Acute Toxicity Evaluation of Phosphatidylcholine Nanoliposomes Containing Nisin in Caenorhabditis elegans.

Liposomes are among the most studied nanostructures. They are effective carriers of active substances both in the clinical field, such as delivering genes and drugs, and in the food industry, such as promoting the controlled release of bioactive substances, including food preservatives. However, toxicological screenings must be performed to ensure the safety of nanoformulations. In this study, the nematode Caenorhabditis elegans was used as an alternative model to investigate the potential in vivo toxicity of nanoliposomes encapsulating the antimicrobial peptide nisin. The effects of liposomes containing nisin, control liposomes, and free nisin were evaluated through the survival rate, lethal dose (LD50), nematode development rate, and oxidative stress status by performing mutant strain, TBARS, and ROS analyses. Due to its low toxicity, it was not possible to experimentally determine the LD50 of liposomes. The survival rates of control liposomes and nisin-loaded liposomes were 94.3 and 73.6%, respectively. The LD50 of free nisin was calculated as 0.239 mg mL-1. Free nisin at a concentration of 0.2 mg mL-1 significantly affected the development of C. elegans, which was 25% smaller than the control and liposome-treated samples. A significant increase in ROS levels was observed after exposure to the highest concentrations of liposomes and free nisin, coinciding with a significant increase in catalase levels. The treatments induced lipid peroxidation as evaluated by TBARS assay. Liposome encapsulation reduces the deleterious effect on C. elegans and can be considered a nontoxic delivery system for nisin.

Antibacterial Peptides Resistance in Staphylococcus aureus: Various Mechanisms and the Association with Pathogenicity.

Staphylococcus aureus is a bacterium that mainly colonizes the nasal cavity and skin. To colonize the host, it is necessary for S. aureus to resist many antibacterial factors derived from human and commensal bacteria. Among them are the bacteria-derived antimicrobial peptides (AMPs) called bacteriocins. It was reported that some two-component systems (TCSs), which are signal transduction systems specific to bacteria, are involved in the resistance to several bacteriocins in S. aureus. However, the TCS-mediated resistance is limited to relatively low concentrations of bacteriocins, while high concentrations of bacteriocins still exhibit antibacterial activity against S. aureus. To determine whether we could obtain highly bacteriocin-resistant mutants, we tried to isolate highly nisin A-resistant mutants by exposing the cells to sub-minimum inhibitory concentrations (MICs) of nisin A. Nisin A is one of the bacteriocins produced by Lactococcus lactis and is utilized as a food preservative worldwide. Finally, we obtained highly nisin A-resistant mutants with mutations in one TCS, BraRS, and in PmtR, which is involved in the expression of pmtABCD. Notably, some highly resistant strains also showed increased pathogenicity. Based on our findings, this review provides up-to-date information on the role of TCSs in the susceptibility to antibacterial peptides. Additionally, the mechanism for high antimicrobial peptides resistance and its association with pathogenicity in S. aureus is elucidated.

A Synergistic New Approach Toward Enhanced Antibacterial Efficacy via Antimicrobial Peptide Immobilization on a Nitric Oxide-Releasing Surface.

Despite technological advancement, nosocomial infections are prevalent due to the rise of antibiotic resistance. A combinatorial approach with multimechanistic antibacterial activity is desired for an effective antibacterial medical device surface strategy. In this study, an antimicrobial peptide, nisin, is immobilized onto biomimetic nitric oxide (NO)-releasing medical-grade silicone rubber (SR) via mussel-inspired polydopamine (PDA) as a bonding agent to reduce the risk of infection. Immobilization of nisin on NO-releasing SR (SR-SNAP-Nisin) and the surface characteristics were characterized by Fourier transform infrared spectroscopy and scanning electron microscopy with energy-dispersive X-ray spectroscopy and contact angle measurements. The NO release profile (7 days) and diffusion of SNAP from SR-SNAP-Nisin were quantified using chemiluminescence-based nitric oxide analyzers and UV-vis spectroscopy, respectively. Nisin quantification showed a greater affinity of nisin immobilization toward SNAP-doped SR. Matrix-assisted laser desorption/ionization mass spectrometry analysis on surface nisin leaching for 120 h under physiological conditions demonstrated the stability of nisin immobilization on PDA coatings. SR-SNAP-Nisin shows versatile in vitro anti-infection efficacy against Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus in the planktonic and adhered states. Furthermore, the combination of NO and nisin has a superior ability to impair biofilm formation on polymer surfaces. SR-SNAP-Nisin leachates did not elicit cytotoxicity toward mouse fibroblast cells and human umbilical vein endothelial cells, indicating the biocompatibility of the material in vitro. The preventative and therapeutic potential of SR-SNAP-Nisin dictated by two bioactive agents may offer a promising antibacterial surface strategy.

Antiviral effects of nisin, lysozyme, lactoferrin and their mixtures against bovine viral diarrhoea virus.

BACKGROUND: Bovine viral diarrhoea virus (BVDV), an enveloped, single-stranded, positive-sense RNA virus from the Flaviviridae family, is a globally distributed bovine pathogen. BVDV infection in cattle, despite having a wide range of clinical manifestations, is invariably responsible for significant economic losses. To counteract these losses, various schemes to control and eradicate BVDV have been implemented, although safe drugs effectively inhibiting the replication of the virus are still lacking. The purpose of this study was to characterize the antiviral effect of naturally occurring proteins and peptide, such as bovine lactoferrin, chicken egg lysozyme, and nisin from Lactococcus lactis, used both individually and in combination, against the cytopathic NADL strain of BVDV in vitro. After determining the cytotoxicity level of each protein or peptide to MDBK cells, its antiviral effects were evaluated using virucidal, cytopathic effect inhibition and viral yield reduction assays. In addition, the influence of the tested compounds on the intracellular viral RNA level was determined. RESULTS: The highest efficacy among the single treatments was achieved by bovine lactoferrin, which was effective both at the early stages of viral infection and during its entire course, although the effect weakened over time. Nisin and lysozyme were effective at later stages of infection, and the intensity of their effect did not diminish with time. Nisin+lactoferrin and lysozyme+lactoferrin combinations demonstrated stronger antiviral effects than did the single substances. The nisin+lactoferrin mixture present during the whole period of infection produced the strongest anti-BVDV effect in our entire research on both the extracellular viral titre (titre reduction up to 2.875 log ≈ 99.9%) and the intracellular viral RNA level (reduction up to 89%), and this effect intensified over the incubation time. CONCLUSIONS: The tested substances could be applied in bovine viral diarrhoea prevention and therapy, especially when used in combination.

Application of iron oxide nanoparticles @ polydopamine-nisin composites to the inactivation of Alicyclobacillus acidoterrestris in apple juice.

Iron oxide nanoparticles (IONPs) modified with polydopamine were covalently immobilized with nisin to inhibit the growth of Alicyclobacillus acidoterrestris in apple juice. The minimum bactericidal concentration (MBC) of IONPs @ pDA-nisin composites against Alicyclobacillus acidoterrestris cells and spores in three kinds of apple juice were 1.25 mg/mL and 2.5 mg/mL, respectively. The concentration of cells and spores decreased from 106 to 10 CFU/mL in apple juice after pretreatment with the composites at the concentration of 20 mg/mL for 10 min. Meanwhile, the investigation of the content of polyphenols, organic acids, volatile compounds and sugars indicated that there was no significant influence of the composites on the apple juices. The results of cytotoxicity (HepG2, Caco 2, SH-SY5Y, and BV2 cells) and acute toxicity test demonstrated the biosecurity and nontoxicity of the composites, which suggested that it was promising to apply the proposed composites to apple juice industry for the control of Alicyclobacillus acidoterrestris pollution.

In Vitro Characterization and Evaluation of the Cytotoxicity Effects of Nisin and Nisin-Loaded PLA-PEG-PLA Nanoparticles on Gastrointestinal (AGS and KYSE-30), Hepatic (HepG2) and Blood (K562) Cancer Cell Lines.

The aim of this study was an in vitro evaluation and comparison of the cytotoxic effects of free nisin and nisin-loaded PLA-PEG-PLA nanoparticles on gastrointestinal (AGS and KYSE-30), hepatic (HepG2), and blood (K562) cancer cell lines. To create this novel anti-cancer drug delivery system, the nanoparticles were synthesized and then loaded with nisin. Subsequently, their biocompatibility, ability to enter cells, and physicochemical properties, including formation, size, and shape, were studied using hemolysis, fluorescein isothiocyanate (FITC), Fourier transform infrared (FTIR) spectroscopy, dynamic light scattering (DLS), and scanning electron microscopy (SEM), respectively. Then, its loading efficiency and release kinetics were examined to assess the potential impact of this formulation for the nanoparticle carrier candidacy. The cytotoxicities of nisin and nisin-loaded nanoparticles were evaluated by using the MTT and Neutral Red (NR) uptake assays. Detections of the apoptotic cells were done via Ethidium Bromide (EB)/Acridine Orange (AO) staining. The FTIR spectra, SEM images, and DLS graph confirmed the formations of the nanoparticles and nisin-loaded nanoparticles with spherical, distinct, and smooth surfaces and average sizes of 100 and 200 nm, respectively. The loading efficiency of the latter nanoparticles was about 85-90%. The hemolysis test represented their non-cytotoxicities and the FITC images indicated their entrance inside the cells. An increase in the percentage of apoptotic cells was observed through EB/AO staining. These results demonstrated that nisin had a cytotoxic effect on AGS, KYSE-30, HepG2, and K562 cancer cell lines, while the cytotoxicity of nisin-loaded nanoparticles was more than that of the free nisin.

VirR-Mediated Resistance of Listeria monocytogenes against Food Antimicrobials and Cross-Protection Induced by Exposure to Organic Acid Salts.

Formulations of ready-to-eat (RTE) foods with antimicrobial compounds constitute an important safety measure against foodborne pathogens such as Listeria monocytogenes. While the efficacy of many commercially available antimicrobial compounds has been demonstrated in a variety of foods, the current understanding of the resistance mechanisms employed by L. monocytogenes to counteract these stresses is limited. In this study, we screened in-frame deletion mutants of two-component system response regulators associated with the cell envelope stress response for increased sensitivity to commercially available antimicrobial compounds (nisin, lauric arginate, ε-polylysine, and chitosan). A virR deletion mutant showed increased sensitivity to all antimicrobials and significantly greater loss of membrane integrity when exposed to nisin, lauric arginate, or ε-polylysine (P < 0.05). The VirR-regulated operon, dltABCD, was shown to be the key contributor to resistance against these antimicrobial compounds, whereas another VirR-regulated gene, mprF, displayed an antimicrobial-specific contribution to resistance. An experiment with a ß-glucuronidase (GUS) reporter fusion with the dlt promoter indicated that nisin does not specifically induce VirR-dependent upregulation of dltABCD. Lastly, prior exposure of L. monocytogenes parent strain H7858 and the ΔvirR mutant to 2% potassium lactate enhanced subsequent resistance against nisin and ε-polylysine (P < 0.05). These data demonstrate that VirRS-mediated regulation of dltABCD is the major resistance mechanism used by L. monocytogenes against cell envelope-damaging food antimicrobials. Further, the potential for cross-protection induced by other food-related stresses (e.g., organic acids) needs to be considered when applying these novel food antimicrobials as a hurdle strategy for RTE foods.

Evaluation of the immunogenicity and in vivo toxicity of the antimicrobial peptide P34.

Immunogenicity and toxicity of antimicrobial peptide P34 were evaluated in vivo. BALB/c mice were inoculated intraperitoneally with peptide P34 alone and associated with Freund's adjuvant. For acute toxicity testing, different concentrations of the peptide P34 (82.5, 165.0, 247.5 and 330.0mg/kg) were orally administered. To evaluate the sub-chronic toxicity the tested dose of 0.825 mg/kg/day of the peptide P34 or nisin were administered for 21 days. There were no hypersensitivity reactions or significant increase in antibody titer during the immunogenicity experiment or death of animals during the acute or sub-chronic toxicity tests. The LD(50) was higher than 332.3 ± 0.76 mg/kg. No significant changes in serum biochemical parameters were observed in the animals treated with the peptide P34 unlike nisin-treated group showed a significant increase in alanine transaminase levels in comparison to controls. The group treated with 0.825 mg/kg/day of nisin showed histological changes in the spleen, skin and liver. In the group treated with peptide P34 histological changes in the spleen were observed, with the presence of megakaryocytes. Few studies report the use of animal models to evaluate the in vivo toxicity of antimicrobial peptides and such investigation is an essential step to ensure it safe use in foods.

Decontamination of collagen biomatrices with combined pulsed electric field and nisin treatment.

Pulsed electric field (PEF) treatment has been proposed as a decontamination method for labile matrices used in tissue engineering applications. Through the application of PEF, a non-thermal treatment that causes bacterial inactivation through the irreversible rupture of microbial cell membranes, inactivation is achieved without loss of scaffold structure and function. However, some microorganisms are less susceptible to PEF treatment. This study shows that treatment with PEF and nisin, a food preservative bacteriocin, has a synergistic effect on the inactivation of Staphylococcus epidermidis in collagen gels. Almost complete inactivation of a 10(3) -10(4) CFU/mL S. epidermidis population was achieved when treated with a combination of PEF and 500 IU/mL nisin, with results demonstrating a 3.4 log(10) reduction, compared with 0.66 log(10) reduction with PEF alone. Nisin, at concentrations up to 3000 IU/mL, had no discernable toxicity to mammalian 3T3 cells when added to the culture medium or incorporated into the collagen gels. This combined decontamination method, involving PEF plus nisin, may provide a non-destructive process for inactivation of PEF-resistant bacteria in labile tissue engineering scaffolds.

A 90-day oral toxicity study of nisin A, an anti-microbial peptide derived from Lactococcus lactis subsp. lactis, in F344 rats.

This study was designed to evaluate and characterize any adverse effect of nisin A, when administered to both sexes of F344/DuCrlCrlj rats (10 males and 10 females in each group) at dietary levels of 0%, 0.2%, 1.0% and 5.0% for 90 days. Animals given NaCl at a dietary level of 3.712% (equivalent to the NaCl content in 5.0% nisin A diet) served as a reference material treated group. There were no deaths, and the treatment had no toxicologically significant effects on clinical signs, body weights, food consumption, ophthalmology, hematology, or gross pathology. Statistically significant increases of water consumption, urine volume, and urinary sodium and chlorine, and decreases of urinary potassium and serum sodium, along with increases of absolute and relative kidney weight, and incidences of minimal squamous cell hyperplasia of limiting ridge in the forestomach, were found in nisin A-treated groups. It was considered that these changes were related to NaCl, since they were also noted in rats given diet containing the reference substance. Thus, no toxicologically significant changes were apparent in both sexes of F344/DuCrlCrlj rats fed diet containing 0%, 0.2%, 1.0% and 5.0% nisin A for 90 days. Therefore, the no-observed-adverse-effect level (NOAEL) for nisin A was concluded to be a dietary level of 5.0% (2996 mg/kg/day for males and 3187 mg/kg/day for females).

Evaluation of the in vitro cytotoxicity of the antimicrobial peptide P34.

The in vitro cytotoxicity of the antimicrobial peptide P34 was evaluated in different eukaryotic cells. The food-grade bacteriocin nisin was also analysed for comparison. Vero cells were treated with different concentrations (0.02-2.5 microg x ml(-1)) of antimicrobial peptide P34 and nisin. Cell viability and plasma membrane integrity were checked by MTT [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide], NRU (Neutral Red dye uptake) and LDH (lactate dehydrogenase) assays. The EC50 values of the peptide P34 in MTT and NRU assays were 0.60 and 1.25 microg x ml(-1) respectively, while values of nisin found were 0.50 and 1.04 microg x ml(-1). In the LDH assay, the EC50 values were 0.65 and 0.62 microg x ml(-1) for P34 and nisin, respectively. The peptide P34 revealed similar haemolytic activity on human erythrocytes (5.8%) when compared with nisin (4.9%). The effects on viability, motility and acrosomal exocytosis of human sperm were also evaluated. Nisin and P34 showed similar effects on sperm parameters. The evaluation of cytotoxicity of antimicrobial peptides is a critical step to guarantee their safe use.

Assessment of cervicovaginal cytokine levels following exposure to microbicide Nisin gel in rabbits.

Topical microbicides is an emerging female controlled strategy for preventing the acquisition and transmission of STIs/HIV infections. Since they are intended for repeated vaginal and/or rectal use it is essential to validate their safety. Nisin, a naturally occurring contraceptive antimicrobial peptide (AMP) is currently the focus of clinical trials. The present in vitro vaginal tissue explants culture studies revealed that Nisin did not effect vaginal cell viability analyzed at 15, 30, 45 and 60min following treatment with different concentrations of Nisin gel prepared in 1% polycarbophil gel (30.3, 60.6, 121.2, 242.4 and 484.8 microM/g tissue) and SDS (0.35, 0.70, 1.4, 2.8 and 5.6 microM/g tissue) gels compared to placebo gel treated groups. The levels of various pro-inflammatory (IL-6, IL-8 and TNF-alpha,) and immuno-regulatory cytokines (IL-10 and GM-CSF) in the explant culture supernatants of the Nisin treated cells were unaffected. Repeated intravaginal application of high dose of Nisin gel (15,150 microM/day/14 days) on cervicovaginal epithelium was evaluated in rabbits and the results were compared with SDS treated (56 microM) and 1% polycarbophil gel (placebo) groups. We examined vaginal cell morphology, structural integrity of vaginal epithelium and local production of cytokines (PICs) in the cervicovaginal lavage (CVL) of Nisin treated animals and compared with placebo and SDS treated groups. The results demonstrated no treatment related abnormalities either in the vaginal cell morphology or structural abnormalities in the mucosal epithelium. There was no change in the cytokine levels in cervicovaginal lavage (CVL) compared to SDS gel treated animals indicating Nisin gel did not induce irritation and/or inflammation in the vaginal epithelium. CVL cytokine levels were in accordance with immunohistochemical (IHC) localization of cytokines and flow cytometric evaluation of CD45 immune cell population in cervicovaginal epithelium. The levels of cytokines in the CVLs appear to be sensitive indicators in identifying and/or screening out suitable candidate microbicides before they enter phase-1 trials. In conclusion, the lack of vaginal toxicity of Nisin gel means that it has clinical potential as a safe, prophylactic contraceptive in addition to its antimicrobial activities to curb sexual transmission of HIV in human.

Evaluation of developmental toxicity of microbicide Nisin in rats.

In the present study, we have investigated the developmental toxicity of a naturally occurring peptide, Nisin in rats in order to determine its suitability as a safe vaginal microbicide. Our earlier studies indicated that, Nisin is a dual function microbicide having contraceptive and antibacterial activities. However, as part of the safety evaluation of any vaginal microbicide, it is essential to determine its teratogenic potential in a suitable animal model before it is found suitable to enter clinical trials. Sixty pregnant rats allocated into four groups were orally administered with 10, 25 and 50 mg Nisin/kg/day from day 6 to day 15 of gestation. Individual food/water consumption and body weight changes were measured daily. Nisin did not cause maternal mortality nor did the treated animals show any clinical signs of toxicity when compared to the control animals. There were no biologically significant differences in maternal liver, kidney, thymus, ovary, gravid and empty uterine weights. Mean number of corpora lutea and implantation sites also did not differ in the treated groups when compared to their respective controls. All the fetuses were weighed, sexed and examined carefully for externally visible malformations. No gross external fetal alterations were observed at any dose tested. When stained by the double staining method, no skeletal malformations and visceral defects were observed in the fetuses. The growth and reproductive performance of the F1 progeny was also unaffected. In conclusion, Nisin shows unique clinical potential as a safe prophylactic microbicide to curb the transmission of STIs/HIV and unintended pregnancies.

D-alanyl ester depletion of teichoic acids in Lactobacillus reuteri 100-23 results in impaired colonization of the mouse gastrointestinal tract.

The dlt operon of Gram-positive bacteria encodes proteins required for the incorporation of D-alanine esters into cell wall-associated teichoic acids (TA). D-alanylation of TA has been shown to be important for acid tolerance, resistance to antimicrobial peptides, adhesion, biofilm formation, and virulence of a variety of pathogenic organisms. The aim of this study was to determine the importance of D-alanylation for colonization of the gastrointestinal tract by Lactobacillus reuteri 100-23. Insertional inactivation of the dltA gene resulted in complete depletion of D-alanine substitution of lipoteichoic acids. The dlt mutant had similar growth characteristics as the wild type under standard in vitro conditions, but formed lower population sizes in the gastrointestinal tract of ex-Lactobacillus-free mice, and was almost eliminated from the habitat in competition experiments with the parental strain. In contrast to the wild type, the dlt mutant was unable to form a biofilm on the forestomach epithelium during gut colonization. Transmission electron microscope observations showed evidence of cell wall damage of mutant bacteria present in the forestomach. The dlt mutant had impaired growth under acidic culture conditions and increased susceptibility to the cationic peptide nisin relative to the wild type. Ex vivo adherence of the dlt mutant to the forestomach epithelium was not impaired. This study showed that D-alanylation is an important cell function of L. reuteri that seems to protect this commensal organism against the hostile conditions prevailing in the murine forestomach.

Investigation of the cytotoxicity of eukaryotic and prokaryotic antimicrobial peptides in intestinal epithelial cells in vitro.

Antimicrobial peptides (AMPs) are a diverse group of proteinaceous compounds ranging in size, complexity and antimicrobial spectrum. The activity of AMPs against gut pathogens warrants the study of the interaction of AMPs with the mammalian gastrointestinal tract. In particular, the investigation of the in vitro cytotoxicity of these peptides is critical before they can be considered in clinical infections. The cytotoxicity of gallidermin, nisin A, natural magainin peptides, and melittin was investigated in two gastrointestinal cell models (HT29 and Caco-2) with the MTT conversion assay, neutral red dye uptake assay and compared with that of vancomycin. The hemolytic activities were also investigated in sheep erythrocytes and the effect of AMPs on paracellular permeability was examined by transepithelial resistance (TEER) and TEM. Gallidermin was the least cytotoxic AMP followed by nisin A, magainin I, magainin II and melittin. Melittin and nisin were the only peptides to result in significant hemolysis. However, while nisin caused hemolysis at concentrations which were 1000-fold higher than those required for antimicrobial activity, melittin was hemolytic at concentrations in the same order of magnitude as its antimicrobial activity. Melittin was the only AMP to affect paracellular permeability. Long term melittin treatment also resulted in loss of microvilli, an increase in cell debris and destruction of intestinal tight junctions and cell-cell adhesion. Gallidermin shows most promise as a therapeutic agent, with relatively low cytotoxicity and potent antimicrobial activities. Melittin, while showing little potential as an antimicrobial agent, may have potential in delivery of poorly bioavailable drugs.

Evaluation of antimicrobial peptide nisin as a safe vaginal contraceptive agent in rabbits: in vitro and in vivo studies.

In the midst of the global epidemics of both unwanted pregnancies and sexually transmitted infections (STIs), options that provide protection are ideal. In the present study, nisin, a known antimicrobial peptide, was evaluated for safety and contraceptive potential in vitro and in vivo in the rabbit. A concentration of 400 microg nisin per ml was found to be spermicidal in vitro, and the effect was dose and time dependent. In vivo studies indicated that intravaginal application of 1 mg nisin blocked conception in rabbits. Repeated application of nisin (50 mg/animal per day) in rabbits for 14 consecutive days did not cause local inflammation or damage to the vaginal epithelium. In addition, the rate of diffusion of nisin into the blood via the vaginal mucosal epithelium, and its clearance from the circulation was found to be rapid. No treatment-related changes were observed in the reproductive performance of rabbits after cessation of treatment. Furthermore, no changes were observed in the gestation period, subsequent growth and survival of neonates in these animals. When male rats were given nisin orally for 13 consecutive weeks, no effect was observed on reproductive performance. The number of pups born, survival and growth of pups were unaltered. The affinity studies of nisin revealed that spermatozoa are more susceptible to nisin than red blood cells and vaginal epithelial cells. We suggest that nisin with spermicidal and antimicrobial properties could serve as a safe vaginal contraceptive for future therapeutic interventions in STIs.

In vitro assessment of the cytotoxicity of nisin, pediocin, and selected colicins on simian virus 40-transfected human colon and Vero monkey kidney cells with trypan blue staining viability assays.

Gram-positive bacterial bacteriocins (nisin and pediocin) and gram-negative bacterial bacteriocins (colicins [Col] E1, E3, E6, E7, and K) were evaluated for cytotoxicity against cultured simian virus 40-transfected human colon (SV40-HC) and Vero monkey kidney (Vero) cells. Bacteriocin-treated cells were assessed for viability by trypan blue staining. Monolayers of SV40-HC and Vero cells were cultured in tissue culture plates (35 degrees C, 10% CO2 in humidified air) with the use of Dulbecco's modified Eagle's medium supplemented with 10% (vol/vol) calf serum. Actively growing cells in the log phase (ca. 10(4) cells per ml) were treated with individual partially purified bacteriocin preparations at 170, 350, and 700 activity units per ml. Duplicate culture plates for each bacteriocin treatment and untreated controls were withdrawn after 16, 32, and 48 h of incubation. Cells were dissociated with trypsin and treated with trypan blue and were then counted in a hemocytometer with the use of a phase-contrast microscope. Viability assays indicated dose-dependent toxicity for some bacteriocins. Nisin, pediocin, and Col E6 were the most cytotoxic bacteriocins; SV40-HC cells demonstrated greater sensitivity than Vero cells did. Some bacteriocins can be toxic to mammalian cells; therefore, bacteriocins intended for use as biopreservatives must be evaluated for toxicity to mammalian cells and for other toxicities. Col E1, Col E3, Col E7, and Col K demonstrated little toxicity at the activities tested, indicating that they are safe and thus have potential for use as food biopreservatives.

Evaluation of a nisin-based germicidal formulation on teat skin of live cows.

A purified preparation of the nontoxic antimicrobial peptide, nisin (AMBICIN N), was used in the formulation of a germicidal sanitizer suitable for use on cow teats. The germicidal activity of the formulation against mastitis pathogens was measured on teat skin of live cows. The nisin-based formulation gave a mean log reduction of 3.90 against Staphylococcus aureus and 4.22 log reduction against Escherichia coli after exposure for 1 min to the germicide. This activity was comparable with that exhibited by a 1% iodophor teat dip but was significantly greater than that exhibited by the .1 and .5% iodophors and by the .5% chlorhexidine digluconate teat dips. The nisin-based formulation showed little or no potential for skin irritation after multiple application to skin, but iodophor and chlorhexidine digluconate teat dips showed significant potential for skin irritation in comparable studies.