Antibacterial Properties of Bacteriocin Purified from Serratia marcescens and Computerized Assessment of its Interaction with Antigen 43 in Escherichia coli.

Bacteriocins are a kind of antimicrobial peptides that kill or inhibit the growth of bacterial strains. The purpose of this study was to investigate the antibacterial effect of Serratia marcescens on several pathogenic bacterial strains. Bacteriocin produced by S. marcescens was purified by chromatography with Sephadex G-75 column, and its antibacterial effect on gram-negative bacteria, including Escherichia coli ATCC 700928, Pseudomonas aeruginosa PTCC 1707, S. marcescens PTCC 1621, Vibrio fischeri PTCC 1693, and Vibrio harveyi PTCC 1755, were evaluated by the disk diffusion method. The structure of bacteriocin was determined by nuclear magnetic resonance spectroscopy. The interaction of bacteriocin with the antigen 43 (Ag43) of E. coli was evaluated by the molecular docking method. Bacteriocin extracted from bacterial isolates had antibacterial activity on E. coli strains but not on other studied strains. Bioinformatics analysis also showed bacteriocin docking with Ag43 with an energy of -159.968 kJ/mol. Natural compounds, such as bacteriocin, can be an alternative to common chemical compounds and antibiotics. To reach a definite conclusion in this regard, there is a need for further research and understanding of their mechanism of action.

Mutagenicity and genotoxicity assessment of a new biopreservative product rich in Enterocin AS-48.

A biopreservative derived from the fermentation of a dairy byproduct by Enterococcus faecalis UGRA10 strains being developed. This product possesses a strong and wide antibacterial spectrum mainly due to the presence of Enterocin AS-48 in its composition. To assess its potential as food additive, the mutagenicicity and genotoxicity has been assayed by means of the bacterial reverse-mutation assay in Salmonella typhimurium TA97A, TA98, TA100, TA102, TA1535 strains (Ames test, OECD 471, 2020) and the micronucleus test (MN) (OECD 487, 2016) in L5178Y/Tk ± cells. The results in the Ames test after exposure to the byproduct (6.75-100 µg/plate) with absence and presence of the metabolic activation system from rat liver (S9 fraction), revealed not mutagenicity at the conditions tested. For the MN test, the exposition to five enterocin AS-48 concentrations (0.2-1 µg/µl) was tested in the absence and presence of S9 fraction, with no evidence of genotoxicity. Negative results in the mutagenicity and genotoxicity assays point out the good safety profile of the byproduct and support its use as additive. Further toxicological studies are required before its approval and commercial application.

Assessment of Chemotherapy-Induced Organ Damage with Ga-68 Labeled Duramycin.

PURPOSE: Evaluation of [68Ga]NODAGA-duramycin as a positron emission tomography (PET) tracer of cell death for whole-body detection of chemotherapy-induced organ toxicity. PROCEDURES: Tracer specificity of Ga-68 labeled NODAGA-duramycin was determined in vitro using competitive binding experiments. Organ uptake was analyzed in untreated and doxorubicin, busulfan, and cisplatin-treated mice 2 h after intravenous injection of [68Ga]NODAGA-duramycin. In vivo data were validated by immunohistology and blood parameters. RESULTS: In vitro experiments confirmed specific binding of [68Ga]NODAGA-duramycin. Organ toxicities were detected successfully using [68Ga]NODAGA-duramycin PET/X-ray computed tomography (CT) and confirmed by immunohistochemistry and blood parameter analysis. Organ toxicities in livers and kidneys showed similar trends in PET/CT and immunohistology. Busulfan and cisplatin-related organ toxicities in heart, liver, and lungs were detected earlier by PET/CT than by blood parameters and immunohistology. CONCLUSION: [68Ga]NODAGA-duramycin PET/CT was successfully applied to non-invasively detect chemotherapy-induced organ toxicity with high sensitivity in mice. It, therefore, represents a promising alternative to standard toxicological analyses with a high translational potential.

Antimicrobial peptide arrays for wide spectrum sensing of pathogenic bacteria.

Fast detection of bacteria in samples presumed to be un-contaminated, such as blood, is of great importance. Indeed, rapid diagnosis allows the set-up of appropriate antibiotic treatment. Besides clinical issues, there are many other domains, such as food processing or drug manufacturing, where the strict absence of any bacteria has to be assessed. Because the bacterial load found in most contaminated samples is often below the limit of detection for currently validated assays, a preliminary enrichment step is required to allow bacterial multiplication before proceeding to the analysis step, whatever it might be - cultural, immunological or molecular methods. In this study, we describe the use of a biosensor for single-step bacteria detection. The whole analysis is performed in less than 20 h, during the growth phase of the micro-organisms, using an array of antimicrobial peptides (AMPs) coupled with a surface plasmon resonance imager (SPRI). A wide range of bacterial strains are assayed, showing differentiated affinity patterns with the immobilized peptides, which are confirmed by multivariate analysis. This work establishes the evidence that antimicrobial peptides, mostly used so far in the antibiotic drug industry, are suited for the wide-spectrum detection of unknown bacteria in samples, even at very low initial loads. Moreover, the small set of AMPs that were assayed provided a specific affinity profile for each pathogen, as confirmed by multivariate analyses. Furthermore, this work opens up the possibility of applying this method in more complex and relevant samples such as foodstuff, urine or blood.

Pharmacological, Toxicological, and Dose Range Assessment of OG716, a Novel Lantibiotic for the Treatment of Clostridium difficile-Associated Infection.

Lantibiotics present an attractive scaffold for the development of novel antibiotics. We report here a novel lantibiotic for the treatment of Clostridium difficile infection. The lead compounds were selected from a library of over 700 single- and multiple-substitution variants of the lantibiotic mutacin 1140 (MU1140). The best performers in vitro and in vivo were further used to challenge Golden Syrian hamsters orally in a Golden Syrian hamster model of Clostridium difficile-associated disease (CDAD) in a dose-response format, resulting in the selection of OG716 as the lead compound. This lantibiotic was characterized by a 50% effective dose of 23.85 mg/kg of body weight/day (10.97 µmol/kg/day) in this model. Upon oral administration of the maximum feasible dose (≥1,918 mg/kg/day), no observable toxicities or side effects were noted, and no effect on intestinal motility was observed. Compartmentalization to the gastrointestinal tract was confirmed. MU1140-derived variants offer a large pipeline for the development of novel antibiotics for the treatment of several indications and are particularly attractive considering their novel mechanism of action. Based on the currently available data, OG716 has an acceptable profile for further development for the treatment of CDAD.

Assessment of hemolytic activity, enzyme production and bacteriocin characterization of Bacillus subtilis LR1 isolated from the gastrointestinal tract of fish.

In the present investigation, probiotic potential (antagonistic activity, enzyme production, hemolytic activity, biosafety, antibiotic sensitivity and bile tolerance level) of Bacillus subtilis LR1 was evaluated. Bacteriocin produced by the bacterial strain B. subtilis LR1 isolated from the gastrointestinal tract of Labeo rohita was purified and characterized. The molecular weight of the purified bacteriocin was ~50 kDa in 12 % Native PAGE and showed inhibitory activity against four fish pathogens such as Bacillus mycoides, Aeromonas salmonicida, Pseudomonas fluorescens and Aeromonas hydrophila. The purified bacteriocin was maximally active at temperature 40 °C and pH 7.0, while none of the tested surfactants affect the bacteriocin activity. Extracellular enzyme activity of the selected bacterial strain was also evaluated. Amylase activity was estimated to be highest (38.23 ± 1.15 µg of maltose liberated mg-1 protein ml-1 of culture filtrate) followed by cellulase and protease activity. The selected bacterium was sensitive to most of the antibiotics used in this experiment, can tolerate 0.25 % bile salt and non-hemolytic in nature. Finally, the efficiency of the proposed probiotic candidate was evaluated in in vivo condition. It was detected that the bacterial strain can effectively reduce bacterial pathogenicity in Indian major carps.

Sakacin-A antimicrobial packaging for decreasing Listeria contamination in thin-cut meat: preliminary assessment.

BACKGROUND: Minimally processed ready-to-eat products are considered a high-risk food because of the possibility of contamination with pathogenic bacteria, including Listeria monocytogenes from the animal reservoir, and the minimal processing they undergo. In this study, a sakacin-A anti-Listeria active package was developed and tested on thin-cut veal meat slices (carpaccio). RESULTS: Enriched food-grade sakacin-A was obtained from a cell-free supernatant of a Lactobacillus sakei culture and applied (0.63 mg cm-2 ) onto the surface of polyethylene-coated paper sheets to obtain an active antimicrobial package. The coating retained antimicrobial features, indicating that the process did not affect sakacin-A functionality, as evidenced in tests carried out in vitro. Thin-cut veal meat slices inoculated with Listeria innocua (a surrogate of pathogenic L. monocytogenes) were laid on active paper sheets. After 48 h incubation at 4 °C, the Listeria population was found to be 1.5 log units lower with respect to controls (3.05 vs 4.46 log colony-forming units (CFU) g-1 ). CONCLUSION: This study demonstrates the possibility of using an antimicrobial coating containing sakacin-A to inhibit or decrease the Listeria population in ready-to-eat products, thus lowering the risk of food-related diseases. © 2016 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

A prevalent peptide-binding domain guides ribosomal natural product biosynthesis.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a rapidly growing class of natural products. RiPP precursor peptides can undergo extensive enzymatic tailoring to yield structurally and functionally diverse products, and their biosynthetic logic makes them attractive bioengineering targets. Recent work suggests that unrelated RiPP-modifying enzymes contain structurally similar precursor peptide-binding domains. Using profile hidden Markov model comparisons, we discovered related and previously unrecognized peptide-binding domains in proteins spanning the majority of known prokaryotic RiPP classes, and we named this conserved domain the RiPP precursor peptide recognition element (RRE). Through binding studies we verified RRE's roles for three distinct RiPP classes: linear azole-containing peptides, thiopeptides and lasso peptides. Because numerous RiPP biosynthetic enzymes act on peptide substrates, our findings have powerful predictive value as to which protein(s) drive substrate binding, thereby laying a foundation for further characterization of RiPP biosynthetic pathways and the rational engineering of new peptide-binding activities.

Characterization of [(99m)Tc]Duramycin as a SPECT Imaging Agent for Early Assessment of Tumor Apoptosis.

PURPOSE: We investigated the usefulness of [(99m)Tc]duramycin for monitoring early response to cancer therapy in mice, with an eye towards clinical translation. PROCEDURES: [(99m)Tc]Duramycin was injected in healthy CD1-/- mice to estimate human [(99m)Tc]duramycin radiation dose. [(99m)Tc]Duramycin single-photon emission computed tomography (SPECT) imaging of apoptosis was evaluated in a mouse model of colorectal cancer treated with irinotecan and validated ex vivo using autoradiography, cleaved caspase-3, and TdT-mediated dUTP nick-end labeling (TUNEL) histology of the tumors. RESULTS: The mean effective dose was estimated to be 3.74 × 10(-3) ± 3.43 × 10(-4) mSv/MBq for non-purified and 3.19 × 10(-3) ± 2.16 × 10(-4) mSv/MBq for purified [(99m)Tc]duramycin. [(99m)Tc]Duramycin uptake in vivo following therapy increased significantly in apoptotic irinotecan-treated tumors (p = 0.008). Radioactivity in the tumors positively correlated with cleaved caspase-3 (r = 0.85, p < 0.001) and TUNEL (r = 0.92, p < 0.001) staining. CONCLUSION: [(99m)Tc]Duramycin can be used to detect early chemotherapy-induced tumor cell death, and thus, may be a prospective candidate for clinical SPECT imaging of tumor response to therapy.

Assessment of the bacteriocinogenic potential of marine bacteria reveals lichenicidin production by seaweed-derived Bacillus spp.

The objectives of this study were (1) to assess the bacteriocinogenic potential of bacteria derived mainly from seaweed, but also sand and seawater, (2) to identify at least some of the bacteriocins produced, if any and (3) to determine if they are unique to the marine environment and/or novel. Fifteen Bacillus licheniformis or pumilus isolates with antimicrobial activity against at least one of the indicator bacteria used were recovered. Some, at least, of the antimicrobials produced were bacteriocins, as they were proteinaceous and the producers displayed immunity. Screening with PCR primers for known Bacillus bacteriocins revealed that three seaweed-derived Bacillus licheniformis harbored the bli04127 gene which encodes one of the peptides of the two-peptide lantibiotic lichenicidin. Production of both lichenicidin peptides was then confirmed by mass spectrometry. This is the first definitive proof of bacteriocin production by seaweed-derived bacteria. The authors acknowledge that the bacteriocin produced has previously been discovered and is not unique to the marine environment. However, the other marine isolates likely produce novel bacteriocins, as none harboured genes for known Bacillus bacteriocins.

Evolution of lanthipeptide synthetases.

Lanthionine-containing peptides (lanthipeptides) are a family of ribosomally synthesized and posttranslationally modified peptides containing (methyl)lanthionine residues. Here we present a phylogenomic study of the four currently known classes of lanthipeptide synthetases (LanB and LanC for class I, LanM for class II, LanKC for class III, and LanL for class IV). Although they possess very similar cyclase domains, class II-IV synthetases have evolved independently, and LanB and LanC enzymes appear to not always have coevolved. LanM enzymes from various phyla that have three cysteines ligated to a zinc ion (as opposed to the more common Cys-Cys-His ligand set) cluster together. Most importantly, the phylogenomic data suggest that for some scaffolds, the ring topology of the final lanthipeptides may be determined in part by the sequence of the precursor peptides and not just by the biosynthetic enzymes. This notion was supported by studies with two chimeric peptides, suggesting that the nisin and prochlorosin biosynthetic enzymes can produce the correct ring topologies of epilancin 15X and lacticin 481, respectively. These results highlight the potential of lanthipeptide synthetases for bioengineering and combinatorial biosynthesis. Our study also demonstrates unexplored areas of sequence space that may be fruitful for genome mining.

New developments and applications of bacteriocins and peptides in foods.

There is an increased desire for sophisticated foods, whereby consumers harbor higher expectations of health-promoting benefits above basic nutrition. Moreover, there is a move from the adulteration of foods with chemical preservatives toward biopreservation. Such expectations have led scientists to identify novel approaches to satisfy both demands, which utilize bacteriocin and peptide-based solutions. The best known examples of biopreservation involve bacteriocins. However, with the exception of nisin, bacteriocins have received limited use in the food industry. Peptides can be added to foods to improve consumer health. Some of the best known examples are angiotensin I-converting enzyme (ACE)-inhibitory peptides, which inhibit ACE, a key enzyme involved in blood pressure (BP) regulation. To be effective, these peptides must be bioavailable, but by their nature, peptides are degraded by digestion with proteolytic enzymes. This review critically discusses the use and potential of peptides and bacteriocins in food systems in terms of safety, quality, and improvement of human health.

A new structure-based classification of gram-positive bacteriocins.

Bacteriocins are ribosomally-synthesized peptides or proteins produced by a wide range of bacteria. The antimicrobial activity of this group of natural substances against foodborne pathogenic and spoilage bacteria has raised considerable interest for their application in food preservation. Classifying these bacteriocins in well defined classes according to their biochemical properties is a major step towards characterizing these anti-infective peptides and understanding their mode of action. Actually, the chosen criteria for bacteriocins' classification lack consistency and coherence. So, various classification schemes of bacteriocins resulted various levels of contradiction and sorting inefficiencies leading to bacteriocins belonging to more than one class at the same time and to a general lack of classification of many bacteriocins. Establishing a coherent and adequate classification scheme for these bacteriocins is sought after by several researchers in the field. It is not straightforward to formulate an efficient classification scheme that encompasses all of the existing bacteriocins. In the light of the structural data, here we revisit the previously proposed contradictory classification and we define new structure-based sequence fingerprints that support a subdivision of the bacteriocins into 12 groups. The paper lays down a resourceful and consistent classification approach that resulted in classifying more than 70% of bacteriocins known to date and with potential to identify distinct classes for the remaining unclassified bacteriocins. Identified groups are characterized by the presence of highly conserved short amino acid motifs. Furthermore, unclassified bacteriocins are expected to form an identified group when there will be sufficient sequences.

Rings, radicals, and regeneration: the early years of a bioorganic laboratory.

This Perspective provides an overview of the progress in two of the original programs in my research group focused on the biosynthesis of the antibiotics nisin, lacticin 481, fosfomycin, and bialaphos. The path from start-up funds to tenure and beyond offers insights into the opportunities realized and missed along the road.

Data mining and characterization of a novel pediocin-like bacteriocin system from the genome of Pediococcus pentosaceus ATCC 25745.

The genome of Pediococcus pentosaceus ATCC 25745 contains a gene cluster that resembles a regulated bacteriocin system. The gene cluster has an operon-like structure consisting of a putative pediocin-like bacteriocin gene (termed penA) and a potential immunity gene (termed peiA). Genetic determinants involved in bacteriocin transport and regulation are also found in proximity to penA and peiA but the so-called accessory gene involved in transport and the inducer gene involved in regulation are missing. Consequently, this bacterium is a poor bacteriocin producer. To analyse the potency of the putative bacteriocin operon, the two genes penA-peiA were heterologously expressed in a Lactobacillus sakei host that contains the complete apparatus for gene activation, maturation and externalization of bacteriocins. It was demonstrated that the heterologous host expressing penA and peiA produced a strong bacteriocin activity; in addition, the host became immune to its own bacteriocin, identifying the gene pair penA-peiA as a potent bacteriocin system. The novel pediocin-like bacteriocin, termed penocin A, has an isotopic mass [M+H]+ of 4684.6 Da as determined by mass spectrometry; this value corresponds well to the expected size of the mature 42 aa peptide containing a disulfide bridge. The bacteriocin is heat-stable but protease-sensitive and has a calculated pI of 9.45. Penocin A has a relatively broad inhibition spectrum, including pathogenic Listeria and Clostridium species. Immediately upstream of the regulatory genes reside some features that resemble remnants of a disrupted inducer gene. This degenerate gene was restored and shown to encode a double-glycine leader-containing peptide. Furthermore, expression of the restored gene triggered high bacteriocin production in P. pentosaceus ATCC 25745, thus confirming its role as an inducer in the pen regulon.

Are antimicrobial peptides an alternative for conventional antibiotics?

Antimicrobial peptides are widespread in living organisms and constitute an important component of innate immunity to microbial infections. By the early 1980s, more than 800 different antimicrobial peptides had been isolated from mammals, amphibians, fish, insects, plants and bacterial species. In humans, they are produced by granulocytes, macrophages and most epithelial and endothelial cells. Newly discovered antibiotics have antibacterial, antifungal, antiviral and even antiprotozoal activity. Occasionally, a single antibiotic may have a very wide spectrum of activity and may show activity towards various kinds of microorganisms. Although antimicrobial activity is the most typical function of peptides, they are also characterized by numerous other properties. They stimulate the immune system, have anti-neoplastic properties and participate in cell signalling and proliferation regulation. As antimicrobial peptides from higher eukaryotes differ structurally from conventional antibiotics produced by bacteria and fungi, they offer novel templates for pharmaceutical compounds, which could be used effectively against the increasing number of resistant microbes.

Computational analysis of the first biheterocyclization site of the antibiotic microcin B17.

Microcin B 17 (MccB17) undergoes an enzyme catalyzed posttranslational modification to form four oxazole and four thiazole rings. Four of these rings form 4,2 - connected biheterocyclic functionalities. In this study, the hexapeptide sequence surrounding the first biheterocyclization site of microcin B17 was examined using computational calculations and database analysis to see if it was preorganized for cyclization in a manner similar to that found in the autocatalytic posttranslational cyclization of Green Fluorescent Protein (GFP). Attention was focused on the intermolecular distances between the sulfur and oxygen atoms of the cysteine and serine residues and the carbonyl carbons which they attack in the ring formation. Conformational searches located some low energy conformations that contained relatively short oxygen to carbonyl carbon distances, which indicated that the oxazole forming fragment in microcin B17 is preorganized for cyclization. However, the lack of any clear patterns for the sulfur to carbon distances show that the side-chain of cysteine does not adopt any low energy conformations that are geometrically preorganized for cyclization. The MccB17 synthetase enzyme complex which catalyzes the cyclization process therefore has both steric and electronic functions. The data obtained in this investigation is in agreement with empirical data which shows that biheterocyclization will only occur if the thiazole forms before the oxazole.