Activity Of Broth Research Articles

Comparison of a Short Linear Antimicrobial Peptide with Its Disulfide-Cyclized and Cyclotide-Grafted Variants against Clinically Relevant Pathogens.

According to the World Health Organization (WHO) the development of resistance against antibiotics by microbes is one of the most pressing health concerns. The situation will intensify since only a few pharmacological companies are currently developing novel antimicrobial compounds. Discovery and development of novel antimicrobial compounds with new modes of action are urgently needed. Antimicrobial peptides (AMPs) are known to be able to kill multidrug-resistant bacteria and, therefore, of interest to be developed into antimicrobial drugs. Proteolytic stability and toxicities of these peptides are challenges to overcome, and one strategy frequently used to address stability is cyclization. Here we introduced a disulfide-bond to cyclize a potent and nontoxic 9mer peptide and, in addition, as a proof-of-concept study, grafted this peptide into loop 6 of the cyclotide MCoTI-II. This is the first time an antimicrobial peptide has been successfully grafted onto the cyclotide scaffold. The disulfide-cyclized and grafted cyclotide showed moderate activity in broth and strong activity in 1/5 broth against clinically relevant resistant pathogens. The linear peptide showed superior activity in both conditions. The half-life time in 100% human serum was determined, for the linear peptide, to be 13 min, for the simple disulfide-cyclized peptide, 9 min, and, for the grafted cyclotide 7 h 15 min. The addition of 10% human serum led to a loss of antimicrobial activity for the different organisms, ranging from 1 to >8-fold for the cyclotide. For the disulfide-cyclized version and the linear version, activity also dropped to different degrees, 2 to 18-fold, and 1 to 30-fold respectively. Despite the massive difference in stability, the linear peptide still showed superior antimicrobial activity. The cyclotide and the disulfide-cyclized version demonstrated a slower bactericidal effect than the linear version. All three peptides were stable at high and low pH, and had very low hemolytic and cytotoxic activity.

Synergistic antimicrobial effect of lactocin AL705 and nisin combined with organic acid salts against Listeria innocua 7 in broth and a hard cheese

SummaryThe effectiveness of antimicrobial mixtures against Listeria innocua 7, used as a L. monocytogenes surrogate, was investigated in broth and a food system. Synergistic effects were found for nisin (Nis), potassium sorbate (PS), calcium propionate (CP) and sodium lactate (SL), Nis + PS being the most effective binary mixture that exhibited listericidal activity in broth. To assess the effect of adding lactocin AL705 (AL705) to Nis + organic acid salt combinations, tridimensional isobolograms were generated. Sub‐MIC combinations of the antimicrobials exerted bactericidal activity against L. innocua 7 after AL705 addition to the binary mixtures. However, when applied on Sardo cheese contaminated with L. innocua 7 (initial inoculum 4.45 ± 0.06 CFU g−1), only Nis + PS + AL705 produced count reductions respect to the control, reaching 3.04 ± 0.35 CFU g−1 counts after 15 days at 15 °C. Ternary combinations containing AL705 showed potential to reduce antimicrobial usages for L. innocua 7 inhibition.

Screening and Molecular Characterization of Cellulase Producing Actinobacteria from Litchi Orchard

Background:Cellulolysis is of considerable economic importance in laundry detergents, textile and pulp and paper industries and in fermentation of biomass into biofuels.Objective:The aim was to screen cellulase producing actinobacteria from the fruit orchard because of its requirement in several chemical reactions.Methods:Strains of actinobacteria were isolated on Sabouraud’s agar medium. Similarities in cultural and biochemical characterization by growing the strains on ISP medium and dissimilarities among them perpetuated to recognise nine groups of actinobacteria. Cellulase activity was measured by the diameter of clear zone around colonies on CMC agar and the amount of reducing sugar liberated from carboxymethyl cellulose in the supernatant of the CMC broth. Further, 16S rRNA gene sequencing and molecular characterization were placed before NCBI for obtaining recognition with accession numbers.Results:Prominent clear zones on spraying Congo Red were found around the cultures of strains of three groups SK703, SK706, SK708 on CMC agar plates. The enzyme assay for carboxymethylcellulase displayed extra cellulase activity in broth: 0.14, 0.82 and 0.66 µmol mL-1 min-1, respectively at optimum conditions of 35°C, pH 7.3 and 96 h of incubation. However, the specific cellulase activities per 1 mg of protein did not differ that way. It was 1.55, 1.71 and 1.83 μmol mL-1 min-1. The growing mycelia possessed short compact chains of 10-20 conidia on aerial branches. These morphological and biochemical characteristics, followed by their verification by Bergey’s Manual, categorically allowed the strains to be placed under actinobacteria. Further, 16S rRNA gene sequencing, molecular characterization and their evolutionary relationship through phylogenetics also confirmed the putative cellulase producing isolates of SK706 and SK708 subgroups to be the strains of Streptomyces. These strains on getting NCBI recognition were christened as Streptomyces glaucescens strain SK91L (KF527284) and Streptomyces rochei strain SK78L (KF515951), respectively.Conclusion:Conclusive evidence on the basis of different parameters established the presence of cellulase producing actinobacteria in the litchi orchard which can convert cellulose into fermentable sugar.

Temperature gradient-based high-cell density fed-batch fermentation for the production of pyruvate oxidase by recombinant E. coli

ABSTRACTPyruvate oxidase (PyOD) is a very powerful enzyme for clinical diagnostic applications and environmental monitoring. Influences of temperature on cell growth, plasmid stability, and PyOD expression during the PyOD fermentation process by recombinant Escherichia coli were investigated. Based on the influences of temperature on the physiological metabolism, a novel high-cell density fed-batch cultivation with gradient temperature decrease strategy for effective PyOD production was achieved, under which the biomass (OD600) of recombinant E. coli could reach to 71 and the highest PyOD activity in broth could reach to 3,307 U/L in 26 hr fermentation.

Characterization and lipolytic activity of lactic acid bacteria isolated from Thai fermented meat

Twenty six strains of lactic acid bacteria (LAB) were isolated from Thai fermented meat products, including 7 samples of Nham (fermented pork), 7 Sai-krog-prieo (fermented sausage) and one Mum (fermented beef). The isolates were identified as Lactobacillus pentosus (5 isolates), Lactobacillus sp. (11 isolates), Pediococcus pentosaceus (5 isolates) and each isolate of Pediococcus lolii, Leuconostoc fallax, Weissella thailandensis, W. cibaria, and W. paramesenteroides based on their phenotypic characteristics and 16S rRNA gene sequence similarities (99.8-100 %). The isolates were primary screened for their lipolytic activity on agar plates. The rod-shaped isolates showed 0.031±0.030-0.938±0.127 U/ml of lipase activity in broth supplemented with Tween 20, Tween 40, Tween 60 or Tween 80. The isolate SS50-1 identified as Lactobacillus pentosus showed the highest activity in Tween 80 (0.938±0.127 U/ml). The coccal isolates showed lipase activity ranged from 0.029±0.006-1.090±0.033 U/ml when Tween 20, Tween 40, Tween 60 or Tween 80 was used as a substrate. The isolate SS48-4 identified as Pediococcus lolii showed the highest activity in Tween 80 (1.090±0.033 U/ml).

Glycerol monolaurate antibacterial activity in broth and biofilm cultures.

BackgroundGlycerol monolaurate (GML) is an antimicrobial agent that has potent activity against gram-positive bacteria. This study examines GML antibacterial activity in comparison to lauric acid, in broth cultures compared to biofilm cultures, and against a wide range of gram-positive, gram-negative, and non-gram staining bacteria.Methodology/Principal FindingsGML is ≥200 times more effective than lauric acid in bactericidal activity, defined as a ≥3 log reduction in colony-forming units (CFU)/ml, against Staphylococcus aureus and Streptococcus pyogenes in broth cultures. Both molecules inhibit superantigen production by these organisms at concentrations that are not bactericidal. GML prevents biofilm formation by Staphylococcus aureus and Haemophilus influenzae, as representative gram-positive and gram-negative organisms, tested in 96 well microtiter plates, and simultaneously is bactericidal for both organisms in mature biofilms. GML is bactericidal for a wide range of potential bacterial pathogens, except for Pseudomonas aeruginosa and Enterobacteriaceae. In the presence of acidic pH and the cation chelator ethylene diamine tetraacetic acid, GML has greatly enhanced bactericidal activity for Pseudomonas aeruginosa and Enterobacteriaceae. Solubilization of GML in a nonaqueous delivery vehicle (related to K-Y Warming®) enhances its bactericidal activity against S. aureus. Both R and S, and 1 and 2 position lauric acid derivatives of GML exhibit bactericidal activity. Despite year-long passage of Staphylococcus aureus on sub-growth inhibitory concentrations of GML (0.5 x minimum bactericidal concentration), resistance to GML did not develop.Conclusions/SignificanceGML may be useful as a broad-spectrum human or animal topical microbicide and may be useful as an environmental surface microbicide for management of bacterial infections and contamination.

Site-directed mutagenesis of an Aspergillus niger xylanase B and its expression, purification and enzymatic characterization in Pichia pastoris

Xylanase is an important industrial enzyme. In this research, to improve the thermostability and biochemical properties of a xylanase from Aspergillus niger F19, five arginine substitutions and a disulfide bond were introduced by site-directed mutagenesis. The wild-type gene xylB and the mutant gene xylCX8 were expressed in Pichia pastoris. Compare to those of the wild-type enzyme, the optimal reaction temperature for the mutant enzyme increased from 45 °C to 50 °C, the half-life of the mutant enzyme extended from 10 min to 180 min, and the specific activity increased from 2127 U/mg to 3330 U/mg. However, the V max and K m of the mutant xylanase decreased. The enzyme activity in broth obtained from shake flask cultures could be induced to 1850 U/mL in 7 days, which is higher than results reported previously. Furthermore, the highest achievable enzyme activity was 7340 U/mL from 140 g/L of biomass in a 3 L fermentor used in our study.

Improved Luciferase Tagging System for Listeria monocytogenes Allows Real-Time Monitoring In Vivo and In Vitro

An improved system for luciferase tagging Listeria monocytogenes was developed by constructing a highly active, constitutive promoter. This construct gave 100-fold-higher activity in broth than any native promoter tested and allowed for imaging of lux-tagged L. monocytogenes in food products, during murine infections, and in tumor targeting studies.

The control of Listeria innocua and Lactobacillus sakei in broth and meat slurry with the bacteriocinogenic strain Lactobacillus casei CRL705

The effect of the bacteriocin-producing strain, Lactobacillus casei CRL705, in the control of Listeria innocua 7 and Lactobacillus sakei CRL1424 in MRS medium and meat slurry during the storage under vacuum at chill temperatures was evaluated. L. sakei CRL 1424 isolated from vacuum-packaged contaminated raw meat was identified as the predominant indigenous lactic acid bacterial flora. Co-inoculation of MRS broth at 8°C with L. casei CRL705 caused growth inhibition of L. sakei CRL1424 and L. innocua 7 after 10 and 4 days of storage, respectively. At 4°C the complete inhibition of both strains occurred within 14 and 8 days for L. sakei CRL1424 and L. innocua 7, respectively. Bacteriocin activity in broth was observed to be maximal at 8°C reaching 2130 AU ml −1 after10 days and 400 AU m1 −1 after 8 days of storage, while at 4°C maximal activities, 690 and 30 AU ml −1 were obtained at 14 and 10 days, respectively. Addition of bacteriocinogenic strain to the meat slurry did not allow the growth of L. sakei and L. innocua, showing a bacteriostatic effect during 21 days of storage at 4°C. In addition, L. casei CRL705, as a protective culture did not change significantly the pH of the meat slurry in the assayed storage conditions. The results presented here confirm that the bacteriocinogenic strain L. casei CRL705 can be used as a useful tool to improve the microbial stability and safety in the commercial meat preservation.

Potent Antifungal Activity of Garlic (Allium Sativum) Against Experimental Murine Dissemenated Cryptococcosis

The adequate management of cryptococcosis is difficult with the available armamentarium of synthetic antifungals. We made an effort to evaluate fresh extracts of garlic ( Allium sativum L.) to assess potential therapeutic use of this herb as an anticryptococcal agent. The in vitro antifungal activity in broth showed greatest activity in the aqueous extract (MIC 125–250 µg/ml; MFC 250 to = 500 µg/ml) compared to the alcoholic, crude or commercial extracts. Treatment of Balb/c mice with garlic extracts in mice (orally) showed that alcoholic extracts (5 mg/kg, q.i.d.) provided 100% protection of mice against disseminated cryptococcosis. A concomitant increase in MST (> 35 days) was observed, along with a significant reduction in cfu (log 10 ) burden of cryptococci in visceral (liver, spleen, kidney, lung and heart) tissues, especially brain (> 4.5 log 10 ). The efficacy of extracts was also evident in the sera of mice. Thus, alcoholic extracts of fresh garlic elicit potential anticryptococcal activity against murine disseminated cryptococcosis.

Computer simulation of l-lactate batch fermentation applying the enzyme inactivation scheme

In our previous work, a batch culture growth model for Lactococcus lactis IO-1 was introduced, but this model gave a deviation from the observed results at the end phase of the fermentation. In this experiment, we found that LDH activity in broth, the key enzyme of this fermentation, decreased with a constant decreasing rate smaller than α, the rate of sterile cell formation. Simulation program using this decreasing rate gave perfect results that agreed with the observed results for the whole phase of the fermentation.

Cefazaflur, a New Parenteral Cephalosporin: In Vitro Studies

Cefazaflur was tested in vitro against 262 strains of bacteria. Inhibitory and bactericidal concentrations were determined with two inoculum sizes of bacterial cells in Mueller-Hinton broth and nutrient broth. Agar dilution studies also were performed. When tested in agar, 5.0 mug or less of cefazaflur per ml inhibited almost all strains of Staphylococcus aureus, Escherichia coli, Klebsiella, and Proteus mirabilis. The drug was less active against Enterobacter and indole-positive Proteus, and 7.5 mug of antibiotic per ml inhibited approximately two-thirds to one-fourth of the strains. A concentration of 50 mug of cefazaflur per ml was required for inhibition of the enterococci. There was negligible activity against Pseudomonas. The drug demonstrated less activity in broth than in agar, and a major inoculum effect was seen with some strains. For example, with a lower inoculum, 2.5 mug of cefazaflur per ml killed all strains of E. coli, whereas with the higher inoculum, 7.5 mug of cefazaflur per ml, inhibited 64% and killed only 8% of strains. The activity of the drug for some strains was greater in Mueller-Hinton broth; for others, it was greater in nutrient broth. There were considerable differences in the results of the broth and agar studies for some species when the same medium was employed. Because of differences in activity found with different media, inocula, and method of testing, an evaluation of the eventual usefulness of cefazaflur must await the results of in vivo studies.

Antibacterial activity of antibiotics in human bile (author's transl)

Marked differences exist between antibacterial activity in human bile and broth. Whereas penicillins and cephalosporines exhibit virtually the same activity in broth and bile, tetracycline shows a great lose, and the activity of the aminoglykosides is increased in bile. Minimal inhibitory concentrations of chlorampenicol and co-trimoxazole are also different in broth and human bile. The differences in activity are related to the pH although not all deviations can be attributed to the effect of the pH. These results should be considered when treating gallbladder infections.

Lysine Decarboxylase Activity in Broth and Agar Media

Four lysine decarboxylase media were studied by testing them with 305 Enterobacteriaceae and 42 nonfermenting bacilli. A comparison was made between lysine decarboxylase broth medium (Moeller base) and Johnson's semisolid agar without lactose and Bachrach's broth medium and lysine-agar slants which contain lactose. The nonlactose media, lysine decarboxylase broth and the semisolid medium of Johnson, were the best media for use with all of the bacteria studied. The exclusion of lactose from lysine decarboxylase medium seems desirable to extend the usefulness of this medium among members of the Enterobacteriaceae. When the results with lysine decarboxylase broth and Johnson's semisolid medium without lactose were compared, a 6% difference existed between the results obtained with lysine decarboxylase broth and Johnson's semisolid agar. When the results with Bachrach's broth and lysine-agar slants with lactose were compared, a 1% difference existed between Bachrach's broth and the agar slant method. At times, reading and interpretation were difficult because of intermediate degrees of color change. The inability of Pseudomonas aeruginosa or Herellea to utilize glucose under the anaerobic condition of the medium makes the lysine decarboxylase test an undesirable procedure for these organisms. Of the four test media used, the lysine-lactose-agar slants seemed to be the least desirable because of the more frequent occurrence of indistinct color reactions and shifts in color.