Microbiota-derived lantibiotic restores resistance against vancomycin-resistant Enterococcus.

Intestinal commensal bacteria can inhibit dense colonization of the gut by vancomycin-resistant Enterococcus faecium (VRE), a leading cause of hospital-acquired infections1,2. A four-strained consortium of commensal bacteria that contains Blautia producta BPSCSK can reverse antibiotic-induced susceptibility to VRE infection3. Here we show that BPSCSK reduces growth of VRE by secreting a lantibiotic that is similar to the nisin-A produced by Lactococcus lactis. Although the growth of VRE is inhibited by BPSCSK and L. lactis in vitro, only BPSCSK colonizes the colon and reduces VRE density in vivo. In comparison to nisin-A, the BPSCSK lantibiotic has reduced activity against intestinal commensal bacteria. In patients at high risk of VRE infection, high abundance of the lantibiotic gene is associated with reduced density of E. faecium. In germ-free mice transplanted with patient-derived faeces, resistance to VRE colonization correlates with abundance of the lantibiotic gene. Lantibiotic-producing commensal strains of the gastrointestinal tract reduce colonization by VRE and represent potential probiotic agents to re-establish resistance to VRE.

Lipidated variants of the antimicrobial peptide nisin produced via incorporation of methionine analogs for click chemistry show improved bioactivity.

The increase in antibiotic resistance calls for accelerated molecular engineering strategies to diversify natural products for drug discovery. The incorporation of non-canonical amino acids (ncAAs) is an elegant strategy for this purpose, offering a diverse pool of building blocks to introduce desired properties into antimicrobial lanthipeptides. We here report an expression system using Lactococcus lactis as a host for non-canonical amino acid incorporation with high efficiency and yield. We show that incorporating the more hydrophobic analog ethionine (instead of methionine) into nisin improves its bioactivity against several Gram-positive strains we tested. New-to-nature variants were further created by click chemistry. By azidohomoalanine (Aha) incorporation and subsequent click chemistry, we obtained lipidated variants at different positions in nisin or in truncated nisin variants. Some of them show improved bioactivity and specificity against several pathogenic bacterial strains. These results highlight the ability of this methodology for lanthipeptide multi-site lipidation, to create new-to-nature antimicrobial products with diverse features, and extend the toolbox for (lanthi)peptide drug improvement and discovery.

Label-Free Monitoring of Coculture System Dynamics: Probing Probiotic and Cancer Cell Interactions via Infrared Spectroscopic Imaging.

Evaluating the dynamic interaction of microorganisms and mammalian cells is challenging due to the lack of suitable platforms for examining interspecies interactions in biologically relevant coculture conditions. In this work, we demonstrate the interaction between probiotic bacteria (Lactococcus lactis and Escherichia coli) and A498 human cancer cells in vitro, utilizing a hydrogel-based platform in a label-free manner by infrared spectroscopy. The L. lactis strain recapitulated in the compartment system secretes polypeptide molecules such as nisin, which has been reported to trigger cell apoptosis. We propose a mid-infrared (IR) spectroscopic imaging approach to monitor the variation of biological components utilizing kidney cells (A498) as a model system cocultured with bacteria. We characterized the biochemical composition (i.e., nucleic acids, protein secondary structures, and lipid conformations) label-free using an unbiased measurement. Several IR spectral features, including unsaturated fatty acids, ß-turns in protein, and nucleic acids, were utilized to predict cellular response. These features were then applied to establish a quantitative relationship through a multivariate regression model to predict cellular dynamics in the coculture system to assess the effect of nisin on A498 kidney cancer cells cocultured with bacteria. Overall, our study sheds light on the potential of using IR spectroscopic imaging as a label-free tool to monitor complex microbe-host cell interactions in biological systems. This integration will enable mechanistic studies of interspecies interactions with insights into their underlying physiological processes.

The capsule of Bacillus anthracis protects it from the bactericidal activity of human defensins and other cationic antimicrobial peptides.

During infection, Bacillus anthracis bacilli encounter potent antimicrobial peptides (AMPs) such as defensins. We examined the role that B. anthracis capsule plays in protecting bacilli from defensins and other cationic AMPs by comparing their effects on a fully virulent encapsulated wild type (WT) strain and an isogenic capsule-deficient capA mutant strain. We identified several human defensins and non-human AMPs that were capable of killing B. anthracis. The human alpha defensins 1-6 (HNP-1-4, HD-5-6), the human beta defensins 1-4 (HBD-1-4), and the non-human AMPs, protegrin, gramicidin D, polymyxin B, nisin, and melittin were all capable of killing both encapsulated WT and non-encapsulated capA mutant B. anthracis. However, non-encapsulated capA mutant bacilli were significantly more susceptible than encapsulated WT bacilli to killing by nearly all of the AMPs tested. We demonstrated that purified capsule bound HBD-2, HBD-3, and HNP-1 in an electrophoretic mobility shift assay. Furthermore, we determined that the capsule layer enveloping WT bacilli bound and trapped HBD-3, substantially reducing the amount reaching the cell wall. To assess whether released capsule might also play a protective role, we pre-incubated HBD-2, HBD-3, or HNP-1 with purified capsule before their addition to non-encapsulated capA mutant bacilli. We found that free capsule completely rescued the capA mutant bacilli from killing by HBD-2 and -3 while killing by HNP-1 was reduced to the level observed with WT bacilli. Together, these results suggest an immune evasion mechanism by which the capsule, both that enveloping the bacilli and released fragments, contributes to virulence by binding to and inhibiting the antimicrobial activity of cationic AMPs.

Strategies for effective high pressure germination or inactivation of Bacillus spores involving nisin.

The major challenge in employing high pressure (HP) at moderate temperature for sterilization is the remarkable resistance of bacterial spores. High isostatic pressure can initiate spore germination, enabling subsequent inactivation under mild conditions. However, not all spores could be triggered to germinate under pressure at temperatures ≤80°C so far. In this study, germination treatment combinations were evaluated for Bacillus spores involving moderate HP (150 MPa, 37°C, 5 min), very HP (vHP, 550 MPa, 60°C, 2.5 or 9 min), simple and complex nutrient germinants [L-valine, L-alanine, and tryptic soy broth (TSB)], nisin, and incubation at atmospheric pressure (37°C). The most effective combinations for Bacillus subtilis resulted in a reduction of culturable dormant spores by 8 log10 units. The combinations involved nisin, a nutrient germinant (L-valine or TSB), a first vHP treatment (550 MPa, 60°C, 2.5 min), incubation at atmospheric pressure (37°C, 6 h), and a second vHP treatment (550 MPa, 60°C, 2.5 min). Such treatment combination with L-valine reduced Bacillus amyloliquefaciens spores by only 2 log10 units. B. amyloliquefaciens, thus, proved to be substantially more HP-resistant compared to B. subtilis, validating previous studies. Despite combining different germination mechanisms, complete germination could not be achieved for either species. The natural bacteriocin nisin did seemingly not promote HP germination initiation under chosen HP conditions, contrary to previous literature. Nevertheless, nisin might be beneficial to inhibit the growth of HP-germinated or remaining ungerminated spores. Future germination experiments might consider that nisin could not be completely removed from spores by washing, thereby affecting plate count enumeration. IMPORTANCE: Extremely resistant spore-forming bacteria are widely distributed in nature. They infiltrate the food chain and processing environments, posing risks of spoilage and food safety. Traditional heat-intensive inactivation methods often negatively affect the product quality. HP germination-inactivation offers a potential solution for better preserving sensitive ingredients while inactivating spores. However, the presence of ungerminated (superdormant) spores hampers the strategy's success and safety. Knowledge of strategies to overcome resistance to HP germination is vital to progress mild spore control technologies. Our study contributes to the evaluation and development of mild preservation processes by evaluating strategies to enhance the HP germination-inactivation efficacy. Mild preservation processes can fulfill the consumers' demand for safe and minimally processed food.

Thiolated chitosan nanoparticles encapsulated nisin and selenium: antimicrobial/antibiofilm/anti-attachment/immunomodulatory multi-functional agent.

BACKGROUND: The increase in the resistance of bacterial strains to antibiotics has led to research into the bactericidal potential of non-antibiotic compounds. This study aimed to evaluate in vitro antibacterial/ antibiofilm properties of nisin and selenium encapsulated in thiolated chitosan nanoparticles (N/Se@TCsNPs) against prevalent enteric pathogens including standard isolates of Vibrio (V.) cholerae O1 El Tor ATCC 14,035, Campylobacter (C.) jejuni ATCC 29,428, Salmonella (S.) enterica subsp. enterica ATCC 19,430, Shigella (S.) dysenteriae PTCC 1188, Escherichia (E.) coli O157:H7 ATCC 25,922, Listeria (L.) monocytogenes ATCC 19,115, and Staphylococcus (S.) aureus ATCC 29,733. METHODS: The synthesis and comprehensive analysis of N/Se@TCsNPs have been completed. Antibacterial and antibiofilm capabilities of N/Se@TCsNPs were evaluated through broth microdilution and crystal violet assays. Furthermore, the study included examining the cytotoxic effects on Caco-2 cells and exploring the immunomodulatory effects of N/Se@TCsNPs. This included assessing the levels of both pro-inflammatory (IL-6 and TNFα) and anti-inflammatory (IL-10 and TGFß) cytokines and determining the gene expression of TLR2 and TLR4. RESULTS: The N/Se@TCsNPs showed an average diameter of 136.26 ± 43.17 nm and a zeta potential of 0.27 ± 0.07 mV. FTIR spectroscopy validated the structural features of N/Se@TCsNPs. Scanning electron microscopy (SEM) images confirmed their spherical shape and uniform distribution. Thermogravimetric Analysis (TGA)/Differential Scanning Calorimetry (DSC) tests demonstrated the thermal stability of N/Se@TCsNPs, showing minimal weight loss of 0.03%±0.06 up to 80 °C. The prepared N/Se@TCsNPs showed a thiol content of 512.66 ± 7.33 µmol/g (p < 0.05), an encapsulation efficiency (EE) of 69.83%±0.04 (p ≤ 0.001), and a drug release rate of 74.32%±3.45 at pH = 7.2 (p ≤ 0.004). The synthesized nanostructure demonstrated potent antibacterial activity against various isolates, with effective concentrations ranging from 1.5 ± 0.08 to 25 ± 4.04 mg/mL. The ability of N/Se@TCsNPs to reduce bacterial adhesion and internalization in Caco-2 cells underscored their antibiofilm properties (p ≤ 0.0001). Immunological studies indicated that treatment with N/Se@TCsNPs led to decreased levels of inflammatory cytokines IL-6 (14.33 ± 2.33 pg/mL) and TNFα (25 ± 0.5 pg/mL) (p ≤ 0.0001), alongside increased levels of anti-inflammatory cytokines IL-10 (46.00 ± 0.57 pg/mL) and TGFß (42.58 ± 2.10 pg/mL) in infected Caco-2 cells (p ≤ 0.0001). Moreover, N/Se@TCsNPs significantly reduced the expression of TLR2 (0.22 ± 0.09) and TLR4 (0.16 ± 0.05) (p < 0.0001). CONCLUSION: In conclusion, N/Se@TCsNPs exhibited significant antibacterial/antibiofilm/anti-attachment/immunomodulatory effectiveness against selected Gram-positive and Gram-negative enteric pathogens. However, additional ex-vivo and in-vivo investigations are needed to fully assess the performance of nanostructured N/Se@TCsNPs.

Suppression of planktonic and biofilm of Escherichia coli by the synergistic lantibiotics-polymyxins combinations.

Escherichia coli are generally resistant to the lantibiotic's action (nisin and warnerin), but we have shown increased sensitivity of E. coli to lantibiotics in the presence of subinhibitory concentrations of polymyxins. Synergistic lantibiotic-polymyxin combinations were found for polymyxins B and M. The killing of cells at the planktonic and biofilm levels was observed for two collection and four clinical multidrug-resistant E. coli strains after treatment with lantibiotic-polymyxin B combinations. Thus, 24-h treatment of E. coli mature biofilms with warnerin-polymyxin B or nisin-polymyxin B leads to five to tenfold decrease in the number of viable cells, depending on the strain. AFM revealed that the warnerin and polymyxin B combination caused the loss of the structural integrity of biofilm and the destruction of cells within the biofilm. It has been shown that pretreatment of cells with polymyxin B leads to an increase of Ca2+ and Mg2+ ions in the culture medium, as detected by atomic absorption spectroscopy. The subsequent exposure to warnerin caused cell death with the loss of K+ ions and cell destruction with DNA and protein release. Thus, polymyxins display synergy with lantibiotics against planktonic and biofilm cells of E. coli, and can be used to overcome the resistance of Gram-negative bacteria to lantibiotics.

Assessing the synergistic potential of bacteriophage endolysins and antimicrobial peptides for eradicating bacterial biofilms.

Phage-encoded endolysins have emerged as a potential substitute to conventional antibiotics due to their exceptional benefits including host specificity, rapid host killing, least risk of resistance. In addition to their antibacterial potency and biofilm eradication properties, endolysins are reported to exhibit synergism with other antimicrobial agents. In this study, the synergistic potency of endolysins was dissected with antimicrobial peptides to enhance their therapeutic effectiveness. Recombinantly expressed and purified bacteriophage endolysin [T7 endolysin (T7L); and T4 endolysin (T4L)] proteins have been used to evaluate the broad-spectrum antibacterial efficacy using different bacterial strains. Antibacterial/biofilm eradication studies were performed in combination with different antimicrobial peptides (AMPs) such as colistin, nisin, and polymyxin B (PMB) to assess the endolysin's antimicrobial efficacy and their synergy with AMPs. In combination with T7L, polymyxin B and colistin effectively eradicated the biofilm of Pseudomonas aeruginosa and exhibited a synergistic effect. Further, a combination of T4L and nisin displayed a synergistic effect against Staphylococcus aureus biofilms. In summary, the obtained results endorse the theme of combinational therapy consisting of endolysins and AMPs as an effective remedy against the drug-resistant bacterial biofilms that are a serious concern in healthcare settings.

Simulation-Guided Molecular Modeling of Nisin and Lipid II Assembly and Membrane Pore Formation.

The lantibiotic pore-forming peptide nisin is a promising candidate in the fight against multidrug-resistant bacteria due to its unique structure, which allows it to disrupt bacteria in two distinct ways─Lipid II trafficking and transmembrane pore formation. However, exactly how nisin and Lipid II assemble into oligomeric pore structures in the bacterial membrane is not known. Spontaneous peptide assembly into pores is difficult to observe in even the very long-time scale molecular dynamics (MD) simulations. In this study, we adopted an MD-guided modeling approach to investigate the nisin-nisin and nisin-Lipid II associations in the membrane environment. Through extensive microsecond-time scale all-atom MD simulations, we established that nisin monomers dimerize by forming ß-sheets in a POPE:POPG lipid bilayer and oligomerize further to form stable transmembrane channels. We determined that these nisin dimers use Lipid II as a dimer interface to incur enhanced stability. Our results provide a clearer understanding of the self-assembly of nisin monomers within the membrane and insights into the role of Lipid II in the structural integrity of oligomeric structures.

Antibacterial characteristics and mechanistic insights of combined tea polyphenols, Nisin, and epsilon-polylysine against feline oral pathogens: a comprehensive transcriptomic and metabolomic analysis.

AIMS: This study evaluates the antibacterial characteristics and mechanisms of combined tea polyphenols (TPs), Nisin, and ε-polylysine (PL) against Streptococcus canis, Streptococcus minor, Streptococcus mutans, and Actinomyces oris, common zoonotic pathogens in companion animals. METHODS AND RESULTS: Pathogenic strains were isolated from feline oral cavities and assessed using minimum inhibitory concentration (MIC) tests, inhibition zone assays, growth kinetics, and biofilm inhibition studies. Among single agents, PL exhibited the lowest MIC values against all four pathogens. TP showed significant resistance against S. minor, and Nisin against S. mutans. The combination treatment (Comb) of TP, Nisin, and PL in a ratio of 13:5:1 demonstrated broad-spectrum antibacterial activity, maintaining low MIC values, forming large inhibition zones, prolonging the bacterial lag phase, reducing growth rates, and inhibiting biofilm formation. RNA sequencing and metabolomic analysis indicated that TP, Nisin, and PL inhibited various membrane-bound carbohydrate-specific transferases through the phosphoenolpyruvate-dependent phosphotransferase system in S. canis, disrupting carbohydrate uptake. They also downregulated glycolysis and the citric acid cycle, inhibiting cellular energy metabolism. Additionally, they modulated the activities of peptidoglycan glycosyltransferases and d-alanyl-d-alanine carboxypeptidase, interfering with peptidoglycan cross-linking and bacterial cell wall stability. CONCLUSIONS: The Comb therapy significantly enhances antibacterial efficacy by targeting multiple bacterial pathways, offering potential applications in food and pharmaceutical antimicrobials.

Facile Halogenation of Antimicrobial Peptides As Demonstrated by Producing Bromotryptophan-Labeled Nisin Variants with Enhanced Antimicrobial Activity.

Antimicrobial peptides (AMPs) have raised significant interest, forming a potential new class of antibiotics in the fight against multi-drug-resistant bacteria. Various AMPs are ribosomally synthesized and post-translationally modified peptides (RiPPs). One post-translational modification found in AMPs is the halogenation of Trp residues. This modification has, for example, been shown to be critical for the activity of the potent AMP NAI-107 from Actinoallomurus. Due to the importance of organohalogens, establishing methods for facile and selective halogen atom installation into AMPs is highly desirable. In this study, we introduce an expression system utilizing the food-grade strain Lactococcus lactis, facilitating the efficient incorporation of bromo-Trp (BrTrp) into (modified) peptides, exemplified by the lantibiotic nisin with a single Trp residue or analogue incorporated at position 1. This provides an alternative to the challenges posed by halogenase enzymes, such as poor substrate selectivity. Our method yields expression levels comparable to that of wild-type nisin, while BrTrp incorporation does not interfere with the post-translational modifications of nisin (dehydration and cyclization). One brominated nisin variant exhibits a 2-fold improvement in antimicrobial activity against two tested pathogens, including a WHO priority pathogen, while maintaining the same lipid II binding and bactericidal activity as wild-type nisin. The work presented here demonstrates the potential of this methodology for peptide halogenation, offering a new avenue for the development of diverse antimicrobial products labeled with BrTrp.

Combination antimicrobial therapy: in vitro synergistic effect of anti-staphylococcal drug oxacillin with antimicrobial peptide nisin against Staphylococcus epidermidis clinical isolates and Staphylococcus aureus biofilms.

The ability of Staphylococcus epidermidis and S. aureus to form strong biofilm on plastic devices makes them the major pathogens associated with device-related infections (DRIs). Biofilm-embedded bacteria are more resistant to antibiotics, making biofilm infections very difficult to effectively treat. Here, we evaluate the in vitro activities of anti-staphylococcal drug oxacillin and antimicrobial peptide nisin, alone and in combination, against methicillin-resistant S. epidermidis (MRSE) clinical isolates and the methicillin-resistant S. aureus ATCC 43,300. The minimum inhibitory concentrations (MIC) and minimum biofilm eradication concentrations (MBEC) of oxacillin and nisin were determined using the microbroth dilution method. The anti-biofilm activities of oxacillin and nisin, alone or in combination, were evaluated. In addition, the effects of antimicrobial agents on the expression of icaA gene were examined by quantitative real-time PCR. MIC values for oxacillin and nisin ranged 4-8 µg/mL and 64-128 µg/mL, respectively. Oxacillin and nisin reduced biofilm biomass in all bacteria in a dose-dependent manner and this inhibitory effect was enhanced with combinatorial treatment. MBEC ranges for oxacillin and nisin were 2048-8192 µg/mL and 2048-4096 µg/mL, respectively. The addition of nisin significantly decreased the oxacillin MBECs from 8- to 32-fold in all bacteria. At the 1× MIC and 1/2× MIC, both oxacillin and nisin decreased significantly the expression of icaA gene in comparison with untreated control. When two antimicrobial agents were combined at 1/2× MIC concentration, the expression of icaA were significantly lower than when were used alone. Nisin/conventional oxacillin combination showed considerable anti-biofilm effects, including inhibition of biofilm formation, eradication of mature biofilm, and down-regulation of biofilm-related genes, proposing its applications for treating or preventing staphylococcal biofilm-associated infections, including device-related infections.

Natural approach of using nisin and its nanoform as food bio-preservatives against methicillin resistant Staphylococcus aureus and E.coli O157:H7 in yoghurt.

BACKGROUND: Natural antimicrobial agents such as nisin were used to control the growth of foodborne pathogens in dairy products. The current study aimed to examine the inhibitory effect of pure nisin and nisin nanoparticles (nisin NPs) against methicillin resistant Staphylococcus aureus (MRSA) and E.coli O157:H7 during the manufacturing and storage of yoghurt. Nisin NPs were prepared using new, natural, and safe nano-precipitation method by acetic acid. The prepared NPs were characterized using zeta-sizer and transmission electron microscopy (TEM). In addition, the cytotoxicity of nisin NPs on vero cells was assessed using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The minimum inhibitory concentrations (MICs) of nisin and its nanoparticles were determined using agar well-diffusion method. Further, fresh buffalo's milk was inoculated with MRSA or E.coli O157:H7 (1 × 106 CFU/ml) with the addition of either nisin or nisin NPs, and then the inoculated milk was used for yoghurt making. The organoleptic properties, pH and bacterial load of the obtained yoghurt were evaluated during storage in comparison to control group. RESULTS: The obtained results showed a strong antibacterial activity of nisin NPs (0.125 mg/mL) against MRSA and E.coli O157:H7 in comparison with control and pure nisin groups. Notably, complete eradication of MRSA and E.coli O157:H7 was observed in yoghurt formulated with nisin NPs after 24 h and 5th day of storage, respectively. The shelf life of yoghurt inoculated with nisin nanoparticles was extended than those manufactured without addition of such nanoparticles. CONCLUSIONS: Overall, the present study indicated that the addition of nisin NPs during processing of yoghurt could be a useful tool for food preservation against MRSA and E.coli O157:H7 in dairy industry.

Impact of a teat disinfectant based on Lactococcus cremoris on the cow milk proteome.

BACKGROUND: Dairy cow milking practices require cleaning and disinfection of the teat skin before and after milking to ensure the safety and quality of milk and prevent intramammary infections. Antimicrobial proteins of natural origin can be valuable alternatives to traditional disinfectants. In a recent field trial, we demonstrated that a teat dip based on a nisin A-producing Lactococcus cremoris (L) had comparable efficacy to conventional iodophor dip (C) in preventing dairy cow mastitis. Here, we present the differential shotgun proteomics investigation of the milk collected during the trial. METHODS: Four groups of quarter milk samples with low (LSCC) and high somatic cell count (HSCC) collected at the beginning (T0) and end (TF) of the trial were analyzed for a total of 28 LSCC (14 LSCC T0 and 14 LSCC TF) and 12 HSCC (6 HSCC T0 and 6 HSCC TF) samples. Milk proteins were digested into peptides, separated by nanoHPLC, and analyzed by tandem mass spectrometry (LC-MS/MS) on an Orbitrap Fusion Tribrid mass spectrometer. The proteins were identified with MaxQuant and interaction networks of the differential proteins were investigated with STRING. The proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD045030. RESULTS: In healthy milk (LSCC), we detected 90 and 80 differential proteins at T0 and TF, respectively. At TF, the Lactococcus group showed higher levels of antimicrobial proteins. In mastitis milk (HSCC), we detected 88 and 106 differential proteins at T0 and TF, respectively. In the Lactococcus group, 14 proteins with antimicrobial and immune defense functions were enriched at TF vs. 4 proteins at T0. Cathelicidins were among the most relevant enriched proteins. Western immunoblotting validation confirmed the differential abundance. CONCLUSIONS: At T0, the proteomic differences observed in healthy milk of the two groups were most likely dependent on physiological variation. On the other hand, antimicrobial and immune defense functions were higher in the milk of cows with mammary gland inflammation of the Lactococcus-treated group. Among other factors, the immunostimulatory action of nisin A might be considered as a contributor.

Antimicrobial peptides and proteins as alternative antibiotics for porcine semen preservation.

BACKGROUND: Antimicrobial resistance (AMR) is nowadays a major emerging challenge for public health worldwide. The over- and misuse of antibiotics, including those for cell culture, are promoting AMR while also encouraging the research and employment of alternative drugs. The addition of antibiotics to the cell media is strongly recommended in sperm preservation, being gentamicin the most used for boar semen. Because of its continued use, several bacterial strains present in boar semen have developed resistance to this antibiotic. Antimicrobial peptides and proteins (AMPPs) are promising candidates as alternative antibiotics because their mechanism of action is less likely to promote AMR. In the present study, we tested two AMPPs (lysozyme and nisin; 50 and 500 µg/mL) as possible substitutes of gentamicin for boar semen preservation up to 48 h of storage. RESULTS: We found that both AMPPs improved sperm plasma membrane and acrosome integrity during semen storage. The highest concentration tested for lysozyme also kept the remaining sperm parameters unaltered, at 48 h of semen storage, and reduced the bacterial load at comparable levels of the samples supplemented with gentamicin (p > 0.05). On the other hand, while nisin (500 µg/mL) reduced the total Enterobacteriaceae counts, it also decreased the rapid and progressive sperm population and the seminal oxidation-reduction potential (p < 0.05). CONCLUSIONS: The protective effect of lysozyme on sperm function together with its antimicrobial activity and inborn presence in body fluids, including semen and cervical mucus, makes this enzyme a promising antimicrobial agent for boar semen preservation.

Subinhibitory concentrations of nisin enhance virulence gene expression in Staphylococcus aureus and increase mortality in Galleria mellonella.

Alternative strategies for controlling Staphylococcus aureus and other pathogens have been continuously investigated, with nisin, a bacteriocin widely used in the food industry as a biopreservative, gaining increasing attention. In addition to its antimicrobial properties, bacteriocins have significant effects on genome functionality even at inhibitory concentrations. This study investigated the impact of subinhibitory concentrations of nisin on S. aureus. Culturing in the presence of 0.625 µmol l-1 nisin, led to the increased relative expression of hla, saeR, and sarA, genes associated with virulence while expression of the sea gene, encoding staphylococcal enterotoxin A (SEA), decreased. In an in vivo experiment, Galleria mellonella larvae inoculated with S. aureus cultured in the presence of nisin exhibited 97% mortality at 72 h post-infection, compared to over 40% of larvae mortality in larvae infected with S. aureus. A comprehensive understanding of the effect of nisin on the transcriptional response of virulence genes and the impact of these changes on the virulence of S. aureus can contribute to assessing the application of this bacteriocin in food and medical contexts.

Combined treatment of pulsed light and nisin-organic acid based antimicrobial wash for inactivation of Escherichia coli O157:H7 in Romaine lettuce, reduction of microbial loads, and retention of quality.

Microbial safety of fresh produce continues to be a major concern. Novel antimicrobial methods are needed to minimize the risk of contamination. This study investigated the antimicrobial efficacy of pulsed light (PL), a novel nisin-organic acid based antimicrobial wash (AW) and the synergy thereof in inactivating E. coli O157:H7 on Romaine lettuce. Treatment effects on background microbiota and produce quality during storage at 4 °C for 7 days was also investigated. A bacterial cocktail containing three outbreak strains of E. coli O157:H7 was used as inoculum. Lettuce leaves were spot inoculated on the surface before treating with PL (1-60 s), AW (2 min) or combinations of PL with AW. PL treatment for 10 s, equivalent to fluence dose of 10.5 J/cm2, was optimal and resulted in 2.3 log CFU/g reduction of E. coli O157:H7, while a 2 min AW treatment, provided a comparable pathogen reduction of 2.2 log CFU/g. Two possible treatment sequences of PL and AW combinations were investigated. For PL-AW combination, inoculated lettuce leaves were initially exposed to optimum PL dose followed by 2 min AW treatment, whereas for AW-PL combination, inoculated lettuce were subjected to 2 min AW treatment prior to 10 s PL treatment. Both combination treatments (PL-AW and AW-PL) resulted in synergistic inactivation as E. coli cells were not detectable after treatment, indicating >5 log pathogen reductions. Combination treatments significantly (P < 0.05) reduced spoilage microbial populations on Romaine lettuce and also hindered their growth in storage for 7 days. The firmness and visual quality appearance of lettuce were not significantly (P > 0.05) influenced due to combination treatments. Overall, the results reveal that PL and AW combination treatments can be implemented as a novel approach to enhance microbial safety, quality and shelf life of Romaine lettuce.

Synthesis and Characterization of a Novel Chitosan-Based Nanoparticle-Hydrogel Composite System Promising for Skin Wound Drug Delivery.

As a natural preservative, nisin is widely used in the food industry, while its application in biomedicine is limited due to its susceptibility to interference from external conditions. In this study, a nanoparticle-hydrogel composite system was designed to encapsulate and release nisin. Nisin nanoparticles were identified with a smooth, spherical visual morphology, particle size of 122.72 ± 4.88 nm, polydispersity coefficient of 0.473 ± 0.063, and zeta potential of 23.89 ± 0.37 mV. Based on the sample state and critical properties, three temperature-sensitive hydrogels based on chitosan were ultimately chosen with a rapid gelation time of 112 s, outstanding reticular structure, and optimal swelling ratio of 239.05 ± 7.15%. The composite system exhibited the same antibacterial properties as nisin, demonstrated by the composite system's inhibition zone diameter of 17.06 ± 0.83 mm, compared to 20.20 ± 0.58 mm for nisin, which was attributed to the prolonged release effect of the hydrogel at the appropriate temperature. The composite system also demonstrated good biocompatibility and safety, making it suitable for application as short-term wound dressings in biomedicine due to its low hemolysis rate of less than 2%. In summary, our nanoparticle-based hydrogel composite system offers a novel application form of nisin while ensuring its stability, thereby deepening and broadening the employment of nisin.

Comparative assessment of antibacterial activity of Matricaria chamomilla L. extract, nisin and of its combination against Alicyclobacillus spp.

Alicyclobacillus spp. is a potential spoiling agent of acidic products and citrus drinks, leading to sensory alterations in contaminated products and consequent economic losses. Treatments such as pasteurization eliminate vegetative cells, but also create a favorable atmosphere for spore germination. To guarantee quality and safety, the application of natural substances as bioconservatives is a considerable and promising alternative for the food industry. This study evaluated the effect of hexane extract of Matricaria chamomilla L. (HE), Nisin (N) and their combination (HE + N). These compounds are present in some studies describing their antibacterial action, but no studies were found on the association of these compounds against the species Alicyclobacillus spp. This study aimed to analyze the antioxidant activity (AA) for the DPPH• (0,23 µmol Trolox/mg) and ABTS (27.93 µmol Trolox/mg), the Checkboard test revealed synergism between HE and N with a fractional inhibitory index (FIC) of 0.068., and to study the antibacterial and sporicidal effect. The antibacterial and sporicidal activity was satisfactory against Alicyclobacillus acidoterrestris with MIC and MBC of 1.95 µg/mL and MSC of 7.81 µg/mL in analyzes using HE + N. The application in orange juice proved to be effective, with an MBC of 0.007 µg/mL. The MIC results served as a parameter for other tests carried out in this study, such as flow cytometry and Scanning Electron Microscopy (SEM), and for the evaluation of sensory characteristics with Electronic Nose (E-nose).

AcrR1, a novel TetR/AcrR family repressor, mediates acid and antibiotic resistance and nisin biosynthesis in Lactococcus lactis F44.

Lactococcus lactis, widely used in the manufacture of dairy products, encounters various environmental stresses both in natural habitats and during industrial processes. It has evolved intricate machinery of stress sensing and defense to survive harsh stress conditions. Here, we identified a novel TetR/AcrR family transcription regulator, designated AcrR1, to be a repressor for acid and antibiotic tolerance that was derepressed in the presence of vancomycin or under acid stress. The survival rates of acrR1 deletion strain ΔAcrR1 under acid and vancomycin stresses were about 28.7-fold (pH 3.0, HCl), 8.57-fold (pH 4.0, lactic acid) and 2.73-fold (300 ng/mL vancomycin) greater than that of original strain F44. We also demonstrated that ΔAcrR1 was better able to maintain intracellular pH homeostasis and had a lower affinity to vancomycin. No evident effects of AcrR1 deletion on the growth and morphology of strain F44 were observed. Subsequently, we characterized that the transcription level of genes associated with amino acids biosynthesis, carbohydrate transport and metabolism, multidrug resistance, and DNA repair proteins significantly upregulated in ΔAcrR1 using transcriptome analysis and quantitative reverse transcription-PCR assays. Additionally, AcrR1 could repress the transcription of the nisin post-translational modification gene, nisC, leading to a 16.3% increase in nisin yield after AcrR1 deletion. Our results not only refined the knowledge of the regulatory mechanism of TetR/AcrR family regulator in L. lactis, but presented a potential strategy to enhance industrial production of nisin.