Structural insights into CodY activation and DNA recognition.

Virulence factors enable pathogenic bacteria to infect host cells, establish infection, and contribute to disease progressions. In Gram-positive pathogens such as Staphylococcus aureus (Sa) and Enterococcus faecalis (Ef), the pleiotropic transcription factor CodY plays a key role in integrating metabolism and virulence factor expression. However, to date, the structural mechanisms of CodY activation and DNA recognition are not understood. Here, we report the crystal structures of CodY from Sa and Ef in their ligand-free form and their ligand-bound form complexed with DNA. Binding of the ligands-branched chain amino acids and GTP-induces conformational changes in the form of helical shifts that propagate to the homodimer interface and reorient the linker helices and DNA binding domains. DNA binding is mediated by a non-canonical recognition mechanism dictated by DNA shape readout. Furthermore, two CodY dimers bind to two overlapping binding sites in a highly cooperative manner facilitated by cross-dimer interactions and minor groove deformation. Our structural and biochemical data explain how CodY can bind a wide range of substrates, a hallmark of many pleiotropic transcription factors. These data contribute to a better understanding of the mechanisms underlying virulence activation in important human pathogens.

1H, 13C, and 15N resonance assignments of a conserved putative cell wall binding domain from Enterococcus faecalis.

Enterococcus faecalis is a major causative agent of hospital acquired infections. The ability of E. faecalis to evade the host immune system is essential during pathogenesis, which has been shown to be dependent on the complete separation of daughter cells by peptidoglycan hydrolases. AtlE is a peptidoglycan hydrolase which is predicted to bind to the cell wall of E. faecalis, via six C-terminal repeat sequences. Here, we report the near complete assignment of one of these six repeats, as well as the predicted backbone structure and dynamics. This data will provide a platform for future NMR studies to explore the ligand recognition motif of AtlE and help to uncover its potential role in E. faecalis virulence.

Isolation and Identification of Dominant Bacteria from Raw Donkey Milk Produced in a Region of Morocco by QIIME 2 and Evaluation of Their Antibacterial Activity.

Recently, the interest in donkey milk has increased considerably because it proved high nutritive and functional values of their ingredients. Its chemical composition is widely studied, but its microbiota, especially lactic acid bacteria, remains less studied. This study focuses on analyzing, isolating, and identifying lactic acid bacteria and evaluating their capacity to produce biomolecules with antibacterial activity. Among 44 strains identified, 43 are Gram-positive, and most are catalase-negative and cocci-shaped. Five strains were selected to evaluate their antibacterial activity against Listeria monocytogenes, Staphylococcus aureus, and Escherichia coli. Different induction methods allowed to amplify the antibacterial effects against these pathogenic strains.

Modeling microbial ethanol production by S. aureus, K. pneumoniae, and E. faecalis under aerobic/anaerobic conditions - applicability to laboratory cultures and real postmortem cases.

A quite intriguing subject being intensively researched in the forensic toxicology field is the source of postmortem determined blood ethanol concentration: antemortem ingestion or postmortem microbial production. Our previous research on microbial ethanol production has reported a quantitative relationship between the ethanol and the higher alcohols and 1-butanol produced by Escherichia coli, Clostridium perfrigens, and Clostridium sporogenes. In this contribution, we continue our research reporting on the following: (i) the patterns of ethanol, higher alcohols, and 1-butanol production by the microbes Klebsiella pneumoniae, Staphylococcus aureus, and Enterococcus faecalis (all being aerobic/facultative anaerobic species, common corpse's colonizers, and ethanol producers), under controlled laboratory conditions, (ii) the mathematical modeling, with simple mathematical equations, of the correlation between ethanol concentration and the other studied alcohols' concentrations, by performing multiple linear regression analysis of the results, and (iii) the applicability of the constructed models in microbial cultures developed under different temperature than that used to build the models, in denatured blood cultures and in real postmortem cases. The aforementioned alcohols were proved to be all indicators of ethanol production, both in qualitative and quantitative terms. 1-Propanol was the most significant alcohol in modeling microbial ethanol production, followed by methyl-butanol. The K. pneumoniae's models achieved the best scoring in applicability (E < 40%) compared to the S. aureus and E. faecalis models, both at laboratory microbial cultures at 37 °C and real postmortem cases. Overall, a noteworthy accuracy in estimating the microbial ethanol in cultures and autopsy blood is achieved by the employed simple linear models.

Purification and characterization of bacteriocins-like inhibitory substances from food isolated Enterococcus faecalis OS13 with activity against nosocomial enterococci.

Nosocomial infections caused by enterococci are an ongoing global threat. Thus, finding therapeutic agents for the treatment of such infections are crucial. Some Enterococcus faecalis strains are able to produce antimicrobial peptides called bacteriocins. We analyzed 65 E. faecalis isolates from 43 food samples and 22 clinical samples in Egypt for 17 common bacteriocin-encoding genes of Enterococcus spp. These genes were absent in 11 isolates that showed antimicrobial activity putatively due to bacteriocins (three from food, including isolate OS13, and eight from clinical isolates). The food-isolated E. faecalis OS13 produced bacteriocin-like inhibitory substances (BLIS) named enterocin OS13, which comprised two peptides (enterocin OS13α OS13ß) that inhibited the growth of antibiotic-resistant nosocomial E. faecalis and E. faecium isolates. The molecular weights of enterocin OS13α and OS13ß were determined as 8079 Da and 7859 Da, respectively, and both were heat-labile. Enterocin OS13α was sensitive to proteinase K, while enterocin OS13ß was resistant. Characterization of E. faecalis OS13 isolate revealed that it belonged to sequence type 116. It was non-hemolytic, bile salt hydrolase-negative, gelatinase-positive, and sensitive to ampicillin, penicillin, vancomycin, erythromycin, kanamycin, and gentamicin. In conclusion, BLIS as enterocin OS13α and OS13ß represent antimicrobial agents with activities against antibiotic-resistant enterococcal isolates.

Regulation of growth, intestinal microflora composition and expression of immune-related genes by dietary supplementation of Streptococcus faecalis in blunt snout bream (Megalobrama amblycephala).

A 10-week feeding trial was performed to investigate the effects of Streptococcus faecalis on the growth, intestinal microflora composition and expression of immune-related genes of blunt snout bream (Megalobrama amblycephala). Fish (46.32 ± 0.09 g) were fed four experimental diets containing 0 cfu/g (SF0, control), 1 × 105 cfu/g (SF1), 1 × 106 cfu/g (SF2) and 1 × 107 cfu/g (SF3) of S. faecalis, respectively. Results showed that daily growth index (DGI), feed efficiency ratio (FER), plasma glucose level, plasma contents of total protein and albumin as well as intestinal serous layer (SL), muscular layer (ML), submucous layer (SML), villi thickness (VT) and lamina propria (LP) were all no significant difference among all the treatments, whereas their (except plasma albumin content and intestinal ML) relatively high values were found in the SF2 group. Meanwhile, the intake of the SF2 diets significantly increased plasma globulin content and intestinal digestive enzymes activities, the opposite was true for the activities of plasma aspartate aminotransferase (AST) and alanine transaminase (ALT). In addition, the analysis of the intestinal microbiota showed that fish fed the SF2 diet have the highest values of intestinal alpha diversity and intestinal abundances of Actinobacteria, Chlamydiae, Firmicutes, Planctomycetes, Verrucomicrobia, Clostridium and Synechococcus, while the opposite was true for intestinal abundances of Acinetobacter, Anoxybacillus, Flavobacterium, Planctomyces, Plesiomonas, Pseudomonas, Staphylococcus and Clostridium perfringens. At the molecular level, the expression levels of tumour necrosis factor α (TNF α), interleukin 1ß (IL 1ß) and heat shock proteins 7 (HSP 70) in head kidney and spleen were all decreased significantly with the increasing S. faecalis levels up to 1 × 106 cfu/g, and then they were increased with further increasing S. faecalis levels. Overall, dietary supplementation of S. faecalis at 1 × 106 cfu/g could improve the intestinal health and innate immunity of blunt snout bream.

Fluorescent reporter plasmids for single-cell and bulk-level composition assays in E. faecalis.

Fluorescent reporters are an important tool for monitoring dynamics of bacterial populations at the single cell and community level. While there are a large range of reporter constructs available-particularly for common model organisms like E. coli-fewer options exist for other species, including E. faecalis, a gram-positive opportunistic pathogen. To expand the potential toolkit available for E. faecalis, we exchanged the original fluorescent reporter in a previously developed plasmid (pBSU101) with one of eight fluorescent reporters and confirmed that all constructs exhibited detectable fluorescence in single E. faecalis cells and mixed biofilm communities. To identify promising constructs for bulk-level experiments, we then measured the fluorescence spectra from E. faecalis populations in microwell plate (liquid) cultures during different phases of aerobic growth. Cultures showed density- and reporter-specific variations in fluorescent signal, though spectral signatures of all reporters become clear in late-exponential and stationary-phase populations. Based on these results, we identified six pairs of reporters that can be combined with simple spectral unmixing to accurately estimate population composition in 2-strain mixtures at or near stationary phase. This approach offers a simple and scalable method for selection and competition experiments in simple two-species populations under aerobic growth conditions. Finally, we incorporated codon-optimized variants of blue (BFP) and red (RFP) reporters and show that they lead to increased fluorescence in exponentially growing cells. As a whole, the results inform the scope of application of different reporters and identify both single reporters and reporter pairs that are promising for fluorescence-based assays at bulk and single-cell levels in E. faecalis.

Effects of single or conjoint administration of lactic acid bacteria as potential probiotics on growth, immune response and disease resistance of snakehead fish (Channa argus).

Lactic acid bacteria (LAB) has been documented to promoting growth, enhancing immunity and disease resistance. In this study, we aimed to evaluate the single or conjoint effects of Lactococcus lactis L19 (Genbank: MT102745.1) and Enterococcus faecalis W24 (Genbank: MT102746.1) isolated from the intestine of Channa argus (C. argus) on growth performance, immune response and disease resistance of C. argus. A total of 720 apparently healthy C. argus (9.50 ± 0.03 g) were randomly divided into four equal groups. Fish were fed with a basal diet (CK) supplemented with L. lactis (L19), E. faecalis (W24), and L. lactis L19 + E. faecalis W24 (L + W) at 1.0 × 108 cfu/g basal diet for 56 days. After feeding, the final body weight (FBW), weight gain (WG), feed efficiency ratio (FER), specific growth rate (SGR) and protein efficiency ratio (PER) had significantly increased (p < 0.05), especially with L19. The results indicated that single or conjoint administration of LAB as potential probiotics can induce high levels of IgM, ACP, AKP, LZM, C3 and C4 activity in serum, which may effectively induce humoral immunity, and L19 induce even higher levels. Meanwhile, when compared to CK group, the results of qPCR showed that LAB administration significantly up-regulated (p < 0.05) the expression of IL-1ß, IL-6, IL-10, TNF-α, IFN-γ, HSP70, HSP90, TGF-ß in the spleen, head kidney, gill, liver and intestine of C. argus. After challenge with Aeromonas veronii, the survival rates in all LAB-fed groups were significantly higher (p < 0.05) than that of the CK group, and the L19 group showed the highest (63.3%) disease resistance. Our data indicated that L. lactis L19 and E. faecalis W24, as a feed additive at 1.0 × 108 cfu/g feed, could promote growth performance, enhance immune response and disease resistance of C. argus, with greatest effects in fish fed L. lactis L19 for 56 days. Hence, these LAB additives could be used as promising probiotics for C. argus. L19 was more effective than W24 or the mixture of the two for promoting growth performance, enhancing immune response and disease resistance of C. argus.

Structural and functional insights into a novel two-component endolysin encoded by a single gene in Enterococcus faecalis phage.

Using bacteriophage-derived endolysins as an alternative strategy for fighting drug-resistant bacteria has recently been garnering renewed interest. However, their application is still hindered by their narrow spectra of activity. In our previous work, we demonstrated that the endolysin LysIME-EF1 possesses efficient bactericidal activity against multiple strains of Enterococcus faecalis (E. faecalis). Herein, we observed an 8 kDa fragment and hypothesized that it contributes to LysIME-EF1 lytic activity. To examine our hypothesis, we determined the structure of LysIME-EF1 at 1.75 Å resolution. LysIME-EF1 exhibits a unique architecture in which one full-length LysIME-EF1 forms a tetramer with three additional C-terminal cell-wall binding domains (CBDs) that correspond to the abovementioned 8 kDa fragment. Furthermore, we identified an internal ribosomal binding site (RBS) and alternative start codon within LysIME-EF1 gene, which are demonstrated to be responsible for the translation of the truncated CBD. To elucidate the molecular mechanism for the lytic activity of LysIME-EF1, we combined mutagenesis, lytic activity assays and in vivo animal infection experiments. The results confirmed that the additional LysIME-EF1 CBDs are important for LysIME-EF1 architecture and its lytic activity. To our knowledge, this is the first determined structure of multimeric endolysin encoded by a single gene in E. faecalis phages. As such, it may provide valuable insights into designing potent endolysins against the opportunistic pathogen E. faecalis.

Influence of graphene oxide nanoparticles on the transport and cotransport of biocolloids in saturated porous media.

This study examines the effect of graphene oxide (GO) nanoparticles (NPs) on the transport (individual species) and cotransport (simultaneous transport) of three biocolloids (Escherichia (E.) coli, Enterococcus (E.) faecalis and Staphylococcus (S.) aureus) in water saturated porous media. Flowthrough experiments were performed in 30-cm long laboratory columns packed with quartz sand. All of the experiments were conducted at room temperature (22 °C), pH = 7, and ionic strength Is = 2 mM. The results from the cotransport experiments indicated that the mass recovery values for all biocolloids, calculated based on total biocolloid concentration in the effluent, were reduced in the presence of GO NPs. The strains E. coli and E. faecalis were shown to be more vulnerable to GO NPs than S. aureus. Temporal moments of the breakthrough concentrations suggested that the presence of GO NPs significantly influenced the fate and transport of the three biocolloids. Extended DLVO theory was used to quantify the various interaction energy profiles, based on electrokinetic and hydrodynamic measurements.

Reisolation and Configurational Reinvestigation of Cottoquinazolines E-G from an Arthropod-Derived Strain of the Fungus Neosartorya fischeri.

The reported fumiquinazoline-related alkaloids cottoquinazolines E-G (1-3) were reisolated from solid cultures of the fungus Neosartorya fischeri, which was isolated from the medicinal arthropod Cryptotympana atrata. The unresolved issues regarding the absolute configurations (for cottoquinazolines E and F) prompted a reinvestigation of the configurations for all three compounds, as enabled by extensive spectroscopic methods, comparisons of experimental electronic circular dichroism data, and X-ray crystallography. In addition, cottoquinazoline F (2) showed significant antibacterial activity against ESBL-producing Escherichia coli, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterococcus faecalis with MIC values of 8, 32, 32, and 16 µg/mL, respectively.

[Evaluation of anti-candida activity of metabolites of enterococcal clinical isolates.]

When studying the effect of the metabolic products of clinical isolates of enterococci on the viability of Candida albicans, it was found that metabolites of all tested strains of Enterococcus faecium, E. faecalis had a fungistatic effect. At the same time a reliable fungicidal effect is a strain-specific feature. It is better to use the method of delayed antagonism on double-layer agar to assess the antifungal effect of enterococcal metabolism products.

Second Harmonic Generation Spectroscopy of Membrane Probe Dynamics in Gram-Positive Bacteria.

Bacterial membranes are complex mixtures with dispersity that is dynamic over scales of both space and time. To capture adsorption onto and transport within these mixtures, we conduct simultaneous second harmonic generation (SHG) and two-photon fluorescence measurements on two different gram-positive bacterial species as the cells uptake membrane-specific probe molecules. Our results show that SHG not only can monitor the movement of small molecules across membrane leaflets but also is sensitive to higher-level ordering of the molecules within the membrane. Further, we show that the membranes of Staphylococcus aureus remain more dynamic after longer times at room temperature in comparison to Enterococcus faecalis. Our findings provide insight into the variability of activities seen between structurally similar molecules in gram-positive bacteria while also demonstrating the power of SHG to examine these dynamics.

Pheromone peptide cOB1 from native Enterococcus faecalis forms amyloid-like structures: A new paradigm for peptide pheromones.

Pheromone peptides are an important component of bacterial quorum-sensing system. The pheromone peptide cOB1 (VAVLVLGA) of native commensal Enterococcus faecalis has also been identified as an antimicrobial peptide (AMP) and reported to kill the prototype clinical isolate strain of E. faecalis V583. In this study, the pheromone peptide cOB1 has shown to form amyloid-like structures, a characteristic which is never reported for a pheromone peptide so far. With in silico analysis, the peptide was predicted to be highly amyloidogenic. Further, under experimental conditions, cOB1 formed aggregates displaying characteristics of amyloid structures such as bathochromic shift in Congo red absorbance, enhancement in thioflavin T fluorescence, and fibrillar morphology under transmission electron microscopy. This novel property of pheromone peptide cOB1 may have some direct effects on the binding of the pheromone to the receptor cells and subsequent conjugative transfer, making this observation more important for the therapeutics, dealing with the generation of virulent and multidrug-resistant pathogenic strains.

Lactic acid bacteria, Enterococcus faecalis Y17 and Pediococcus pentosaceus G11, improved growth performance, and immunity of mud crab (Scylla paramamosain).

Mud crabs (Scylla paramamosain), a commercially important cultured species in the southeastern region of China, is usually infected by Vibriosis or parasites, causing great economic losses in cultured farms. Previous studies have demonstrated that probiotics benefited in enhancing the immune response against invading pathogens in aquatic animals. In this study, the effects of dietary administration of lactic acid bacteria (LAB) (Enterococcus faecalis Y17 and Pediococcus pentosaceus G11) on growth performance and immune responses of mud crab were assessed. Both strains (Y17 and G11) showed an inhibitory activity against bacterial pathogens (Aeromonas hydrophila, Vibrio parahaemolyticus, Vibrio alginolyticus, Staphylococcus aureus, and ß Streptococcus), and a wide pH tolerance range of 2-10. In vivo, mud crabs were fed a control diet and experimental diets supplemented with 109 cfu g-1 diet either Y17 or G11 for 6 weeks before subjecting to a challenge test with V. parahaemolyticus for 12 h. The probiotic-supplemented diets had significant effects on weight gain and specific growth rate during the feeding trial. Increased serum enzyme activities of phenoloxidase, lysozyme, and SOD were observed in the hemolymph of mud crab in Y17 and G11-supplemented groups compared to that in the controls (P < 0.01). The significantly up-regulated expression of gene CAT, LYS, proPO, and SOD could be seen in hepatopancreas in G11-supplemented groups. After the pathogenicity test, the survival rate of Y17 + and G11 + V. parahaemolyticus groups was 66.67% and 80.00%, respectively, compared with 53.33% for the control groups. Taken together, dietary supplementation of Y17 and G11 strains were beneficial in mud crab, which could increase growth performance, modulate immune system and protect the host against V. parahaemolyticus infection.

Effect of the additional cysteine 503 of vancomycin-resistant Enterococcus faecalis (V583) alkylhydroperoxide reductase subunit F (AhpF) and the mechanism of AhpF and subunit C assembling.

The vancomycin-resistant Enterococcus faecalis alkyl hydroperoxide reductase complex (AhpR) with its subunits AhpC (EfAhpC) and AhpF (EfAhpF) is of paramount importance to restore redox homeostasis. Therefore, knowledge about this defense system is essential to understand its antibiotic-resistance and survival in hosts. Recently, we described the crystallographic structures of EfAhpC, the two-fold thioredoxin-like domain of EfAhpF, the novel phenomenon of swapping of the catalytic domains of EfAhpF as well as the unique linker length, connecting the catalytically active N-and C-terminal domains of EfAhpF. Here, using mutagenesis and enzymatic studies, we reveal the effect of an additional third cysteine (C503) in EfAhpF, which might optimize the functional adaptation of the E. faecalis enzyme under various physiological conditions. The crystal structure and solution NMR data of the engineered C503A mutant of the thioredoxin-like domain of EfAhpF were used to describe alterations in the environment of the additional cysteine residue during modulation of the redox-state. To glean insight into the epitope and mechanism of EfAhpF and -AhpC interaction as well as the electron transfer from the thioredoxin-like domain of EfAhpF to AhpC, NMR-titration experiments were performed, showing a coordinated disappearance of peaks in the thioredoxin-like domain of EfAhpF in the presence of full length EfAhpC, and indicating a stable EfAhpF-AhpC-complex. Combined with docking studies, the interacting residues of EfAhpF were identified and a mechanism of electron transfer of the EfAhpF donor to the electron acceptor EfAhpC is described.

Colorimetric and Electrochemical Detection of Escherichia coli and Antibiotic Resistance Based on a p-Benzoquinone-Mediated Bioassay.

The facile and economical identification of pathogenic bacteria, especially their antibiotic-resistance, is crucial in the realm of human health and safety. The presence of Escherichia coli ( E. coli) is considered as an indicator of water contamination and is closely related to human health. Herein, inspired by the biocatalysis of bacterial surfaces, we developed a simple and cost-effective colorimetric- and electrochemical-based bioassay that is capable of analyzing both the presence of E. coli and its relative level of antibiotic resistance. In this approach, p-benzoquinone is used as a redox mediator to monitor the bacterial concentration and specifically distinguish E. coli from four other common clinical bacteria, namely, Staphylococcus aureus ( S. aureus), Enterococcus faecalis ( E. faecalis), Salmonella pullorum ( S. pullorum), and Streptococcus mutans ( S. mutans). A visible color change, captured with a smartphone using a "light box", without relying on any complex instruments, can reflect the concentration of bacteria. The accurate quantification of E. coli was investigated with an electrochemical system in the concentration ranges of 1.0 × 103 to 1.0 × 109 CFU/mL. We further demonstrated the capability of the presented biosensor in identifying drug-resistant bacteria with two artificially induced antibiotic-resistant bacteria. Therefore, the presented bioassay is not only capable of detecting E. coli with high sensitivity and specificity but also provides a rapid solution to evaluate E. coli antibiotic resistance.

Insights into Chagas treatment based on the potential of bacteriocin AS-48.

Chagas disease caused by the protozoan parasite Trypanosoma cruzi represents a significant public health problem in Latin America, affecting around 8 million cases worldwide. Nowadays is urgent the identification of new antichagasic agents as the only therapeutic options available, Nifurtimox and Benznidazole, are in use for >40 years, and present high toxicity, limited efficacy and frequent treatment failures in the chronic phase of the disease. Recently, it has been described the antiparasitic effect of AS-48, a bacteriocin produced by Enterococcus faecalis, against Trypanosoma brucei and Leishmania spp. In this work, we have demonstrated the in vitro potential of the AS-48 bacteriocin against T. cruzi. Interesting, AS-48 was more effective against the three morphological forms of different T. cruzi strains, and displayed lower cytotoxicity than the reference drug Benznidazole. In addition, AS-48 combines the criteria established as a potential antichagasic agent, resulting in a promising therapeutic alternative. According to the action mechanism, AS-48 trypanocidal activity could be explained in a mitochondrion-dependent manner through a reactive oxygen species production and mitochondrial depolarization, causing a fast and severe bioenergetic collapse.

Effects of Enterococcus faecalis on egg production, egg quality and caecal microbiota of hens during the late laying period.

This study was conducted to determine the effects of diet supplementation of laying hens with Enterococcus faecalis (EF) on egg production, egg quality and caecal microbiota. A total of 360 Hy-Line Brown laying hens (72 weeks old) were divided into three groups with four replicates of 30 birds each. The laying hens were fed with the basal diet (Control), the basal diet + 3.75 · 108 cfu EF/kg (Group I) or the basal diet + 7.5 · 108 cfu EF/kg (Group II). The experiment lasted for 45 d. Eggs and caecal samples were collected at the end of the experiment. Results showed that dietary supplementation with EF did not affect the average daily egg weight, cracked egg rate, mortality and egg quality. However, EF supplementation caused a significantly increased laying rate and decreased feed/egg ratio (p < 0.05). The differences in caecal microbiota between Group II and the Control were significant. The relative abundance of Verrucomicrobia and Cyanobacteria at the phylum level, Rikenellaceae, Christensenellaceae and Veillonellaceae at the family level, and the Faecalibacterium, Christensenellaceae R-7 group and Eubacterium coprostanoligenes group at the genus level changed significantly in Group II compared with the Control (p < 0.05). In conclusion, the tested dietary supplementations with EF improved product performance and affected the caecal microbial community structure of laying hens during the late laying period.

In Vitro Synthesis of Bioactive Glycovariants of Enterocin 96, an Antimicrobial Peptide from Enterococcus faecalis.

Antimicrobial peptides (AMPs) are novel agents for therapeutic application for their inherent broad spectrum of activities against bacteria, fungi, and viruses, as well as anti-inflammatory, anticancerous, and immunomodulatory activities. This chapter presents an enzymatic method to generate glycovariants of one such antimicrobial peptide, namely enterocin 96, using a bacterial protein O- and S-glycosyltransferase, in vitro.