed that all earthworm extracts showed the inhibition of all tested bacterial pathogens except P. aeruginosa (1), P. aeruginosa (2), S. warneri, and L. curvatus. The maximum zone of inhibition of E. coli was recorded as 14.66 ± 0.57 mm by A. corticis, 25.0 ± 0.0 mm by P. posthuma, 20.0 ± 0.0 mm by E. fetida, and 20.0 ± 0.0 mm by A. trapezoid. Cell proliferation, biofilm inhibition, the synergistic effect of extracts along with antibiotics, and direct bioautography supported the results of agar well diffusion assay. Similarly, P. hawayana, A. corticis, A. caliginosa, and A. trapezoids increase the prothrombin time more efficiently compared to other earthworms. A. corticis, A. gracilis, A. rosea, A. chlorotica, P. elongata, and A. trapezoides showed maximum DPPH scavenging potential effect. CONCLUSIONS: The coelomic fluid of earthworms possessed several bioactive compounds/enzymes/antioxidants that play an important role in the bacterial inhibition and act as anticoagulant agents. Therefore, the development of new therapeutic drugs from invertebrates could be effective and potential for the prevention of the emergence of multidrug-resistant bacteria.

Motility effects biofilm formation in Pseudomonas aeruginosa and Enterobacter cloacae.

Chronic infections caused by gram negative bacteria are the mains reasons to have morbidity and death in patients, despite using high doses of antibiotics applied to cure diseases producing by them. This study was designed to identify the role of flagella in biofilm formation Ten pure strains were collected from our lab. Morphological variation and motility assays led us to study two strains in detail. They were characterized biochemically, physiologically and genetically. Biofilm formation analysis was performed using test tube assay, congo red assay and liquid-interface coverslip assay. In order to disrupt flagella of studied strains, blending was induced for 5, 10 and 15 minutes followed by centrifugation and observing motility using motility test. Biofilm quantification of wild type (parental) and blended strains was done using test tube and liquid interface coverslip assays. 16S rRNA sequencing identified strains as Pseudomonas aeroginosa and Enterobacter cloacae. Significant biofilm formation (p>0.05) by was observed after 72 and 18 hours using test tube and liquid-interface coverslip assays respectively. Flagellar disruption showed that 15 minutes blending caused significant reduction in both strains, hence demonstrated that flagellar mediated motility could be a potent strategy to stabilize aggregate and invest resources for biofilm formation in P. aeruginosa and E. cloacae.

Flagellar motility plays important role in Biofilm formation of Bacillus cereus and Yersinia enterocolitica.

Bacteria live either independently as planktonic cells or in organized surface associated colonies called as biofilms. Biofilms play an important role in increased pathogenesis of bacteria and it is assumed that motility is one of the contributing factors towards biofilm initiation. This study was planned to identify the role of flagella in biofilm formation by constructing flagellated (wild type) and physically disrupted variants (non-motile). Total 10 clinical bacterial strains were isolated and characterized. Morphological and biochemical study identified these strains as Enterobacter spp., Pseudomonas spp., Yersinia spp., Escherichia spp., Salmonella spp., Proteus spp., Staphylococcus spp., Streptococcus spp., Lactobacillus spp. and Bacillus spp. Among all strains, two strains including Yersinia spp and Bacillus spp. showed higher antibiotic resistance, hence studied at molecular and physiological level. Biofilm formation capacity of strains was analyzed using three methods including Congo red assay, Test tube assay and Liquid-interface coverslip assay. Afterwards, flagellar disintegration was induced by blending and centrifugation for 5, 10 and 15 minutes. 16S rRNA sequencing showed two strains as Bacillus cereus and Yersinia enterocolitica. Both strains produced significant biofilm by all three above mentioned methods. A motility test of these blended variants showed partial/diminished motility with increased blending time. The significant loss in biofilm formation after 15 minutes blending confirmed the important flagellar contribution to the initiation of biofilm formation. This biofilm defect observed in flagella paralysed/minus variants presumably may be due to defects in attachments to surface at early stages. This study indicated that flagellar motility is crucial initially for surface attachment and subsequently for biofilm formation.

Antibacterial activity of different plant extracts and antibiotics on pathogenic bacterial isolates from wheat field water.

The present work was carried out to study bacterial pathogens isolated from wheat field water and also effect of some plant extracts on these bacterial pathogens. Five bacterial strains were isolated from wheat field water. Different morphological and biochemical tests were performed to identify and characterize bacterial pathogens. Among isolated strains two belonged to genus Staphylococcus sp., other two were Pseudomonas spp. and one strain belonged to genus Salmonella sp. Effect of various parameters such as temperature, pH, antibiotics and heavy metals of these pathogens were also studied. Optimum temperature for all bacterial strains was 37oC and optimum pH was 7 except strain 3 which had pH 6. Different antibiotics with different potency were applied to check the resistance of bacterial strains against them. Among these antibiotics Cloxacillin and Teicoplanin were most potent while Oxacillin was as less potent antibiotic because three bacterial strains were resistant against it. While remaining antibiotics proved as potent. Seven heavy metals which were zinc (Zn+2), copper (Cu+2), Ferrous (Fe+2), mercury (Hg+2), Nickel (Ni+2), chromium (Cr+2) and cobalt (Co+2) with different concentrations were applied to bacterial strains. Minimum inhibitory concentration of heavy metals for all bacterial was different. Different plant extracts (Artemezia incise, Colebrookia oppositifolia, Rhynchosia pseudocajan) checked for their antibacterial activity against these pathogens. These plant extracts showed antibacterial activity against antibiotic and metal resistant bacterial isolates.

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.

Efficacy of cow and buffalo dung on vermiremediation and phytoremediation of heavy metals via Fourier-transform infrared spectroscopy and comet assay.

Heavy metal contamination raised significant concerns throughout the world. The current research aimed to evaluate the impact of organic manure (cow dung and buffalo dung) on vermiremediation and phytoremediation and to remediate heavy metals, i.e., cadmium, lead, and chromium, from artificial contaminated soil via both remediation techniques. The impact of livestock manure was evaluated for the first time which could be effective in in situ as well as ex situ studies. Eisenia fetida, Pheretima lignicola, and Spinacia oleracea were used for the remediation process. Results revealed that E. fetida tolerated lead at 280 mg, cadmium at 150 mg, and chromium at 860 mg compared to P. lignicola. The growth and reproduction of E. fetida were efficient in the cow dung manure compared to buffalo dung. Similarly, seed germination and growth of Spinacia oleracea were better in cow dung media compared to buffalo dung. Bioaccumulation factor showed that E. fetida showed a higher accumulation of heavy metals in their tissues when vermi + phytoremediation was jointly applied (9.50 mg/l of Pb, 24.166 of Cd, and 6.695 of Cr). Fourier-transform infrared spectroscopy indicated that heavy metals had no drastic effects on E. fetida and S. oleracea. Similarly, comet assay revealed that heavy metals had no genotoxic effect on the E. fetida and S. oleracea. It was concluded that both E. fetida and S. oleracea are appropriate for heavy metals remediation in cow dung manure.

Acute toxicity, anti-diabetic, and anti-cancerous potential of Trillium govanianum-conjugated silver nanoparticles in Balb/c mice.

BACKGROUND: The current study aimed to develop an economic plant-based therapeutic agent to improve the treatment strategies for diseases at the nano-scale because Cancer and Diabetes mellitus are major concerns in developing countries. Therefore, in vitro and in vivo anti-diabetic and anti-cancerous activities of Trillium govanianum conjugated silver nanoparticles were assessed. METHODS: In the current study synthesis of silver nanoparticles using Trillium govanianum and characterization were done using a scanning electron microscope, UV-visible spectrophotometer, and FTIR analysis. The in vitro and in vivo anti-diabetic and anti-cancerous potential (200 mg/kg and 400 mg/kg) were carried out. RESULTS: It was discovered that Balb/c mice did not show any major alterations during observation of acute oral toxicity when administered orally both TGaqu (1000 mg/kg) and TGAgNPs (1000 mg/kg), and results revealed that 1000 mg/kg is not lethal dose as did not find any abnormalities in epidermal and dermal layers when exposed to TGAgNPs. In vitro studies showed that TGAgNPs could not only inhibit alpha-glucosidase and protein kinases but were also potent against the brine shrimp. Though, a significant reduction in blood glucose levels and significant anti-cancerous effects was recorded when alloxan-treated and CCl4-induced mice were treated with TGAgNPs and TGaqu. CONCLUSION: Both in vivo and in vitro studies revealed that TGaqu and TGAgNPs are not toxic at 200 mg/kg, 400 mg/kg, and 1000 mg/kg doses and possess strong anti-diabetic and anti-cancerous effects due to the presence of phyto-constituents. Further, suggesting that green synthesized silver nanoparticles could be used in pharmaceutical industries to develop potent therapeutic agents.

Phylogenetic Illustration of Eisenia fetida Associated Vermi-bacteria Involved in Heavy Metals Remediation and Retaining Plant Growth Promoting Traits.

Heavy metals contamination in the soil is a major threat to wildlife, the environment, and human health. Microbial remediation is an emerging and promising technology to reduce heavy metals toxicity. Therefore, the present research aimed to isolate and to identify the heavy metals tolerated bacteria from the Eisenia fetida for the first time, and to screen the bacto-remediation capabilities and plant growth promoting traits of vermi-bacterial isolates. Vermi-bacteria was isolated from the gut of E. fetida, identified through staining, culturing, biochemical tests, and ribotyping. Plant growth-promoting traits were also evaluated. Phylogenetic results revealed that isolated Vermi-bacterial strains showed resemblance with Bacillus thuringiensis, Bacillus aryabhattai, Staphylococcus hominis, Bacillus toyonensis, Bacillus cabrialesii, Bacillus tequilensis, Bacillus mojavensis, Bacillus amyloliquefaciens, Bacillus toyonensis, Bacillus anthracis, and Bacillus paranthracis. All identified Vermi-bacterial species are Gram-positive (rod and cocci) in nature, not only indicated the efficient biosorption of lead, cadmium, and chromium but also produce all plant growth stimulating traits such as indole acetic acid (IAA), amylase, protease, lipase, hydrogen cyanide, ammonia, and siderophore production, and also act as a phosphate solubilizers. Bacillus anthracis showed significant production of siderophore (33.0±0.0 mm), phosphate solubilizing (33.0±0.0 mm), proteolytic (15.0±0.0 mm), and lipolytic activities (20.0±0.0 mm) compared to other vermi-bacterial isolates. Bioaccumulation factor results revealed that Bacillus anthracis showed more accumulation of Cd (12.00±0.01 ppm), Cr (5.38±0.01 ppm), and Pb (4.38±0.01 ppm). Therefore, the current findings showed that all identified vermi-bacteria could be used as potential bactoremediation agents in heavy metals polluted environments and could be used as microbial biofertilizers to enhance crop production in a polluted area.

Bacteriocin-mediated inhibition of some common pathogens by wild and mutant Lactobacillus species and in vitro amplification of bacteriocin encoding genes.

Lactobacilli are the most common probiotics used in food and other industries because of their capability of producing bacteriocins. Bacteriocins are compounds that are used to kill pathogenic microorganisms. As most bacteria have become resistant to synthetic antibacterial tools, the importance of using probiotics as antibacterial agents has increased. This work was done to check the bacteriocin effect on some common pathogens and the influence of mutation on the bacteriocin activity of Lactobacilli was also investigated. Four strains were isolated, identified from meat and pickles samples via culturing methods, staining, biochemical tests, and ribotyping. Preliminary tests, including Gram staining and catalase test, were done for the confirmation of Lactobacillus species. All strains were gram-positive and catalase-negative. Antibacterial activity was checked against Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus thuringiensis, Escherichia coli, and Salmonella enteritis via agar well diffusion method. The mutations were done using ethidium bromide and the influence of wild and mutants were also checked. Interestingly, mutants developed more virulence than wild ones. It was also observed that they all were sensitive to pepsin. Protein estimation was done via Bradford method. Ribotyping of GCU-W-PS1 revealed 99 % homology with Lactobacillus plantarum and GCU-W-MS1 to Lactobacillus curvatus (99 % homology). Curvacin A, sakacin P, and plantaricin A genes were also amplified using specific primers. Gene sequence showed the presence of curvacin A gene in GCU-W-MS1. It was concluded that lactic acid bacteria could be used as antibacterial tools against common pathogens.

Bacteriocin Production by Lactobacilli and Their Role as Antibacterial Tool against Common Pathogens.

Lactobacilli are commonly found in raw and fermented foods. These bacteria are of common interest because of their capability of bacteriocin production. Bacteriocins are compounds that may be used as natural preservatives in place of chemical additives. In the present study five different samples of poultry, meat and pickle were used to isolate lactic acid bacteria (LAB) by using the MRS culture medium. Antibacterial activity was checked against Pseudomonas aeruginosa, E. coli and Staphylococcus aureus. Five strains from the poultry meat and six strains from pickle had shown antibacterial activity out of eight strains against pathogenic bacteria. Three strains of poultry meat i.e. M3, M7, M11, had shown antibacterial activity against all three indicator strains. While one strain M1 was active against Pseudomonas aeruginosa and E. coli but it was found not to be active against Staphylococcus aureus. E. coli and Staphylococcus aureus were not susceptible to M2. In the case of pickle strains H1, H2, NAT1, NAT2, were found to be active against Pseudomonas aeruginosa and E. coli but Staphylococcus aureus was not susceptible to bacteriocins of these strains. NAT3 and SHZa strains showed antibacterial activity against all the three used pathogens. The highest activity (6 mm) had shown by the lNAT3 strain of pickle against Pseudomonas aeruginosa. The least antimicrobial activity had shown by SHZa strain. M3 had an inhibition zone of 5 mm against Pseudomonas aeruginosa. M11 had an inhibition zone of 5 mm against E. coli. Bacteriocins of all isolates were sensitive towards enzyme pepsin showing their proteinaceous nature. Detection of the bacteriocin gene by PCR the strains M2, M7 were amplified for Curvacin and plantaricin genes. From current study, it is concluded that different species of Lactobacilli can be used as antibacterial tool against common bacterial pathogens.

Molecular characterization of plant growth-promoting vermi-bacteria associated with Eisenia fetida gastrointestinal tract.

Earthworms are highly productive invertebrates and play a vital role in organic farming and improving soil structure and function. The gastrointestinal tract of earthworms possessed agricultural important bacteria. So, the current research aimed was to examine, screen, and identify the plant growth promoting bacteria existing in the digestive tract of Eisenia fetida called plant growth promoting vermi-bacteria. The plant growth promoting traits such as siderophore, phytohormone, and hydrolytic enzymes production, and phosphate solubiliation were assessed. Eleven vermi-bacteria i.e. Bacillus mycoides, B. aryabhattai, B. megaterium, Staphylococcus hominis, B. subtilis, B. spizizenii, B. licheniformis, B. mojavensis, B. toyonensis, B. anthracis, B. cereus, B. thuringiensis, and B. paranthracis were isolated and identified based on microscopic studies, biochemical tests, ribotyping, and agricultural traits. All vermi-bacteria are Gram-positive rods except Staphylococcus hominis and produce different compounds such as siderophore, indole acetic acid, catalase, oxidase, proteases, amylases, and lipases. All vermi-bacteria also act as phosphate solubilizers. Therefore, all isolated vermi-bacteria could be used as potential microbial biofertilizers to enhance crops production in Pakistan.

Synthesis of Cinnamum zeylanicum and Acacia nilotica Extracts and Their Antibacterial Activity against Staphylococcus aureus and Streptococcus pyogenes.

Different plants are used medically and thofese therapeutic plants have great importance for healing contagious wounds. This herbal treatment is actually also a substitute of different antibiotics and having less side effects on intestinal systems of animals. The foremost concern of this study was to observe the antibacterial activity of Cinnamum zeylanicum and Acacia nilotica. Pathogenic bacteria obtained from wound samples and later identified by biochemical and molecular characterization. Methanol (an organic solvent) was used to extract Cinnamum zeylanicum and Acacia nilotica to check their antimicrobial exertion by using agar diffusion method. Different antibiotics such as, ampicillin, oflaxocin, ticarcillin and cefexime, showed their susceptibility toward antibiotics. The zone of inhibitions for antibiotic and plant extracts' antibacterial activity were measured. Pathogenic bacteria were identified as Staphylococcus aureus and Streptococcus pyogenesby molecular characterization. These bacteria showed susceptibility to antibiotics and also the plant extracts. Antibiotic oflaxocin showed maximum activity against these two pathogens (12.25 ± 0.44 and 12.375 ± 0.47) while antibiotic cefixime showed minimum effect (1.25 ± 0.28 and 0.625 ± 0.25). Plant extracts showed significant antibacterial activity with maximum activity (14 ± 0.9 by Acacia nilotica and 12 ± 0.5 by Cinnamum zeylanicum) in 100% solution. It can be concluded thatmethanolic extract of traditional therapeutic plants proved to be a promising source of antimicrobial agents against antibiotic resistant bacteria. Cinnamum zeylanicum and Acacia nilotica were observed to be competent as antibacterial tool against pathogenic bacterial strains.

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.

Optimization of fermentation media and growth conditions for microbial xylanase production.

Efficiency of cellulase-free xylanases is one of the determining factors in paper and pulp industries. Use of microbes which can produce cellulase-free xylanases may help to overcome the current challenges in kraft pulp processing. Isolation and screening of microorganisms from local samples offers a possibility for obtaining the potential microbes for this purpose. This research was therefore aimed to collect, screen, characterize and identify potential cellulase-free xylanase producers. A total of 313 microbial isolates were collected while using selective media (EBAM and XAM) to determine the xylanolytic potential of microbes. Qualitative and quantitative analyses were performed and finally 11 bacterial and 6 fungal strains were selected for characterization and identification. The potential isolates were identified as Bacillus pumilus (388.82 U/mg), Bacillus safensis (385.26 U/mg), Aspergillus flavus (493.33 U/mg) and Aspergillus niger (419.33 U/mg). Optimization of the microbial strains while using agro-industrial waste is suggested.

Multienzyme and Antibacterial Potential of Bacteria Isolated from gut of Asian Honey Bee (Apis cerana Indica), Lahore Using Culture Dependent Method

Abstract The bacteria residing in the gut of honey bees (HB) has demonstrated a significant role in protecting bees against various pathogens, production of honey and wax. However, no information exists about the antibacterial potential of bacterial isolates from gut of Asian HB, Apis cerana Indica F. (Hymenoptera: Apidae), against human pathogens. This study aims to investigate the antibacterial and multienzyme potential of aerobic bacteria from A. cerana gut using culture dependent approach. A total of 12 HB gut bacteria were characterized morphologically and biochemically. These strains were further screened for their antimicrobial activity against pathogenic human microorganisms Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumonia, Bacillus licheniformis and Bacillus subtilis using cross streak (primary screening) and agar well diffusion methods (secondary screening). Preliminary characterization of cell-free supernatant (CFS) of two promising isolates was performed by measuring lactic acid concentrations, enzymatic digestion of antimicrobial compounds, stability over a range of temperature, pH and amplification of spaS (subtilin) and spoA (subtilosin) genes. In primary screening, among 12 HB isolates, eight strains showed statistically significant highest zones of inhibition (p≤0.05) against E. coli, K. pneumoniae and P. aeruginosa. 16S rRNA sequencing revealed that these isolates belong to Bacillus genus, identified as B. tequilensis, B. pumilus, B. xiamenensis, B. subtilis, B. amyloliquefaciens, B. safensis, B. licheniformis, B. altitudinis (Accession numbers: MT186230-MT186237). Secondary screening revealed that among eight isolates, B. subtilis and B. amyloliquefaciens showed statistically significantly strong inhibition (p≤0.05) against all tested pathogens. Antibiotic susceptibility testing revealed that both isolates were resistant to antibiotics and possesses proteolytic, lipolytic and cellulolytic activities. The nature of the compound causing inhibitory activity was found to be proteinaceous and showed stability over a wide range of temperature as well as pH. PCR study confirmed the presence of bacteriocins by successful amplification of important antimicrobial peptide biosynthesis genes spaS and spoA. These results suggest that the HB gut is a home to bacteria that possess antimicrobial activity and important enzymes with antimicrobial potential. To our knowledge, this is the first report demonstrating the antimicrobial potential of bacteria isolated from gut of HB (A. cerana) against human pathogens.