Journal of Pure and Applied Microbiology

Karst habitat, as a kind of fragile ecology environment, has some typical characters such as shallow and less soil, high rate of bare stone, serious soil erosion and few soil nutrient content. However, D. luodianense can distribute and grow well in Karst regions.. In this study, ecological characteristics of D. luodianense were studied in the Karst microhabitat by field investigation and comparative and system analysis. The results showed that there was a series of adaptive features for Karst environment in D. luodianense population. It has high shoot-emergence number and mortality rate and low growing rate, and these features mainly result from nutrition shortage. Our finding may not only provide theoretical direction for the protection and development of D. luodianense, but also benefit species selection and reasonable collocation in the process of rocky desertification in Karst area.

Several scolocidal agents have been used to eradicate the infective protoscoleces, however recurrences occur. The objective of this study was determining of the scolicidal effects of 3% Hydrogen peroxide, 0.5% Silver nitrate and 20% hypertonic saline on protoscoleces of hydatid cyst. Protoscoleces were collected aseptically from sheep and cattle livers containing hydatid cyst. Hypertonic saline was used as positive control, while normal saline was used as negative control. These scolocidal solutions were used for 5,10,15 and 20 minutes in the experiments. Viability of protoscoleces was assessed by 0.1% eosin staining . Normal saline had no significant protoscolicidal effect. However, 20% Hypertonic saline and 0.5%Silver nitrate solution revealed higher protoscolicidal effect than 3% Hydrogen proxide. The results showed that hypertonic saline 20% and 0.5% Silver nitrate solution is highly effective in killing protoscoleces of Echinococcus granulosus in vitro.

A bacterium which produced novel extracellular fibrinolytic enzyme for digesting bovine blood clots was isolated from soil, and identified by 16s rRNA sequencing as Bacillus altitudinis, given strain name was S-CSR 0020 (accession number KT369312). Fibrin proved the best nitrogen source with an enzyme activity of 750 U/mL, followed by casein after incubation at 37 °C for 4 days. The cultural conditions were optimised using Response Surface Methodology (RSM) and Box-Behnken Design (BBD). Based on 3D surface plot and contour plots, the optimized temperature, pH and substrate concentrations were 47 °C, 10.5 and 4 g/L respectively, resulted in increase in enzyme activity of 306.88 U/mL and specific activity of 780 U/mg which was 2-fold; compared to initial level of 400 U/mg after 2 days of incubation. The crude enzyme has got potent activity and digested human blood clot completely within 1 hr.

The main aim of this work was optimization of physical conditions as well as medium components for the maximum production of fibrinolytic enzyme. A fibrinolytic enzyme producing organism was isolated from soil and identified as Bacillus altitudinis strain S-CSR 0020. Various physical parameters such as temperature, pH, incubation time and medium components such as inoculum size, substrate (nitrogen and carbon) concentrations were optimized. A cultivation medium was designed using optimized conditions by Box Behnken Design for mass production of fibrinolytic enzyme and specific activity of enzyme was analyzed. The maximum enzyme production seen at 50 °C temperature, 10.0 pH, 4% substrate concentration with 3 ml inoculum size and 96 h. of incubation time. Among the different carbon sources tested, fructose (8%) showed maximum enzyme activity of 325 U/ml with a specific activity of 812.5 U/ml. Box Behnken Design was used to optimize the percentage of best nitrogen sources (casein, peptone, soya bean and yeast extract) with an enzyme activity of 1089.8 U/ml. Experiments were repeated with optimized condition manually and found with an enzyme activity of 1072.12 U/ml. Mass production of extracellular fibrinolytic enzyme was done with all these conditions and it showed an enzyme activity of 2070 U/ml with an initial activity of 400 U/ml without medium optimization. There was a fivefold increase in fibrinolytic activity after medium optimization that proved the reliability of optimization manually as well as using the Box Behnken design. © 2018 Journal of Pure and Applied Microbiology. All rights reserved.

Streptomyces sp. strain ERIMA-01 was isolated from marine sponge. The strain was grown in ISP 2 medium to study the morphology and biochemical characteristics. The strain was subjected to 16S rRNA and identified as Steptomyces sp. The nucleotide sequence of the 16S rRNA gene of Streptomyces sp. strain ERIMA-01 exhibited close similarity with other Streptomyces. The accession number was FJ865352. Antifungal metabolite production of ERIMA-01 was evaluated using five different fermentation media. Most active broth (MNGA) was extracted with ethyl acetate. The ethyl acetate extract inhibited the growth of all the tested fungi (Trichophyton mentagrophytes at 62.5μg/ml, Trichophyton rubrum at 31.25 μg/ml, Trichophyton simii at 125 μg/ml, Scopulariopsis sp at 125 μg/ml, Aspergillus flavus at 250 μg/ml, Aspergillus Niger at 62.5 μg/ml, Botrytis cinerea at 125 μg/ml, Candida albicans at 15.2 μg/ml, Candida krusae at 31.25 μg/ml, Candida tropicalis at 125 μg/ml and Candida parapsilosis at 125 μg/ml. Most active ethyl acetate extract was subjected to GC-MS analysis which showed 38 components. The major compound was 1-tetradecanol. The present paper reports the isolated Streptomyces sp. Showed good antifungal activity.

The presence of E. coli enterohemorragic O157:H7 was inspected in 756 samples of frozen bovine meat imported from different countries, Argentina, Australia, Brazil, Ireland and Uruguay, using the microbiological standard method of culture and serotyping technique. The pathogen strain of E. coli was detected in five samples (0.66 %). All the isolates were tested using the method of KIRBY and BAUER according to the nccls standards to view resistance to antibiotics and was considered sensitive to the sixteen antimicrobial agents tested. All isolates were further characterized by serotyping and molecular methodology. The PCR technique showed that all the strains carried the stx2 and the ehxA genes 1 isolate carried stx1 genes, 1 both stx1 and stx2. Intimin (eae c) virulence genes were detected in 4 isolates. The interaction with lactic acid bacteria revealed that all E. coli O157:H7 isolates were inhibited.

Due to the fact that the Zataria multiflora essential oil has antimicrobial effects on some food pathogenic microbes, the aim of the study is to seek to determine the effects of different Zataria multiflora essential oil concentrations of (0, 0.005, 0.015 and 0.03%) on E. coli 0157:H7 growth and survival in MDM (mechanical deboned meat) on different days (10 days) storage in refrigerator. The plants essential oil was prepared using the distilled water and the Essential oil analysis was performed by gas Chromatograph connected to mass spectrograph (GC/MS). The antimicrobial effects of different Zataria multiflora essential oil concentrations were evaluated for the bacterium growth in a sample culturing condition in a laboratory. The results showed that by increasing the essential oil concentration, the bacterium growth rate in the storage period would take a downward trend and decrease; and the concentration level of %0/03 had the highest antibacterial effects which was statistically significant (P < 0.01). It was also stated that the E. coli 0157:H7 bacterium population in MDM (mechanical deboned meat) with different Zataria multiflora essential oil concentrations kept at 4 °C showed a higher degree of reduction compared with the 10 °C which is considered to be unfavorable temperature condition and shows the storage key role for maintaining a better condition.

A thermophilic esterase isolated from Rhodococcus sp. LKE-021. This enzyme was purified with purification fold 60 from the crude extracts of enzyme and recovery of enzyme obtained approximately 21%. The specific activity of the LKE-021 esteraseis 795.1 U/mg. SDS-PAGE analysis determined the molecular weight of LKE-21 esteraseis around 32,000Da/32KDa. The enzyme activity of LKE-021 esterase exhibited over a wide range of temperature i.e. 30° to 80°C and the enzyme remained stable when incubated on 60° for 2h. This indicates that the isolated LKE-021 esterase is thermostable. The isolated enzyme exhibits activity on various pH ranges from 2.0 to 12.0 and the highest activity observed on 11.0 pH.The LKE-021 esterase was active after proteinase K treatment and shows over 75 % specific activity i.e. 50 U/µg Proteinase K.

Rhodococcus sp. LKE-021 from soil samples of the region of Gangotri (10,000 feet of average height) of Uttarakhand Himalayas, India, produced a thermophilic esterase. The physiological and morphological characteristics of the isolated Rhodococcus sp. LKE-021 detected as Gram Positive, rod shape, catalase positive, indole negative, positive to glucose and xylose fermentation test, and can grow on the Nutrient Broth medium. Esterase production confirmed on the basis of spectrophotometric enzyme assay. Taxonomic characteristics Rhodococcus confirmed by 16s rRNA gene sequencing.

Foodborne pathogens are the main threat and cause of food poisoning. The majority of food infections have been related to the biofilm formation of foodborne pathogens in the food industry. Shewanella putrefaciens (KX355803, GRD 03), a Gram-negative pathogen isolated from mackerel fish, was identified and recognized as a food spoilage bacterium and a strong biofilm producer. The adhesion or attachment ability of Shewanella putrefaciens was determined on steel, plastic, glass, PVC and wood. NB (Nutrient broth), LB (Luria-Bertani broth), TSB (Tryptic soy broth) and BHI (Brain heart infusion broth) were enriched with glucose and shows optimum for bacterial adhesion. In the microtiter plate method (MTP), the strong attachment was observed at 48 and 72 hours of incubation and significant differences were obtained at p < 0.05. As the incubation period increases, the OD value (Optical density) of samples also increase. Biofilm formation is the major cause cross-contamination, and shows resistance to certain disinfectants, which leads to environmental stress tolerance. This study suggested with optimum biofilm production of isolate from fish by using glucose enriched media on different substrates, also comparing different growth media provide a detailed idea about biofilm-forming ability at different incubation time intervals.

Optically pure (S)-1-phenyl-1,2-ethanodiol(PED) is a versatile chiral building block for the synthesis of pharmaceuticals, pheromones and liquid crystals. An alternative and convenient biocatalytic process was developed for the preparation of (S)-PED by Gluconobacter oxydans-catalyzed optical resolution of the corresponding racemate. Whole cells of G. oxydans DSM2003 were found to catalyze the regio- And stereo-selective concurrent oxidation of racemic PED to give (S)-PED with the optical purity of 99% and yield of 38% in 10h, meanwhile (R)-mandelic acid with moderate ee value produced. This oxidation is carried out by two sets of enzymes, the one made of membrane-bound alcohol dehydrogenase is responsible for the PED oxidation to hydroxyl aldehyde intermediate and the other composed of membrane-bound aldehyde dehydrogenase and other enzymes is responsible for the mandelic acid production.

Novel series of 1-substituted-2-thio-(1H)-4-(3-phenylthiocarbamido-1- yl)-6-(1-substituted-guanidino-3-yl)-1,2-dihydro-s-triazine [4a(i) to 4f(ii)] has been obtained by the isomerisation of 2-(1- substitutedguanidino-3-yl)-4-(3-phenylthiocarbamido-1-yl)-6- substitutediniino-1,3,5-thiadiazine [3a(i) to 3f(ii)] in presence of ethanolic sodium bicarbonate solution, which have been obtained by basification of their hydrochlorides [2a(i) to 2f(ii)] which are synthesized by the interaction of 1-Formamidino- (N-substitutedthioamido)-5-phenyl-2-thio-4-iminobiuret (1a-f) and N-aryl/alkylisocyanodichlorides. The latter were prepared initially by the condensation of N-aryl/alkylisothiocyanate with N-phenylformamidinoformamidino-thiocarbamide. The structure of all these compounds was established on the basis of elemental analysis, IR and PMR spectral data. All the synthesized compounds have been screened for their antimicrobial activity against both gram-positive and gram-negative human pathogen.

The main purpose of the work was to screen intracellular lipase-producing microbial with high selective synthesis of 1,3-diolein, a healthy lipid. The screening model is a combination of Rhodamine B-olive oil agar plat, para-nitrophenyl palmitate (ρ-NPP) for ester synthetase activity and HPLC analysis of products of glycerolysis of triolein. The results show that the strain GZUF36 (CCTCC No. M2012538) can be used to catalyze the selective synthesis of 1,3-diolein. 1,3-Diolein yield is 11.12% (w/w) by whole-cell of GZUF36 catalyzed glycerolysis and it is accounted for 72% of total diolein (including 1,3- and 1,2-isomer). Then, GZUF36 was identified as Aspergillus niger by morphology and 18S rDNA sequence analysis. It is also suggested that the model can be applied to screen other lipases with high ester synthetase activities.

Novel series 2-(2-imino-4-thio-5-substitutedbiureto-1-yl)-4-(3-substituted thiocarbamido-1-yl)-6-substitutedimino-1,3,5-thiadiazine [3a(i) to 3f(iii)] have been obtained by basification of their hydrochlorides (2a(i) to 2f(iii)] in presence of ammonium hydroxide solution, which are synthesized by the interaction of 1,3-bis-(N-substitutedamidinothiocarbamido)-thiocarbamide(1a-1f) and aryl/alkylisocyanodichlorides. The latter were prepared initially by the condensation of aryl/alkylisothiocyanate with 1,3-Diformamidinothiacarbamide. The structure of all these compounds was established on the basis of IR and PMR spectrum data. All the synthesized compounds have been assayed for their antibacterial activity against both gram-positive and gram-negative human pathogens.

Pectobacterium wasabiae is a dreadful causal agent of potato soft rot. It relied mainly on the production a wide range of plant cell wall-degrading enzymes to cause disease. Of the different enzymes, the endo-b-1,4-glucanase may be important in pathogenesis, being responsible for hydrolyzing the cellulose. In the process of pathogen infection, cellulase secreted by the pathogen is major determinant of pathogenicity, which played an important role in softening and decomposition of cell wall. But so far, the related research very rarely. However, most characteristic of the cellulase in the soft rot bacteria was not clear so far. Here, a putative endo-b-1,4-glucanase gene from P. wasabiae SCC3193 was previously synthesized according the bias codon of yeast, heteroexpressed in Pichia pastoris, and further investigated for its characteristic. Pectobacterium wasabiae SCC3193 is a genetically well characterized model in soft rot research. SDS-PAGE analysis showed that the recombinant endo-b-1,4-glucanase (PcegAI) produced in P. pastoris has a molecular mass of approximately 30kDa. When CMC was used as substrate, it exhibited maximal activity at pH 4.0 with an optimum temperature of 40°C, and stable at pH 5-8. The Km and specific activity values for CMC are 64.5 mM and 7 ×103 U mg-1 respectively. Most metallic ions had no influence on the activity of endo-β-1,4-glucanase at a concentration of 1 mM except for Cu²⁺ and Mn²⁺. These characteristics suggested that PcegAI may play a major role in degrading of cellulose polymers. The high activity of this enzyme also has potential in biomass opening, such as the bioconversion of lignocellulosic materials and bioethanol production. © 2018 Journal of Pure and Applied Microbiology. All rights reserved.

Anaerobic bacteria producing cellulases have not been extensively studied though they are the efficient producers of cellulase. Clostridium papyrosolvens CFR-1010, an anaerobic cellulolytic bacterial strain was identified as a potent cellulase producer and selected for the purification and kinetic studies of Exo-β-l,4-glucanase and Endo-β-1,4-glucanase. The molecular masses of the enzymes were 65 and 60 kDa, respectively. The enzymes showed maximum activities at pH 5.0 and at the temperature of 50 °C. The activities increased in the presence of MnCl2, whereas, N-bromosuccinimide decreased enzyme activities by 68 and 75% respectively, thus suggesting the presence of tryptophan residues at the active sites of enzymes. Exoglucanase had Kmof 20 mg/ml and Vmaxof 22 units/min/mg of protein whereas the endoglucanase exhibited Km of 6.66mg/ml and V max of 11.76 units/min/mg of protein. Results of the present studies suggest the use of C. papyrosolvens for cellulase production in shorter periods of time and they also add significance for the exploration of this organism for industrial applications.

Bacteria living as biofilm are frequently reported to exhibit the resistances to the common antimicrobial agents. Hence, the in vitro antimicrobial activities of the compound 1,8-cineole were evaluated to explore the novel agent. The bioactivities were estimated by the MIC and MBC determinations on 8 species of bacteria and fungi, as well as the confocal laser scanning on 3 types of bacterial biofilms. It was found that the essential oil has good inhibiting activities against fungi rather than bacteria, especially Candida albicans, with the lowest MIC value (0.156 %). Both of gram-positive and gramnegative bacterial biofilms are sensitive to 1,8-cineole. In addition, there is linear relationship between inhibition activities and concentration of 1,8-cineole. Further analysis revealed that 1,8-cineole can prompt the biofilm-surface bacteria to die off or live as planktonic, in order to help the immune system to remove the bacteria. Thus, 1,8- cineole could be judged as a kind of potential drug with rather antimicrobial activities.

Angiotensin-I-converting enzyme (ACE) is an important enzyme in the regulation of blood pressure. In recent years, several ACE inhibitory peptides had been isolated from the enzymatic digests of milk proteins, or fermented milk prepared with lactic acid bacteria. In this study, to analyze the inhibition mechanism of ACE by Lactobacillus casei 1.2435-fermented milk, the method of Cushman-Cheung was used to examine the change of ACE activity before and after being treated with the supernatant of fermented milk. In addition, the promoter of human ACE was also cloned and inserted into pGL3 luciferase reporter plasmid, and then the effect of the supernatant of fermented milk on the transcription of ACE was detected by luciferase reporter assay in COS-7 cells. The results showed that the ACE inhibitory rate of L. casei 1.2435-fermented milk was about 97.6%, and it would decrease to 73.6y and 75.7y after being treated with chloroform and boil, respectively. Besides, it seemed that the fermented milk and the lysis product of the bacteria also had an influence on the activity of ACE promoter.

Epidemiology and resistance patterns of bacterial pathogens in pediatric urinary tract infections (UTIs) show large inter-regional variability, and rates of bacterial resistance are changing due to different antibiotic treatment policy. The aim of this study was to determine the etiology and antibiotic resistance pattern UTI in an Iranian hospital. All children with culture proven UTI from April 2010 to March 2011 were included in our study. Urine culture was deemed positive with a pure growth >105CFU/ML (single organism).Identification of all isolates were performed by convectional bacteriology methods.Susceptibility testing was performed by disk diffusion methods as recommended by Clinical laboratory standard institutes. (CLSI) During our study in total 60951 urine specimen were cultured in our laboratory. Of 60951 urine cultures, 2676 (4.3%) were obtained from children under 12 years old. A total of 322 positive urine cultures were yielded. E.coli with 137 (42.54%) isolates was the predominant organisms. The second common organism was K.pneumoniae with 72(22.36) isolates.Among gram- positive organisms entrococci with 38 (11.80) isolate was the predominant organisms. .E. coli was found to be most sensitive to amikacin, nitrofurantoin,ofloxacin,ciprofloxacin and least sensitive to most commonly used drugs like ampicillin,cefazoline, nalidixic acid, Co-trimoxazole.Drug resistance among K.pneumoniae isolates were prevalent in comparison E.coli isolates. Vancomycin resistance among enterococci isolates was 7.4%.Nitrfuantoin was the second most effective antibiotic against entrococci isolates. Resistance rate of entrococci to tereacyclin ampicillin, nofloxacin was 70.37%, 48.14 and 33.33% respectively.

Biomineralization phenomenon of bacteria proved to have various biotechnological and environmental applications. Production of magnesium ammonium phosphate (struvite) crystals by the agarolytic bacterium Exiguobacterium aestuarii St. SR 101 isolated from red seaweed, Gracilaria corticata was reported for the first time in the present study. Struvite crystallization occurred in the agar culture medium in the presence of the bacterium. Crystal nucleation and growth occurred apparently as a consequence of the localized ion supersaturation, produced by the microbial metabolites and also by the microbial supply of heterogeneous nuclei resulted in crystallization. The crystals were visible between 10 to 15 days after inoculation. The crystal structure of the struvite characterized by optical microscopy, IR spectroscopy, thermogravimetry, powder X-ray diffractometry, and single crystal X-ray diffractometry. The orthorhombic crystal is with the space group Pmn21 and unit-cell parameters a = 6.9447 Å, b =6.1329 Å, c = 11.2026Å. Exiguobacterium aestuarii St. SR 101 showed to have the capacity of producing struvite based fertilizer by bioremediation of industrial phosphate wastes.

Effect of pectinase enzyme treatment on the physicochemical properties of guava wine including pH, ethanol yield and total soluble solid (TSS) was investigated. Saccharomyces cerevisiae IARI 1035 strain and native strain were used in the production of guava fruit wine. The results indicated that the enzyme treatment promoted juice clarification, increased ethanol yield and decreased TSS and pH of guava wine than that without enzyme. In conclusion, the development of guava wine quality can be achieved by pre-treatment with pectinase enzyme.

The present study was undertaken with the primary objective, PCR amplification of DNA isolated from a Pectinolytic fungus Aspergillus foetidus, which was identified and characterized by IMTECH, Chandigarh as MTCC 10367. Genomic DNA was extracted by using phenol: chloroform method. The quality of DNA was assessed by UV spectrophotometry. PCR was carried out in a final reaction volume of 25 μl reaction mixture by using 18S rDNA fungal primers. Increase in the stringency during initial cycles of PCR reaction increased the sharpness and brightness of the bands. Approximately an amplified product of 350 bp size was obtained for Pectinase enzyme with the genomic DNA of Aspergillus foetidus MTCC 10367.

Six novel Gram-stain-positive, rod shaped strains of bacteria designated as 10nlgT, 25nlgT, SPT, S5T, S7T and S9T which were isolated from marine ecosystems and soda lake from India, represents novel members of the family Bacillaceae. Apart from 10nlgT, 25nlgT and SPT all the other three strains represented members of novel genus from the family. Here we report the draft genome of the strains 10nlgT, 25nlgT, SPT, S5T, S7T and S9T. Strains 10nlgT, 25nlgT and SPT genomes comprised ~ 2.93 Mb, ~ 2.58 Mb and ~ 5.87 Mb with the G + C content of 47.13 %, 43.26 % 44.85 %, respectively. A total of 2772 protein-coding genes in strain 10nlgT, 2581 protein-coding genes in strain 25nlgT and 5199 protein-coding genes in strain SPT were reported. Strain S5T genome comprised ~ 2.52 Mb with the G + C content of 37.08 % and a total of 2407 protein-coding genes. Strains S7T and S9T genomes comprised ~ 3.61 Mb, ~ 4.43 Mb with the G + C content of 47.65 %, 42.42 % and a total of 3662, 4232 protein-coding genes, respectively.

Interleukin (IL)-11 is a multifunctional cytokine that stimulates hematopoietic progenitor cells and exerts a series of important immunomodulatory effects. Recombinant human interleukin-11 (rhIL-11) has been shown to increase platelet counts in animals and humans and is the only drug approved for use in chemotherapy-induced thrombocytopenia (CIT). rhIL-11 is 19 kDa protein (1} that lacks the N-terminal proline in compare with wild type and has been produced in recombinant E.coli. A synthetic gene of human interleukin 11 was designed, codon optimized, cloned and expressed in E.coli BL21 (DE3) and E. coli Rosetta (DE3) using a pET-15b expression vector. In this study we compared two strategies for better expression of heterologus protein by using two different E. coli systems. As the results show, expression of codon optimized 6His-interleukin 11 in E. coli Rosetta (DE3) was better in comparison with E. coli BL21 (DE3). Not only codon optimization, but appropriate strain selection has a great effect on recombinant protein production in E.coli.

Production of poly-(3-hydroxybutyrate) by Bacillus sp. 112A isolated from activated sludge sample using flours of ten different pulses and cereals, such as, pigeon pea, red lentil, black gram, bengal gram, green gram, corn, soya bean, wheat, rice and sorghum available in abundant as spills was in a range of 1.20 g/L to 0.14 g/L. Bacillus sp. 112A was able to accumulate a maximum of 1.20 g/L PHB within 30 h of incubation when corn flour (25 g/L) was used as carbon substrate suggesting a very faster rate of polymer synthesis when compared to all other substrates.