Antibacterial Properties of Melanoidins Produced from Various Combinations of Maillard Reaction against Pathogenic Bacteria.

Novel melanoidins are produced by the Maillard reaction. Here, melanoidins with high antibacterial activity were tested by examining various combinations of reducing sugars and amino acids as reaction substrates. Twenty-two types of melanoidins were examined by combining two reducing sugars (glucose and xylose) and eleven l-isomers of amino acids (alanine, arginine, glutamine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, and valine) to confirm the effects of these melanoidins on the growth of Listeria monocytogenes at 25°C. The melanoidins produced from the combination of d-xylose with either l-phenylalanine (Xyl-Phe) or l-proline (Xyl-Pro), for which absorbance at 420 nm was 3.5 ± 0.2, completely inhibited the growth of L. monocytogenes at 25°C for 48 h. Both of the melanoidins exhibited growth inhibition of L. monocytogenes which was equivalent to the effect of nisin (350 IU/mL). The antimicrobial spectrum of both melanoidins was also investigated for 10 different species of bacteria, including both Gram-positive and Gram-negative species. While Xyl-Phe-based melanoidin successfully inhibited the growth of Bacillus cereus and Brevibacillus brevis, Xyl-Pro-based melanoidin inhibited the growth of Salmonella enterica Typhimurium. However, no clear trend in the antimicrobial spectrum of the melanoidins against different bacterial species was observed. The findings in the present study suggest that melanoidins generated from xylose with phenylalanine and/or proline could be used as potential novel alternative food preservatives derived from food ingredients to control pathogenic bacteria. IMPORTANCE Although the antimicrobial effect of melanoidins has been reported in some foods, there have been few comprehensive investigations on the antimicrobial activity of combinations of reaction substrates of the Maillard reaction. The present study comprehensively investigated the potential of various combinations of reducing sugars and amino acids. Because the melanoidins examined in this study were produced simply by heating in an autoclave at 121°C for 60 min, the targeted melanoidins can be easily produced. The melanoidins produced from combinations of xylose with either phenylalanine or proline exhibited a wide spectrum of antibiotic effects against various pathogens, including Listeria monocytogenes, Bacillus cereus, and Salmonella enterica Typhimurium. Since the antibacterial effect of the melanoidins on L. monocytogenes was equivalent to that of a nisin solution (350 IU/mL), we might expect a practical application of melanoidins as novel food preservatives.

Optimization of staining with SYTO 9/propidium iodide: interplay, kinetics and impact on Brevibacillus brevis.

Fluorophores SYTO 9 and propidium iodide (PI) are extensively applied in medicine, food industry and environmental monitoring to assess the viability of bacteria. However, the actual performance of these dyes remains largely unknown. In addition, their effects on the physiology of cells have not been elucidated. Here we characterized the effects of these two dyes on Brevibacillus brevis under optimized staining. We found that SYTO 9 entered cells continuously while PI tended to adhere to the cell wall before entering the cell. In addition, results showed that a high amount of the dyes altered the physicochemical properties of membranes, improving their breakthrough. These results provide new perspectives and ideas for improving the characterization of bacterial viability using flow cytometry.

Marine bacteria associated with shallow hydrothermal systems in the Gulf of California with the capacity to produce biofilm inhibiting compounds.

Shallow hydrothermal systems are extreme environments. The sediments and fluids emitted from the vents present unusual physical and chemical conditions compared to other marine areas, which promotes unique biodiversity that has been of great interest for biotechnology for some years. In this work, a bioprospective study was carried out to evaluate the capacity of bacteria associated with shallow hydrothermal vents to produce biofilm-inhibiting compounds. Degradation assays of N-acyl homoserine lactone (AHL) autoinducers (C6HSL) involved in the quorum sensing process were carried out on 161 strains of bacteria isolated from three shallow hydrothermal systems located in Baja California Sur (BCS), Mexico. The biosensor Chromobacterium violaceum CV026 was used. Twenty-three strains showed activity, and organic extracts were obtained with ethyl acetate. The potential of the extracts to inhibit the formation of biofilms was tested against two human pathogenic strains (Pseudomonas aeruginosa PAO1 and Aeromonas caviae ScH3), a shrimp pathogen (Vibrio parahaemolyticus M8), and two marine strains identified as producing biofilms on submerged surfaces (Virgibacillus sp C29 and Vibrio alginolyticus C96). The results showed that Vibrio alginolyticus and Brevibacillus thermoruber, as well as some thermotolerant strains (mostly Bacillus), produce compounds that inhibit bacterial biofilms (B. licheniformis, B. paralicheniformis, B. firmus, B. oceanizedimenis, B. aerius and B. sonorensis).

Toluene degradation via a unique metabolic route in indigenous bacterial species.

Tanneries are the primary source of toluene pollution in the environment and toluene due to its hazardous effects has been categorized as persistent organic pollutant. Present study was initiated to trace out metabolic fingerprints of three toluene-degrading bacteria isolated from tannery effluents of Southern Punjab. Using selective enrichment and serial dilution methods followed by biochemical, molecular and antibiotic resistance analysis, isolated bacteria were subjected to metabolomics analysis. GC-MS/LC-MS analysis of bacterial metabolites helped to identify toluene transformation products and underlying pathways. Three toluene-metabolizing bacteria identified as Bacillus paralicheniformis strain KJ-16 (IUBT4 and IUBT24) and Brevibacillus agri strain NBRC 15538 (IUBT19) were found tolerant to toluene and capable of degrading toluene. Toluene-degrading potential of these isolates was detected to be IUBT4 (10.35 ± 0.084 mg/h), IUBT19 (14.07 ± 3.14 mg/h) and IUBT24 (11.1 ± 0.282 mg/h). Results of GC-MS analysis revealed that biotransformation of toluene is accomplished not only through known metabolic routes such as toluene 3-monooxygenase (T3MO), toluene 2-monooxygenase (T2MO), toluene 4-monooxygenase (T4MO), toluene methyl monooxygenase (TOL), toluene dioxygenase (Tod), meta- and ortho-ring fission pathways. But additionally, confirmed existence of a unique metabolic pathway that involved conversion of toluene into intermediates such as cyclohexene, cyclohexane, cyclohexanone and cyclohexanol. LC-MS analysis indicated the presence of fatty acid amides, stigmine, emmotin A and 2, 2-dinitropropanol in supernatants of bacterial cultures. As the isolated bacteria transformed toluene into relatively less toxic molecules and thus can be preferably exploited for the eco-friendly remediation of toluene.

Brevibacillus laterosporus strains BGSP7, BGSP9 and BGSP11 isolated from silage produce broad spectrum multi-antimicrobials.

Bacteria active against multi-drug resistant pathogens, isolated by direct selection of colonies from clover silage samples, produce zones of inhibition against two Gram-negative (Klebsiella pneumoniae Ni9 and Pseudomonas aeruginosa MMA83) and two Gram-positive (Staphylococcus aureus ATCC25923 and Listeria monocytogenes ATCC19111) pathogens. Isolates BGSP7, BGSP9, BGSP11 and BGSP12 produced the largest zones of inhibition against all four pathogens when grown in LB broth with aeration at 37°C. Isolates BGSP7, BGSP9, BGSP11 and BGSP12 were identified as Brevibacillus laterosporus and pulsed field gel electrophoresis and extracellular protein profiles showed that three different strains (BGSP7, BGSP9 and BGSP11) were isolated. A semi-native SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) gel overlay assay showed that BGSP7 and BGSP9 produce small antimicrobial molecules of about 1.5 kDa, while BGSP11 produces antimicrobial molecules of 1.5 and 6 kDa active against S. aureus ATCC25923. Amino acid analysis of two antimicrobial molecules (1583.73 Da; from BGSP7 and 1556.31 Da; from BGSP11) revealed that they have a similar composition and differ only by virtue of the presence of a methionine which is present only in BGSP11 molecule. Genome sequencing of the three isolates revealed the presence of gene clusters associated with the production of non-ribosomally synthesized peptides (brevibacillin, bogorol, gramicidin S, plipastatin and tyrocin) and bacteriocins (laterosporulin, a lactococcin 972-like bacteriocin, as well as putative linocin M18, sactipeptide, UviB and lantipeptide-like molecules). Ultimately, the purification of a number of antimicrobial molecules from each isolate suggests that they can be considered as potent biocontrol strains that produce an arsenal of antimicrobial molecules active against Gram-positive and Gram-negative multi-resistant pathogens, fungi and insects.

Bioremediation of triphenyl phosphate in river water microcosms: Proteome alteration of Brevibacillus brevis and cytotoxicity assessments.

Triphenyl phosphate (TPHP), an organophosphate flame retardant, was detected in river water samples collected from an electronic waste recycling area in Guiyu, Southern China. The concentrations of TPHP ranged from not detected to 347.2 ng/L, with an average of 138.8 ng/L. The bioaugmentation potential of Brevibacillus brevis on TPHP biodegradation by aerobic microcosms contained in river water from Guiyu was assessed. The results showed that TPHP degradation efficiency was significantly improved to 97.9% by bioaugmentation with B. brevis after 96 h incubation. A total of 182 significantly changed proteins in B. brevis were identified and quantified by isobaric tags for relative and absolute quantification (iTRAQ) in response to TPHP stress. The differentially expressed proteins were mainly associated with energy metabolism, lipid metabolism, cell wall biosynthesis, amino acid transport, and metabolism. The identification that proteins of B. brevis respond to TPHP existence provides novel insights into biodegradation mechanisms of bacteria under environmental stress. Additionally, cytotoxicity assays indicated that the degrading intermediates of TPHP, namely diphenyl phosphate and phenyl phosphate, were less cytotoxic to human HepG2 cells compared with TPHP. Collectively, these findings suggest that aerobic bioaugmentation with degrading microorganisms is a potential strategy for in situ treatment of TPHP-contaminated sites.

Tolerance of triazole-based fungicides by biocontrol agents used to control Fusarium head blight in wheat in Argentina.

Fusarium head blight (FHB) caused by Fusarium graminearum species complex is a devastating disease that causes extensive yield and quality losses to wheat around the world. Fungicide application and breeding for resistance are among the most important tools to counteract FHB. Biological control is an additional tool that can be used as part of an integrated management of FHB. Bacillus velezensisRC 218, Brevibacillus sp. RC 263 and Streptomyces sp. RC 87B were selected by their potential to control FHB and deoxynivalenol production. The aim of this work was to test the tolerance of these biocontrol agents to triazole-based fungicides such as prothioconazole, tebuconazole and metconazole. Bacterial growth was evaluated in Petri dishes using the spread plating technique containing the different fungicides. Bacillus velezensisRC 218 and Streptomyces sp. RC 87B showed better tolerance to fungicides than Brevibacillus sp. RC 263. Complete growth inhibition was observed at concentrations of 20 µg ml-1 for metconazole, 40 µg ml-1 for tebuconazole and 80 µg ml-1 for prothioconazole. The results obtained indicate the possibility of using these biocontrol agents in combination with fungicides as part of an integrated management to control FHB of wheat. SIGNIFICANCE AND IMPACT OF THE STUDY: This study evaluates the possibility to use biocontrol agents (Bacillus velezensisRC 218, Brevibacillus sp. RC 263 and Streptomyces sp. RC 87B) in combination with triazole-based fungicides to control Fusarium head blight in wheat. The evaluation of biocontrol agents' growth under in vitro conditions was carried out in Petri dishes containing either prothioconazole, tebuconazole or metconazole. Viability studies demonstrated that B. velezensisRC 218 and Streptomyces sp. RC 87B were more tolerant to the fungicides evaluated. Results obtained reflect the possibility to use fungicides at low doses combined with biocontrol agents.

The complex resistomes of Paenibacillaceae reflect diverse antibiotic chemical ecologies.

The ecology of antibiotic resistance involves the interplay of a long natural history of antibiotic production in the environment, and the modern selection of resistance in pathogens through human use of these drugs. Important components of the resistome are intrinsic resistance genes of environmental bacteria, evolved and acquired over millennia, and their mobilization, which drives dissemination in pathogens. Understanding the dynamics and evolution of resistance across bacterial taxa is essential to address the current crisis in drug-resistant infections. Here we report the exploration of antibiotic resistance in the Paenibacillaceae prompted by our discovery of an ancient intrinsic resistome in Paenibacillus sp. LC231, recovered from the isolated Lechuguilla cave environment. Using biochemical and gene expression analysis, we have mined the resistome of the second member of the Paenibacillaceae family, Brevibacillus brevis VM4, which produces several antimicrobial secondary metabolites. Using phylogenomics, we show that Paenibacillaceae resistomes are in flux, evolve mostly independent of secondary metabolite biosynthetic diversity, and are characterized by cryptic, redundant, pseudoparalogous, and orthologous genes. We find that in contrast to pathogens, mobile genetic elements are not significantly responsible for resistome remodeling. This offers divergent modes of resistome development in pathogens and environmental bacteria.

The ability of consortium wastewater protozoan and bacterial species to remove COD in the presence of nanomaterials under varying pH conditions.

The aim of this study was to ascertain the survival limit and capability of commonly found wastewater protozoan (Aspidisca sp, Trachelophyllum sp and Peranema sp) and bacterial (Bacillus licheniformis, Brevibacillus laterosporus and Pseudomonas putida) species to remove COD while exposed to commercial nanomaterials under varying pH conditions. The experimental study was carried out in modified mixed liquor media adjusted to various pH levels (pH 2, 7 and 10) and a comparative study was performed to determine the difference between the cytotoxicity effects of commercial zinc oxide (nZnO) and silver (nAg) nanomaterials (NMs) on the target wastewater microbial communities using standard methods. The selected microbial communities were exposed to lethal concentrations ranging from 0.015 g/L to 40 g/L for nZnO and from 0.015 g/L to 2 g/L for nAg for a period of 5 days of incubation at 30°C (100 r/min). Compared with the absence of NMs in wastewater mixed liquor, the relevant environmental concentration ranging between 10 µg/L and 100 µg/L, for both nZnO and nAg caused no adverse effects, but the presence of 20 g of nZnO/L and 0.65 g of nAg/L significantly inhibited microbial growth. Statistical evidence showed that nAg was significantly more toxic compared to nZnO, but there was an insignificant difference in toxicity between microbial communities and pH variations. A significant decrease in the removal of COD by microbial populations was observed in the presence of NMs with a moderate correlation of r = 0.3 to r = 0.7 at all pH levels. It was evident that there was a physical interaction between commercial NMs and target wastewater microbial communities; although not quantitatively assessed, cell morphology and cell death were observed. Such phenomena suggest the high resilience of the microbial community, but it is the accumulation of NMs that will have adverse effects on the performance in terms of COD removal.

Characterization of a new oxygen-insensitive azoreductase from Brevibacillus laterosporus TISTR1911: toward dye decolorization using a packed-bed metal affinity reactor.

This study reports the identification of a new bacterial azoreductase from Brevibacillus laterosporus TISTR1911, its heterologous production in Escherichia coli, the biochemical characterization and immobilization for use in dye biodegradation processes. The recombinant azoreductase (BrAzo) is a monomeric FMN oxygen-insensitive enzyme with a molecular mass of 23 kDa showing a broad specificity for the reduction of synthetic azo dyes. Double hexahistidine-tagged BrAzo was immobilized onto a nickel chelating column and methyl orange was used to assess its degradation potential using a packed-bed reactor. The dye degradation is described by an exponential model in a downstream batchwise continuous flow mode operated with recycling. The complete degradation of methyl orange (170 µM at 600 mL/h) was achieved in 3 h and continued over 9 cycles. Coupling the immobilized BrAzo with glucose dehydrogenase for NADH regeneration yielded a shorter 1.5 h-degradation period that was maintained throughout 16 cycles.