Synergistic antimicrobial interactions of nisin A with biopolymers and solubilising agents for oral drug delivery.

In order to develop bacteriocins, like the lantibiotic nisin A, into effective alternatives to existing antibiotics, their biophysical and physicochemical properties must first be assessed, from solubility, to susceptibility and absorption. It has been well established that formulation strategies at early drug development stages can be crucial for successful outcomes during preclinical and clinical phases of development, particularly for molecules with challenging physicochemical properties. This work elucidates the physicochemical challenges of nisin A in terms of its susceptibility to digestive enzymes like pepsin, pancreatin and proteinase K and its poor solubility at physiological pHs. Low solution concentrations, below the minimum inhibitory concentration against Staphylococcus aureus, were obtained in phosphate buffered saline (PBS, pH 7.4) and in fasted state simulated intestinal fluid (FaSSIF, pH 6.5), while higher solubilities at more acidic pH's such as in a KCl/HCl buffer (pH 2) and in fasted state simulated gastric fluid (FaSSGF, pH 1.6) are observed. Tween® 80 (0.01% v/v) significantly increased the solution concentration of nisin A in PBS (pH 7.4, 24 hr). Pancreatin doubled nisin A's solution concentration at pH 7.4 (PBS) but reduced its' inhibitory activity to âˆ¼ 20%, and pepsin almost completely degraded nisin (after 24 hr), but retained activity at biologically relevant exposure times (∼15 min). Harnessing synergism between nisin A and either glycol chitosan or ε-poly lysine, combined with the solubilizing effect of Tween®, increased the antimicrobial activity of nisin A six fold in an in vitro oral administration model.

Management of oral biofilms by nisin delivery in adhesive microdevices.

Numerous beneficial microbes thrive in the oral cavity where they form biofilms on dental and mucosal surfaces to get access to nutrients, and to avoid being carried away with the saliva. However, biofilm formation is also a virulence factor as it also protects pathogenic bacteria, providing them with an environment for proliferation causing oral infections. Oral hygiene relies on mechanical removal of biofilms. Some oral care products also contain antimicrobials, but effective eradication of biofilms with antimicrobials requires both a high concentration and long exposure time. In the present communication, we investigate the potential of using miniaturized drug delivery devices, known as microcontainers (MCs), to deliver the antimicrobial peptide, nisin to an oral multi-species biofilm. MCs are loaded with nisin and X-ray micro-computed tomography reveals a full release of nisin through a chitosan lid within 15 min. Chitosan-coated MCs display substantial bioadhesion to the buccal mucosa compared to non-coated MCs (68.6 ± 14.3% vs 33.8 ± 5.2%). Confocal monitoring of multi-species biofilms reveals antibacterial effects of nisin-loaded chitosan-coated MCs with a faster onset (after 3 h) compared to solution-based delivery (after 9 h). Our study shows the potential of using MCs for treatment of multi-species oral biofilms and is encouraging for further design of drug delivery devices to treat oral diseases.

Effects of sub-lethal doses of nisin on the virulence of Salmonella enterica in Galleria mellonella larvae.

Salmonella enterica is a pathogen that induces self-limiting gastroenteritis and is of worldwide concern. Nisin, an antimicrobial peptide, has emerged as an alternative for the control of microbial growth but its effect on the virulence of pathogenic bacteria is not yet well-explored. This work aimed to evaluate the virulence of S. enterica in the presence of sub-inhibitory nisin using the experimental model Galleria mellonella. Sub-inhibitory concentrations of nisin of 11.72 and 46.88 µM did not affect the cellular viability of S. enterica but promoted changes in gene expression within 1 h of treatment, with increases of up to 3-fold of pagC, 1.8-fold of invA and 2.3-fold of invF. Larvae of G. mellonella inoculated with S. enterica combined with nisin at 46.88 µM presented mortality, and TL50 noticeably increased to 50% and 80% at 24 and 48 h post-infection, respectively. Defence responses, such as melanisation, nodulation, pseudopodia, immune response, and expression of defence proteins of the larvae G. mellonella were enhanced when the treatments with S. enterica were combined with 11.72 or 46.88 µM nisin. These results show an increase in virulence of S. enterica by sub-MIC concentration of nisin that needs to be explored.

Augmenting the Pressure-Based Pasteurization of Listeria monocytogenes by Synergism with Nisin and Mild Heat.

The current study investigated Listeria monocytogenes inactivation using mild heat with elevated hydrostatic pressure and nisin under buffered condition. A four-strain pathogen mixture was exposed to 0 (control) and up to 9 min of (1) 4 °C elevated pressure; (2) 4 °C elevated pressure and nisin; (3) 4 °C nisin; (4) heat at 40 °C; (5) 40 °C elevated pressure; (6) 40 °C elevated pressure and nisin; and (7) 40 °C nisin. Elevated hydrostatic pressure at 400 MPa (Hub880 Explorer, Pressure BioScience Inc., Easton, MA, USA) and nisin concentration of 5000 IU/mL were used in the trials. Analyses of variance were conducted, followed by Dunnett's- and Tukey-adjusted means separations. Under conditions of these experiments, nisin augmented (p < 0.05) decontamination efficacy of 40 °C heat and elevated hydrostatic pressure treatments, particularly at treatment interval of 3 min. This synergism with nisin faded away (p ≥ 0.05) as the treatment time for thermal, high-pressure, and thermal-assisted pressure processing increased. The results of our study, thus, exhibit that practitioners and stakeholders of pressure-based technologies could benefit from synergism of mild heat and nisin for short-term, high-pressure pasteurization treatments to achieve microbial safety and economic feasibility comparable to traditional heat-treated products.

Augmented therapeutic efficacy of 5-fluorouracil in conjunction with lantibiotic nisin against skin cancer.

Chemotherapy, a gold standard for treating most of the cancers, involves drastic side-effects and multidrug resistance. An attractive alternative is development of combination therapy employing antimicrobial peptides with chemotherapeutic drugs. In vivo studies: Anti-cancer therapeutic efficacy of 5-fluororuacil (5-FU) in conjunction with nisin (50 mg/kg body weight) was evaluated against murine skin cancer, in terms of tumor biostatistics, histopathology, electron microscopy, infrared spectroscopy and transcriptional studies. In vitro studies: Dose and time dependent cytotoxicity of agents were assessed against A431 cell line using MTT assay, LDH assay and acridine orange/ethidium bromide dual staining. Significant percentage decrease(s) in mean tumor volume and tumor burden were observed in nisin+ 5-FU combination treated groups as compared to alone treated groups. Histoarchitecture of treated skins demonstrated restoration towards normal skin tissue (being highest in the combination group). Modulation of apoptotic, angiogenic and proliferative genes were observed in treated groups. IC50 of combination was found to be 2 µg/ml as compared to nisin alone (32µg/ml) and 5-FU alone (16µg/ml) with combination index of 0.188. Dual staining showed that rate of induction of apoptosis was higher in the combination group as compared to single agents. Nisin and 5-FU in combination were found to be synergistic both in vivo and in vitro.

Diabetic foot infections: Application of a nisin-biogel to complement the activity of conventional antibiotics and antiseptics against Staphylococcus aureus biofilms.

BACKGROUND: Diabetic foot infections (DFIs) are a frequent complication of Diabetes mellitus and a major cause of nontraumatic limb amputations. The Gram-positive bacterium Staphylococcus aureus, known for its resilient biofilms and antibiotic resistant profile, is the most frequent DFI pathogen. It is urgent to develop innovative treatments for these infections, being the antimicrobial peptide (AMP) nisin a potential candidate. We have previously proposed the use of a guar gum biogel as a delivery system for nisin. Here, we evaluated the potential of the nisin-biogel to enhance the efficacy of conventional antibiotics and antiseptics against DFIs S. aureus clinical isolates. METHODS: A collection of 23 S. aureus strains isolated from DFI patients, including multidrug- and methicillin-resistant strains, was used. The antimicrobial activity of the nisin-biogel was tested alone and in different combinations with the antiseptic chlorhexidine and the antibiotics clindamycin, gentamicin and vancomycin. Isolates' in vitro susceptibility to the different protocols was assessed using broth microdilution methods in order to determine their ability to inhibit and/or eradicate established S. aureus biofilms. Antimicrobials were added to the 96-well plates every 8 h to simulate a typical DFI treatment protocol. Statistical analysis was conducted using RCBD ANOVA in SPSS. RESULTS: The nisin-biogel showed a high antibacterial activity against biofilms formed by DFI S. aureus. The combined protocol using nisin-biogel and chlorhexidine presented the highest efficacy in biofilm formation inhibition, significantly higher (p<0.05) than the ones presented by the antibiotics-based protocols tested. Regarding biofilm eradication, there were no significant differences (p>0.05) between the activity of the combination nisin-biogel plus chlorhexidine and the conventional antibiotic-based protocols. CONCLUSIONS: Results provide a valuable contribution for the development of complementary strategies to conventional antibiotics protocols. A combined protocol including chlorhexidine and nisin-biogel could be potentially applied in medical centres, contributing for the reduction of antibiotic administration, selection pressure on DFI pathogens and resistance strains dissemination.

Encapsulação de antimicrobianos naturais em nanolipossomas para inibição de bactérias causadoras de surtos alimentares / Encapsulation of natural antimicrobials in nanoliposomas for the inhibition of bacterials causing foodborne disease

Avaliou-se ação bactericida e caracterização físico-química de duas diferentes formulações de nanolipossomas preparados utilizando técnica de hidratação do filme lipídico. Nanolipossomas contendo carvacrol apresentaram diâmetro médio de 270,83 nm, PDI de 0,20 e potencial zeta de + 8,64 mV. Nanolipossomas contendo carvacrol e nisina apresentaram diâmetro médio de 205,25 nm, PDI de 0,31 e potencial zeta de - 22,48 mV. Para ambas formulações a concentração bactericida mínima foi de 3,53 mg/mL para Salmonella e 5,3 mg/mL para Escherichia coli e Staphylococcus aureus. Não ocorreu efeito sinérgico entre carvacrol e nisina, porém os nanolipossomas contendo carvacrol apresentaram ação bactericida contra os microrganismos testados, demonstrando potencial para sua utilização no desenvolvimento de um sanitizante para uso em indústrias de alimentos.

In vitro immunomodulatory effect of nisin on porcine leucocytes.

Nisin, a lantibiotic bacteriocin, has been used for years as a natural food preservative. In addition to its antimicrobial activity, nisin also shows immunomodulatory properties, and the nisin-producing Lactococcus lactis strain has been successfully tested as a probiotic in weaned piglets. However, the impact of nisin on porcine immune cells has not yet been explored. The objective of the present study was to examine the in vitro immunomodulatory effect of nisin on porcine peripheral blood leucocytes. The whole heparinized blood samples or freshly isolated peripheral blood mononuclear cells (PBMCs) were incubated with different nisin concentrations (0, 1.56, 3.125, 6.25, 12.5, 25 or 50 µg/ml) for 1, 24, 48 or 72 hr. Escherichia coli bacteria were used to stimulate blood phagocytes, while concanavalin A and lipopolysaccharide from E. coli were used as mitogens. Control cells remained unstimulated. MTT colorimetric assay was used to evaluate PBMCs viability and mitogenic response. Phagocyte activity and T-cell proliferation were measured by flow cytometry. Flow cytometer was also used for immunophenotyping of T cells. Cytokine levels in the culture media were determined using commercial immunoassay (ELISA) kits. The highest concentration of nisin exhibited proliferative activity (p Ë‚ 0.05), stimulated interleukin-1 beta (IL-1ß) and interleukin-6 (IL-6) production (both at p Ë‚ 0.001), and increased the percentage of CD4+ CD8+ T cells (p Ë‚ 0.001) among unstimulated leucocytes. After cell stimulation, however, the highest nisin concentration showed antiproliferative activity (p Ë‚ 0.05), decreased phagocytic functions (p Ë‚ 0.05) and inhibited the synthesis of IL-6 (time- and concentration-dependent effect). As a typical bacterial product, nisin had a stronger impact on innate immune cells, and its effect on T cells was likely a consequence of the modulation of the activity of antigen-presenting cells. Nisin may be a good candidate as an immunomodulator in pig breeding.

Precise management of chronic wound by nisin with antibacterial selectivity.

Traditional broad-spectrum antibacterial agents are limited by high toxicity and the destruction to the bacterial flora balance in the wound site. Herein, we propose an opinion that one or several especial antibacterial peptides are adopted to kill the target bacteria in order to precisely manage the bacteria infected chronic wound under the premise of biobalance, and specially employ nisin to treat S. aureus infected chronic wound as model with positive effects. The results showed that without cytotoxicity to the normal cells, only 25 ppm nisin could contrapuntally kill S. aureus and have little inhibitory to other bacteria. Mechanism of antibacterial selectivity indicated the superior biomolecular interaction between nisin and S. aureus compared with E. coli and normal cells. Furthermore, nisin significantly accelerated the healing process of S. aureus-infected rabbit full thickness burn wound, but had no effect on the E. coli-infected wound as a comparison. Therefore, it has been demonstrated that special peptides with antibacterial selectivity can be adopted to precise management for bacteria infected chronic wound under good biobalance.

Potential of two delivery systems for nisin topical application to dental plaque biofilms in dogs.

BACKGROUND: Periodontal disease (PD) is caused by the development of a microbial biofilm (dental plaque) in the periodontium, affecting approximately 80% of dogs. Several bacterial species present in the canine oral cavity can be implicated in the development of this disease, including Enterococcus spp. To decrease antibiotic administration, a possible control strategy for dog's enterococcal PD may involve the use of the antimicrobial peptide (AMP) nisin. Nisin's inhibitory activity was evaluated against a collection of previously characterized enterococci obtained from the oral cavity of dogs with PD (n = 20), as well as the potential of a guar-gum gel and a veterinary toothpaste as topical delivery systems for this AMP. The Minimum Inhibitory (MIC) and Bactericidal Concentrations (MBC) and the Minimum Biofilm Eradication (MBEC) and Inhibitory Concentrations (MBIC) were determined for nisin and for the supplemented guar-gum gel. For the supplemented veterinary toothpaste an agar-well diffusion assay was used to evaluate its inhibitory potential. RESULTS: Nisin was effective against all isolates. Independently of being or not incorporated in the guar-gum gel, its inhibitory activity on biofilms was higher, with MBIC (12.46 ± 5.16 and 13.60 ± 4.31 µg/mL, respectively) and MBEC values (21.87 ± 11.33 and 42.34 ± 16.61 µg/mL) being lower than MIC (24.61 ± 4.64 and 14.90 ± 4.10 µg/mL) and MBC (63.09 ± 13.22 and 66.63 ± 19.55 µg/mL) values. The supplemented toothpaste was also effective, showing inhibitory activity against 95% of the isolates. CONCLUSIONS: The inhibitory ability of nisin when incorporated in the two delivery systems was maintained or increased, demonstrating the potential of these supplemented vehicles to be applied to PD control in dogs.

Cardioprotective Effects of Curcumin-Nisin Based Poly Lactic Acid Nanoparticle on Myocardial Infarction in Guinea Pigs.

Myocardial infarction (MI) is the most prevalent cause of cardiovascular death. A possible way of preventing MI maybe by dietary supplements. The present study was thus designed to ascertain the cardio-protective effect of a formulated curcumin and nisin based poly lactic acid nanoparticle (CurNisNp) on isoproterenol (ISO) induced MI in guinea pigs. Animals were pretreated for 7 days as follows; Groups A and B animals were given 0.5 mL/kg of normal saline, group C metoprolol (2 mg/kg), groups D and E CurNisNp 10 and 21 mg/kg respectively (n = 5). MI was induced on the 7th day in groups B-E animals. On the 9th day electrocardiogram (ECG) was recorded, blood samples and tissue biopsies were collected for analyses. Toxicity studies on CurNisNp were carried out. MI induction caused atrial fibrillation which was prevented by pretreatment of metoprolol or CurNisNp. MI induction was also associated with increased expressions of cardiac troponin I (CTnI) and kidney injury molecule-1 (KIM-1) which were significantly reduced in guinea pig's pretreated with metoprolol or CurNisNp (P < 0.05). The LC50 of CurNisNp was 3258.2 µg/mL. This study demonstrated that the formulated curcumin-nisin based nanoparticle confers a significant level of cardio-protection in the guinea pig and is nontoxic.

Nisin, a potent bacteriocin and anti-bacterial peptide, attenuates expression of metastatic genes in colorectal cancer cell lines.

Colorectal cancer is the third most common cause of cancer-related death in the world which genetic and environmental agents are responsible for cancer. When cells detach from the tumor and invade surrounding tissues, the tumor is malignant and may form secondary tumors at other locations in a process called metastasis. Probiotics are the largest group of inhabitation bacteria in the colon. Gut microbiota has a central role in prevented the risk colon cancer. Probiotics are beneficial microorganisms, like Lactic acid bacteria and Lactobacilli bacteria which are using in the dairy industry. Probiotics nisin are having the most important category of safe usage. In this study LS180, SW48, HT29 and Caco2 was cultured and treated with different dose of nisin. Cell proliferation was assayed with MTT. The expression of CEA, CEAM6 and MMP2F genes was analyzed with Real-time PCR. Protein expression of CEA was evacuated with ELISA. Our result was shown that the 40-50 IU/mL nisin could suppress proliferation of LS180. Cell proliferation of SW48, HT29, Caco2 cells was decreased in 250-350 IU/mL concentration of nisin. The gene expression of CEA, CEAM6, MMP2F was significantly down-regulated with nisin treatment (p < 0.001, p < 0.01). Also, after cells treated with nisin, CEA protein expression was down regulated (p < 0.01). In conclusion, nisin could suppressed metastatic process via down-regulation of CEA, CEAM6, MMP2F, MMP9F genes. We suggested the new treatment strategies beyond Probiotics, which play a role in the prevention local tumor invasion, metastasis and recurrence.

Reverse micelles as nanocarriers of nisin against foodborne pathogens.

Reverse micelles (RMs) as nanocarriers of nisin were optimized for the highest water and bacteriocin content. RMs formulated with either refined olive oil or sunflower oil, distilled monoglycerides, ethanol, and water were effectively designed. Structural characterization of the RMs was assessed using Electron Paramagnetic Resonance Spectroscopy and Small Angle X-ray Scattering in the presence and absence of nisin. No conformational changes occurred in the presence of nisin for the nanocarriers. To assess efficacy of the loaded systems, their antimicrobial activity against Staphylococcus aureus and Listeria monocytogenes was tested in lettuce leaves and minced meat, respectively. Antimicrobial activity was evident in both cases. Interestingly, a synergistic antimicrobial effect was observed in lettuce leaves and to a lesser extent in minced meat between nisin and some of the nanocarriers' constituents (probably ethanol). Our findings suggest complex interactions that take place when RMs are applied in different food matrices.

Nisin-induced expression of recombinant T cell epitopes of major Japanese cedar pollen allergens in Lactococcus lactis.

Japanese cedar pollinosis is a seasonal allergic disease caused by two major pollen allergens: Cry j 1 and Cry j 2 antigens. To develop an oral vaccine to treat pollinosis, we constructed recombinant Lactococcus lactis harboring the gene encoding fused T cell epitopes from the Cry j 1 and Cry j 2 antigens. The recombinant T cell epitope peptide was designed to contain the fused cholera toxin B subunit as an adjuvant and a FLAG tag at the C-terminus. An expression plasmid was constructed by inserting the T cell epitope peptide gene into the multiple cloning sites of plasmid pNZ8148, an Escherichia coli-L. lactis shuttle vector. The constructed plasmid was transformed into L. lactis NZ9000 for expression induced by nisin, an antibacterial peptide from L. lactis. The expression of the epitope peptide was induced with 10-40 ng/mL nisin, and the expressed T cell epitope peptide was detected by western blot analysis using an anti-FLAG antibody and an antibody against the T cell epitopes. The concentration of the epitope peptide was estimated to be ~ 22 mg/L of culture in the presence of 40 ng/mL nisin, although it varied depending on the nisin concentration, the culture time, and the bacterial concentration when nisin was added. The expression of the recombinant epitope peptide in L. lactis, an organism generally recognized as safe, as demonstrated in this study, may contribute to the development of an oral vaccine for the treatment of pollinosis.

The apoptotic impact of nisin as a potent bacteriocin on the colon cancer cells.

Nisin is a polycyclic peptide containing 34 amino acids produced by Lactococcus lactis during fermentation. Recently, researchers considered nisin as an anticancer peptide. Herein, the authors aim to evaluate the nisin effects on the apoptosis stimulation in the colon cancer cell line. The SW480 cells were exposed to discrepant concentrations of nisin and the cell viability as well as the expression of bcl-2 and bax genes and proteins were surveyed by the MTT assay, Real-Time PCR and western blotting method, respectively. Furthermore, the Ethidium bromide/Acridine orange staining was performed to visualize apoptotic cells. 4000, 3000, 2500 and 2000 µg/ml of nisin led to significant anti-proliferative impact and augmentation apoptotic index (bax/bcl-2 ratio) both at mRNA and protein levels (p < 0.05). Furthermore, the apoptotic impacts were demonstrated after Ethidium bromide/Acridine orange (EB/AO) staining to have a dose dependent manner. Our outcome suggested that nisin could induce apoptosis via intrinsic pathways and lead to cancerous cell death.

Core-shell PLA-PVA porous microparticles as carriers for bacteriocin nisin.

This work is focused on preparation of novel porous type of core-shell-structured microparticles based on polylactide (shell) and poly(vinyl alcohol) cross-linked with glutaric acid (GA) (core) prepared by water-in-oil-in-water solvent evaporation technique. The microparticle systems were used as delivery systems for immobilisation of model antibacterial agent - nisin. The effect of cross-linking and the initial amount of nisin on their morphology was investigated using scanning electron microscopy, BET analysis, zeta potential measurement and Fourier transform infra-red spectroscopy. Encapsulation efficiency and release profile of nisin from the microparticles were studied by high performance liquid chromatography. Antibacterial activity of the prepared systems was tested by dilution and spread plate technique. Results showed the microparticles in the size range of 9-16 µm in diameter with spherical multi-hollow core-shell structure. The presence of cross-linking agent GA influences the release profile of the peptide and has synergistic effect on Listeria monocytogenes growth reduction.

High-purity Nisin Alone or in Combination with Sodium Hypochlorite Is Effective against Planktonic and Biofilm Populations of Enterococcus faecalis.

INTRODUCTION: Nisin, a broad-spectrum bacteriocin, has recently been highlighted for its biomedical applications. To date, no studies have examined the antimicrobial and antibiofilm properties of high-purity (>95%) nisin (nisin ZP) on Enterococcus faecalis and biofilms formed by this species. We hypothesize that nisin can inhibit E. faecalis and reduce biofilm biomass, and combinations of nisin and sodium hypochlorite (NaOCl) will enhance the antibiofilm properties against E. faecalis biofilms. METHODS: Using broth cultures, disc diffusion assays, and biofilm assays, we examined the effects of nisin on various E. faecalis growth parameters and biofilm properties (biovolume, thickness, and roughness). Confocal microscopy was used in conjunction with Imaris and Comstat2 software (Kongens Lyngby, Copenhagen, Denmark) to measure and analyze the biofilm properties. RESULTS: Nisin significantly decreased the growth of planktonic E. faecalis dose dependently. The minimum inhibitory concentrations against E. faecalis strains OG-1 and ATCC 29212 were 15 and 50 µg/mL, and the minimum bactericidal concentrations were 150 and 200 µg/mL, respectively. A reduction in biofilm biovolume and thickness was observed for biofilms treated with nisin at ≥10 µg/mL for 10 minutes. In addition, the combination of nisin with low doses of NaOCl enhanced the antibiofilm properties of both antimicrobial agents. CONCLUSIONS: Nisin alone or in combination with low concentrations of NaOCl reduces the planktonic growth of E. faecalis and disrupts E. faecalis biofilm structure. Our results suggest that nisin has potential as an adjunctive endodontic therapeutic agent and as an alternative to conventional NaOCl irrigation.

Efficacy of vaccination and nisin Z treatments to eliminate intramammary Staphylococcus aureus infection in lactating cows.

This study evaluated the effect of a Staphylococcus aureus bacterin and nisin on bovine subclinical mastitis. A total of 75 Holstein subclinically mastitic cows were randomly allocated to three groups with 25 cows per group. In group I, an intramammary infusion of nisin Z at a dose of 2.50×106 IU was carried out once daily for three days, and an autogenous S. aureus bacterin was inoculated into the supramammary lymph node one week before and one week after nisin treatment. In group II, nisin was administered in the same way as in group I, but no bacterin was inoculated. Group III received no treatment and served as a control. Milk was aseptically sampled from the affected quarters before and 2, 4, and 6 weeks after treatment, for bacteriological examination and analyses of N-acetyl-ß-D-glucosaminidase (NAGase) activity, somatic cell count (SCC), and milk protein and fat contents. Results indicated that, compared to the nisin-treated group, nisin-bacterin treatment significantly reduced intramammary S. aureus infections, reduced the number of quarters with milk SCCs of more than 5×105 cells/ml, and increased the protein and fat contents of the milk. Therefore, nisin-bacterin therapy is suggested when subclinical mastitis occurs in lactating cows.

Long-term antimicrobial effect of nisin released from electrospun triaxial fiber membranes.

Electrospun membranes encapsulating nisin in the core of multi-layer coaxial fibers, with a hydrophobic PCL intermediate layer and a hygroscopic cellulose acetate sheath, have been demonstrated to provide long-term antimicrobial activity combined with a hygroscopic outer layer. Antimicrobial performance has been evaluated using modified versions of the antimicrobial textile test AATCC 100 and AATCC 147 against Staphylococcus aureus. The AATCC 147 tests indicate that antimicrobial activity persists up to 7days. The quantitative analysis from the AATCC 100 test indicates that tri-layer coaxial ("triaxial") electrospun fiber membranes provide >99.99% bacteria kill (4logkill) for up to five days. This indicates that the nisin-incorporated triaxial fibers have excellent biocidal activities for up to 5days and then provide biostatic activity for 2 or more days. Compared with other types of electrospun membranes, such as core-sheath coaxial ("coaxial") and single homogenous fibers, triaxial fiber membranes provided more robust and more sustained antimicrobial activity. Single fibers with nisin showed relatively weak activity and only for one day. Coaxial fiber membranes exhibited antimicrobial activity for a long period, but their biocidal activity was much weaker than that of triaxial fiber membranes, and only exhibited >99% bacteria kill (2logkill) after 1day of exposure. STATEMENT OF SIGNIFICANCE: The increase in drug resistant pathogens has driven the need for alternative treatments that are effective against resistant bacteria and do not contribute to drug resistance. Nisin is an excellent model bacteriocin for antimicrobials because of its size and mode of action, and has been extensively used as FDA-approved food preservatives without any problematic resistance growth in bacteria during past decades. Nisin-containing fibers have been previously reported using conventional electrospinning but sustained antimicrobial effect has not been obtained. Here, we report the encapsulation of nisin into a multi-layered nanofiber construct using triaxial electrospinning in order to obtain a long-term antimicrobial activity. This will be highly beneficial in many applications, such as protective textiles, food packaging and cancer therapy.

Short communication: Inhibitory activities of the lantibiotic nisin combined with phenolic compounds against Staphylococcus aureus and Listeria monocytogenes in cow milk.

We aimed to investigate the antibacterial activities of carvacrol, thymol, eugenol, cinnamaldehyde, and lantibiotic nisin against standard bacterial strains of the milk pathogens Staphylococcus aureus ATCC 25923 and Listeria monocytogenes ATCC 15313 in cow milk. The minimum inhibitory concentrations (MIC) of these substances were recorded. The synergistic effects were also assessed in culture medium (time kill curve) and in a food model (cow milk) during the storage period (4 °C for 6 d) after inoculation with S. aureus and L. monocytogenes individually by combining nisin and the phenolic compounds at proportions of 1/4 + 1/4 the MIC (determined in a previous in vitro assay) in the culture medium and 1/4 + 1/4 of MIC in the food model. Inhibitory activities of nisin and the tested compounds, as well as synergism in the combinations, were found against both bacteria assayed. Bacteriostatic effects were found with all combinations and a significant difference in L. monocytogenes reduction was found compared with the control assays. Thus, the antibacterial activity of nisin combined with phenolic compounds was confirmed against these pathogenic bacteria that are important in the milk industry, or more broadly in food science, with potential applications for milk preservation.