Activity Of Nisin Research Articles

The effect of silver nanoparticles on the antimicrobial activity of cloned nisin against extensively drug-resistant Acinetobacter baumannii”

BackgroundAntibiotic resistance is a global threat to human health that leads to disasters. Acinetobacter baumannii cannot be controlled by the existing antibiotics, and it became challenging. Therefore, novel antibacterial agents are required to combat such threats. The aim of this project is to find a novel antimicrobial agent to treat this multi-drug resistant bacterium. MethodsThe NisA gene was isolated from Lactococcus lactis spp. lactis and cloned into the pET-3a plasmid using Gibson cloning assembly. Purified Nisin from cloning was conjugated with silver nanoparticles. Finally, an assessment of antibacterial activity for each of the purified Nisin, Silver nanoparticles, and Nisin-Silver nanoparticles conjugate against the extensively drug-resistant A. baumannii was performed. ResultsNisin was successfully purified from cloned bacteria, and the concentration was 416 µg/ml. The conjugation of nisin and silver nanoparticles was analyzed by electron microscopy. The minimum inhibitory concentration of Nisin and silver nanoparticles against A. baumannii were 104 µg/ml and 125 µg/ml, respectively. While Nisin-silver nanoparticle conjugates showed potent antimicrobial activity with MIC 125–52 µg/ml in which silver nanoparticles increased the antimicrobial activity of nisin beyond its optimum concentration (104 µg/ml). ‎ ConclusionThe development of new antibacterial agents is necessary to control extensively drug-resistant bacteria. Nisin-silver conjugates showed more potent antimicrobial activity than when applied separately and gave hope to combat the multi-drug resistant A. baumannii.

Screening, characterization and mechanism of a potential stabiliser for nisin nanoliposomes with high encapsulation efficiency

The encapsulation efficiency (EE%) reflects the amount of bioactive components that can be loaded into nanoliposomes. Obtaining a suitable nanoliposome stabiliser may be the key to improving their EE%. In this study, three polyphenols were screened as stabilisers of nanoliposomes with high nisin EE%, with curcumin nanoliposomes (Cu-NLs) exhibiting the best performance (EE% = 95.94%). Characterizations of particle size, PDI and zeta potential indicate that the Cu-NLs had good uniformity and stability. TEM found that nisin accumulated at the edges of the Cu-NLs' phospholipid layer. DSC and FT-IR revealed that curcumin was involved in the formation of the phospholipid layer and altered its structure. FT-IR and molecular docking simulations indicate that the interactions between curcumin and nisin are mainly hydrogen bonding and hydrophobic. In whole milk, Cu-NLs effectively protected nisin activity. This study provides an effective strategy for improving the EE% of nanoliposomes loaded with nisin and other bacteriocins.

Nisin Inhibition of Gram-Negative Bacteria.

Aims: This study investigates the activity of the broad-spectrum bacteriocin nisin against a large panel of Gram-negative bacterial isolates, including relevant plant, animal, and human pathogens. The aim is to generate supportive evidence towards the use/inclusion of bacteriocin-based therapeutics and open avenues for their continued development. Methods and Results: Nisin inhibitory activity was screened against a panel of 575 strains of Gram-negative bacteria, encompassing 17 genera. Nisin inhibition was observed in 309 out of 575 strains, challenging the prevailing belief that nisin lacks effectiveness against Gram-negative bacteria. The genera Acinetobacter, Helicobacter, Erwinia, and Xanthomonas exhibited particularly high nisin sensitivity. Conclusions: The findings of this study highlight the promising potential of nisin as a therapeutic agent for several key Gram-negative plant, animal, and human pathogens. These results challenge the prevailing notion that nisin is less effective or ineffective against Gram-negative pathogens when compared to Gram-positive pathogens and support future pursuits of nisin as a complementary therapy to existing antibiotics. Significance and Impact of Study: This research supports further exploration of nisin as a promising therapeutic agent for numerous human, animal, and plant health applications, offering a complementary tool for infection control in the face of multidrug-resistant bacteria.

The two-component regulatory systems GraRS and SrrAB mediate Staphylococcus aureus susceptibility to Pep5 produced by clinical isolate of Staphylococcus epidermidis.

Staphylococcus aureus is a common bacterium on the skin and in the nose that sometimes causes severe illness. Bacteriocins, antimicrobial peptides, or proteins produced by bacteria are candidates for the treatment of S. aureus infection. In this study, we found that a clinical Staphylococcus epidermidis strain, KSE112, produced the lantibiotic Pep5, which showed anti-S. aureus activity. The complete nucleotide sequence of the Pep5-encoding plasmid was determined. Several S. aureus two-component regulatory systems (TCSs) are known to be involved in bacteriocin susceptibility. Therefore, susceptibility tests were performed using TCS-inactivated S. aureus mutants to determine which TCS is responsible for Pep5 susceptibility; the ΔgraRS mutant exhibited increased susceptibility to Pep5, while the ΔsrrAB mutant exhibited decreased susceptibility. GraRS is known to regulate dltABCD and mprF in concert with vraFG, and Pep5 susceptibility was significantly increased in the ΔdltABCD, ΔmprF, and ΔvraFG mutants. Regarding the ΔsrrAB mutant, cross-resistance to aminoglycosides was observed. As aminoglycoside activity is known to be affected by aerobic respiration, we focused on qoxABCD and cydAB, which are quinol oxidase genes that are necessary for aerobic respiration and have downregulated the expression in the ΔsrrAB mutant. We constructed ΔqoxABCD and ΔcydAB mutants and found that qoxABCD inactivation decreased susceptibility to Pep5 and aminoglycosides. These results indicate that reduced aerobic respiration due to the reduced qoxABCD expression in the ΔsrrAB mutant decreased Pep5 activity.IMPORTANCEThe emergence of drug-resistant bacteria, including MRSA, is a severe health problem worldwide. Thus, the development of novel antimicrobial agents, including bacteriocins, is needed. In this report, we found a Pep5-producing strain with anti-S. aureus activity. We determined the complete sequence of the Pep5-encoding plasmid for the first time. However, in S. aureus, GraRS and its effectors conferred decreased susceptibility to Pep5. We also revealed that another TCS, SrrAB, affects susceptibility Pep5 and other lantibiotics by controlling aerobic respiration. In our study, we investigated the efficacy of Pep5 against S. aureus and other Gram-positive bacteria and revealed that respiratory constancy regulated by TCS is required for the antimicrobial activity of nisin, nukacin, and Pep5. These findings provide important information for the clinical application of bacteriocins and suggest that they have different properties among similar pore-forming lantibiotics.

Acid-Heat-Induced Fabrication of Nisin-Loaded Egg White Protein Nanoparticles: Enhanced Structural and Antibacterial Stability.

As a natural cationic peptide, Nisin is capable of widely inhibiting the growth of Gram-positive bacteria. However, it also has drawbacks such as its antimicrobial activity being susceptible to environmental factors. Nano-encapsulation can improve the defects of nisin in food applications. In this study, nisin-loaded egg white protein nanoparticles (AH-NEn) were prepared in fixed ultrasound-mediated under pH 3.0 and 90 °C. Compared with the controls, AH-NEn exhibited smaller particle size (112.5 ± 2.85 nm), smaller PDI (0.25 ± 0.01), larger Zeta potential (24 ± 1.18 mV), and higher encapsulation efficiency (91.82%) and loading capacity (45.91%). The turbidity and Fourier transform infrared spectroscopy (FTIR) results indicated that there are other non-covalent bonding interactions between the molecules of AH-NEn besides the electrostatic forces, which accounts for the fact that it is structurally more stable than the controls. In addition, by the results of fluorescence intensity, differential scanning calorimetry (DSC), and X-ray diffraction (XRD), it was shown that thermal induction could improve the solubility, heat resistance, and encapsulation of nisin in the samples. In terms of antimicrobial function, acid-heat induction did not recede the antimicrobial activity of nisin encapsulated in egg white protein (EWP). Compared with free nisin, the loss rate of bactericidal activity of AH-NEn was reduced by 75.0% and 14.0% following treatment with trypsin or a thermal treatment at 90 °C for 30 min, respectively.

The Effect of Nanoscale Modification of Nisin by Different Milk-Derived Proteins on Its Physicochemical Properties and Antibacterial Activity.

Nisin is used as a natural food preservative because of its broad-spectrum antimicrobial activity against Gram-positive bacteria. However, free nisin is susceptible to various factors that reduce its antimicrobial activity. Milk protein, a protein derived from milk, has self-assembly properties and is a good carrier of bioactive substances. In this study, lactoferrin-nisin nanoparticles (L-N), bovine serum albumin-nisin nanoparticles (B-N), and casein-nisin nanoparticles (C-N) were successfully prepared by a self-assembly technique, and then their properties were investigated. The studies revealed that lactoferrin (LF) and nisin formed L-N mainly through hydrophobic interactions and hydrogen bonding, and L-N had the best performance. The small particle size (29.83 ± 2.42 nm), dense reticular structure, and good thermal stability, storage stability, and emulsification of L-N laid a certain foundation for its application in food. Further bacteriostatic studies showed that L-N enhanced the bacteriostatic activity of nisin, with prominent inhibitory properties against Listeria monocytogenes, Staphylococcus aureus, and Bacillus cereus, which mainly disrupted the cell membrane of the bacteria. The above results broaden our understanding of milk protein-nisin nanoparticles, while the excellent antibacterial activity of L-N makes it promising for application as a novel food preservative, which will help to improve the bioavailability of nisin in food systems.

Antifungal activity of nisin against clinical isolates of azole-resistant Candida tropicalis.

The rapid emergence of invasive infections caused by azole-resistant Candida tropicalis has become a public health concern, and there is an urgent need for alternative treatment strategies. Studies have demonstrated the antibacterial effects of nisin, a well-known peptide naturally produced by Lactococcus lactis subsp. lactis. However, there is scant information about the antifungal effect of nisin against C. tropicalis. The present study aims to investigate the in vitro antifungal activity of nisin against clinical isolates of azole-resistant C. tropicalis strains, as well as its inhibitory effect on biofilm formation. A total of 35 C. tropicalis strains isolated from patients with invasive fungal infections were divided into the azole-resistant group and the azole-sensitive group, containing 21 and 14 strains, respectively. The relative expression levels of the ERG11 and UPC2 genes in the azole-resistant group were higher than those in the azole-sensitive group (p < 0.0001), while no significant differences were observed in the expression levels of the MDR1 and CDR1 genes. The minimum inhibitory concentration of nisin against C. tropicalis ranged from 2 to 8 μg/mL. Nisin treatment inhibited the growth of azole-resistant C. tropicalis, with over a four-fold reduction in OD600 nm values observed at the 8-h time point, while it promoted the transition of C. tropicalis from the spore phase to the hyphal phase, as observed on cryo-scanning electron microscopy. The results of biofilm quantification using crystal violet staining indicated a significant decrease in OD570 nm values in the nisin-treated group compared to the controls (p < 0.0001). Among the 21 azole-resistant C. tropicalis strains, the biofilm formation was inhibited in 17 strains (17/21, 81%), and more than 85% inhibition of biofilm formation was observed in the representative strains. With regard to the molecular mechanisms, the expression of the BCR1 and UPC2 genes in the azole-resistant strains was down-regulated on nisin treatment (p < 0.05). In conclusion, we demonstrated, for the first time, that nisin has antifungal activity and significant anti-biofilm activity against clinical isolates of azole-resistant C. tropicalis strains. Based on the findings, nisin could be a promising alternative antifungal agent for combating azole-resistant C. tropicalis infections.

Rerouting the Metabolic Pathway in Lactococcus lactis ATCC 11454 to Improve Nisin Productivity by Disrupting Lactate Dehydrogenase (ldh)

ABSTRACT Nisin is an effective biopreservative, however its wide application in food is restricted due to its low productivity. Therefore, the present work aimed to increase its yield by disrupting lactate dehydrogenase (ldh) and re-directing the carbon flow towards nisin production. The ldh-deficient Lactococcus lactis ATCC 11454 (ldhΔ) strain was created by inserting ampicillin (amp) within the ldh to disrupt its function. Under controlled fermentation conditions (30°C, pH 6, 1.0% glucose), the ldhΔ strain exhibited negligible decrease in pH (2.5%) over 48 h, in contrast to the wild-type (WT) strain, which experienced a 23.6% decrease in pH (pH 4.2). Overall, the ldhΔ strain showed a significant (p < 0.05) increase in growth and nisin activity by 24.9% and 62% compared to the WT strain, respectively. This strategy could serve as a practical approach to further enhance the production of bacteriocin or other compounds by modulating their metabolic pathways.

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.

Sensitivity of Salmonella Typhimurium to nisin in vitro and in orange juice under refrigeration.

The influence of environmental factors on Salmonella sensitivity to nisin in vitro and in refrigerated orange juice were investigated. Nisin activity was observed in the different conditions, but the highest efficiency was achieved at lower pH (4.0) and with higher bacteriocin concentration (174µM). Moreover, the bactericidal action was directly proportional to the incubation period. When tested in orange juice, nisin caused a reduction of up to 4.05 logarithm cycles in the Salmonella population. So, environmental factors such as low pH and low temperature favored the sensitization of Salmonella cells to the bactericidal action of nisin. Therefore, this may represent an alternative to control Salmonella in refrigerated foods.

Improved Antibacterial Activity of Peptide Nisin with Pyrrole-Based Ionic Liquids Having Bis(trifluoromethylsulfonyl)imide as a Counterion: A Synergistic Approach to Combat Bacterial Infections.

Bacterial resistance against antimicrobial drugs is a forthcoming threat to the prevention and treatment of developing bacterial infections. Hence, the development of new antimicrobial therapy or therapeutic drugs is desperately needed. A combination of antibiotics exhibits synergistic antibacterial effects. As the combination approach of antibiotics has always shown better results against pathogens compared to monotherapy with an antibiotic, we focused on creating a new combination that may reduce the chances of strains attaining resistance, consequently lowering the toxicity factor associated with the consumption of high amounts of antibiotics. Nisin, a food preservative and potential antibiotic, shows antibacterial activity against Gram-positive strains. Since the past decade, ionic liquids (ILs) have proven to be an important class of potential antibacterial agents. In our study, we studied the effect of pyrrolidinium-based ILs and arrived at a noncovalent conjugate formed by combining nisin with ILs. The conjugates were tested against a couple of clinically relevant microorganisms, namely, Escherichia coli and Staphylococcus aureus. We reached a novel discovery that the combination of sodium/iodide symporter (NIS) and IL exhibited inhibitory effects against Gram-negative bacteria, which was not observed with NIS alone. The results showed remarkable improvement in the minimum inhibitory concentration (MIC) value of NIS in the presence of ILs targeted against both microorganisms. Further, flow cytometry and confocal microscopy results revealed the membrane disruption efficiency of the best combination obtained, leading to cell death. Additionally, the complexation of nisin and ILs was studied using various techniques, such as surface tension, dynamic light scattering, absorption spectroscopy, and molecular docking.

Mechanisms of Regulation Allergic and Autoimmune Reactions by Bacterial Origin Bioregulators

Relevance. The increase in allergic and autoimmune diseases observed in recent decades highlights the need for therapy and prevention, which requires detailed research into the mechanisms of their occurrence. The onset and progression of allergic and autoimmune diseases are influenced by genetic predisposition, lifestyle, environmental factors, and disruptions in the coordinated operation of the immune system, and as a consequence of immune homeostasis. Treatment of these diseases is primarily symptomatic and often accompanied by undesirable side effects. Immune system disorders in various pathologies have their own characteristics for each type of disease, and at the same time have common mechanisms. Considering the presence of a large number of various microorganisms in the human body, taking their influence into account is of paramount importance. Microorganisms are a source of biologically active molecules, the action of which can either prevent and reduce the severity of the disease or exacerbate it. The aim of this study was to analyze the cytokine profile of the effects of fragments of cell walls of Gram-negative and Gram-positive bacteria - lipopolysaccharide (LPS) and muramyl peptide (MP), as well as nisin - an antimicrobial peptide of bacterial origin on human mononuclear cells. Materials and Methods. Mononuclear cells were obtained from peripheral blood of healthy volunteers using Cell separation media Lympholyte CL 5015, and were cultured in the presence of LPS, GMDP and bacteriocin nisin. The cytokine activity of LPS, GMDP and bacteriocin nisin was examined using the multiplex cytokine analysis; the analysis of surface markers was determined flow cytometry. Results and Discussion. It was shown that bacterial cell wall fragments to a much greater extent than nisin induce the production of cytokines, chemokines, and growth factors. It was established that LPS and MP increase the expression of CD11c on dendritic cells, while bacteriocin nisin does not affect the increase of CD11c+ DCs. LPS and MP in the conducted ex vivo studies did not affect the emergence of CCR7. Conclusion. Bacterial origin bioregulators trigger a negative feedback mechanism by inducing the synthesis of anti-inflammatory factors, that can prevent the inflammatory process. Understanding the molecular mechanisms of the influence of bacterial origin bioregulators on the human body opens new approaches in the prevention and development of personalized therapy strategies.

The modification of nisin with homocysteine thiolactone and its effect on antimicrobial activity.

The aim of the present study is to make an important contribution to the literature by focusing on the preparation of the N-homocysteine conjugate of nisin and evaluating the effect of the N-homocysteinylation reaction on its antimicriobial activity. The modification process was monitored using both acetic acid urea polyacrylamide gel electrophoresis (AAU-PAGE) and tricine sodium dodecyl sulphate polyacrylamide gel electrophoresis (tricine SDS-PAGE). The antibacterial effectiveness of modified nisin was assessed against Staphylococcus aureus ATCC 6538, Enterococcus faecium ATCC 9097, Bacillus subtilis ATCC 6633, Lactococcus lactis ssp. cremoris AÜ, Listeria monocytogenes NCTC 5348, and Escherichia coli RSKK. Optimal conditions for achieving the highest N-homocysteinylation degree (6.30%) were determined as 6 mg/mL nisin, 150 mM homocysteine thiolactone, 150 rpm shaking rate, pH of 3.0, and a reaction time of 6 h. The modified nisin obtained did not have a significant inhibitory effect on the strains tested except E. faecium. E. faecium was inhibited by the modified nisin and its antibacterial activity was determined as approximately 10% of the antibacterial activity of unmodified nisin. On the other hand, hydrolysis of nisin by trypsin and thermolysin resulted in significant specific side chain modifications induced by the homocysteine-thiolactone reaction, especially at Lys12 and Lys22. The results provide valuable insights into the potential of N-homocysteinylation to improve the antibacterial properties of nisin and also suggest that the effects of specific modifications identified during the modification process should be investigated.

Bio Evaluation of Lantibiotic Nisin Loaded Polyvinyl Alcohol – Sodium Alginate Crosslinked Hydrogel As Novel Dental Temporary Filling Material

The incidence of oro-dental disorders has emerged as a serious threat to the healthcare sector owing to the increasing complexity of the oral microbiome. Significant advances in biomaterial research have led to the advent of a plethora of drug delivery systems including nanocarriers, dendrimers, hydrogels and other kinds of stimuli-responsive polymeric biomaterials. Anti-microbial peptides (AMPs) have engendered considerable interest in the past decades as potential alternatives to traditional disinfecting agents and also emerged as potent antibiofilm agents. Among the most viable approaches in targeted drug delivery, hydrogels incorporated with AMPs are emerging as bio-functional platforms yielding increased stability and bioavailability. The antibacterial and antibiofilm activities of Nisin are studied using microbiological methods followed by the synthesis of Nisin loaded PVA-Alginate hydrogel for dental treatment. The physicochemical characteristics of Nisin loaded hydrogel were done by swelling behavior, release kinetics assay, FTIR spectroscopic methods and cytotoxicity studies. Nisin showed antibacterial activity towards clinical isolates of drug-resistant bacteria and the antibiofilm and anti-adhesion studies demonstrated that Nisin could control the bacterial count in the test sample. The polymerization of Nisin into hydrogels was done and the physico-chemical characterization of Nisin loaded hydrogel network could be envisaged as a potential drug delivery platform for oral infections. Nisin loaded PVA-Alginate biocompatible hydrogel exhibited apparent swellable, flexible, nonhaemolytic materials and active antimicrobial and nontoxic materials. Physicochemical properties of these Nisin loaded PVA-Alginate biocompatible hydrogels have great potential in biomaterial-based drug delivery systems in controlling the growth and proliferation of major oro-dental pathogens. This could be exploited for the temporary biocompatible dental filling materials to treat the caries. Exploring potential nisin loaded hydrogel delivery systems will provide a brighter future of more friendly, effective and personalized treatment to deal with dental caries.

Effect of the interaction of nisin Z with various polysaccharides on its antibacterial activity

The effect of various polysaccharides commonly used as thickening or gelling agents in foods on the antibacterial activity of nisin Z was investigated. Addition of 1 g.L−1 sodium alginate in tryptic soy broth resulted in a four-fold increase of minimal inhibitory (MIC) and bactericidal (MBC) concentrations of nisin Z against Listeria innocua ATCC 33090 and Kocuria rhizophila ATCC 9341, while the addition of citrus pectin or various exopolysaccharides (Leuconostoc mesenteroides P35 exopolysaccharides, low (9–11 kDa) or medium (60–76 kDa) molecular weight dextran from Ln. mesenteroides and 2 kefirans (extracted from kefir grains provided by Crokfun or Kefiralia)) did not increase MIC and MBC of nisin Z against these bacteria. Zeta potential determination at pH 7 of the nisin Z, bacteria and polysaccharides allowed us to observe that only nisin Z had a slightly positive zeta potential, while the bacteria, sodium alginate and highly methoxylated citrus pectin zeta potentials were lower than −20 mV and zeta potentials of all exopolysaccharides were between 0 and - 10 mV. It was thus proposed that anionic sodium alginate, which is the only polysaccharide having a lower zeta potential than both bacteria, forms complexes stabilized by electrostatic interactions with oppositely charged nisin Z, thereby limiting the quantity of “free” nisin Z interacting with nisin-susceptible bacteria. This hypothesis was substantiated by the observation of nisin Z - sodium alginate aggregation at pH 7 and the estimation of the apparent affinity of sodium alginate for “free” nisin Z and immobilized nisin Z by partition experiments and by surface plasmon resonance analysis, respectively. This study thus provides useful information for food formulation regarding the effect of different polysaccharides on nisin Z antibacterial activity and some understanding of how interactions between nisin Z, polysaccharides, and nisin Z - sensitive bacteria are explanatory.

Cesin, a short natural variant of nisin, displays potent antimicrobial activity against major pathogens despite lacking two C-terminal macrocycles.

Nisin is a widely used lantibiotic owing to its potent antimicrobial activity and its food-grade status. Its mode of action includes cell wall synthesis inhibition and pore formation, which are attributed to the lipid II binding and pore-forming domains, respectively. We discovered cesin, a short natural variant of nisin, produced by the psychrophilic anaerobe Clostridium estertheticum. Unlike other natural nisin variants, cesin lacks the two terminal macrocycles constituting the pore-forming domain. The current study aimed at heterologous expression and characterization of the antimicrobial activity and physicochemical properties of cesin. Following the successful heterologous expression of cesin in Lactococcus lactis, the lantibiotic demonstrated a broad and potent antimicrobial profile comparable to that of nisin. Determination of its mode of action using lipid II and lipoteichoic acid binding assays linked the potent antimicrobial activity to lipid II binding and electrostatic interactions with teichoic acids. Fluorescence microscopy showed that cesin lacks pore-forming ability in its natural form. Stability tests have shown the lantibiotic is highly stable at different pH values and temperature conditions, but that it can be degraded by trypsin. However, a bioengineered analog, cesin R15G, overcame the trypsin degradation, while keeping full antimicrobial activity. This study shows that cesin is a novel (small) nisin variant that efficiently kills target bacteria by inhibiting cell wall synthesis without pore formation. IMPORTANCE The current increase in antibiotic-resistant pathogens necessitates the discovery and application of novel antimicrobials. In this regard, we recently discovered cesin, which is a short natural variant of nisin produced by the psychrophilic Clostridium estertheticum. However, its suitability as an antimicrobial compound was in doubt due to its structural resemblance to nisin(1-22), a bioengineered short variant of nisin with low antimicrobial activity. Here, we show by heterologous expression, purification, and characterization that the potency of cesin is not only much higher than that of nisin(1-22), but that it is even comparable to the full-length nisin, despite lacking two C-terminal rings that are essential for nisin's activity. We show that cesin is a suitable scaffold for bioengineering to improve its applicability, such as resistance to trypsin. This study demonstrates the suitability of cesin for future application in food and/or for health as a potent and stable antimicrobial compound.

Engineering of Nisin as a Means for Improvement of Its Pharmacological Properties: A Review.

Lantibiotics are believed to have a conceivable potential to be used as therapeutics, especially against clinically resistant bacterial strains. However, their low solubility and poor stability under physiological conditions limit their availability for clinical studies and further pharmaceutical commercialization. Nisin is a readily available and cheap lanthipeptide and thus serves as a good model in the search for the tools to engineer lantibiotics with improved pharmacological properties. This review aims to address technologies that can be applied to alter and enhance the antimicrobial activity, antibacterial spectrum and physicochemical properties (solubility, solution stability and protease resistance) of nisin. There are basically two general means to obtain nisin analogs-protein engineering and chemical functionalization of this antibiotic. Although bioengineering techniques have been well developed and enable the creation of nisin mutants of variable structures and properties, they are lacking spectacular effects so far. Chemical modifications of nisin based on utilization of the reactivity of its free amino and carboxylic moieties, as well as reactivity of the double bonds of its dehydroamino acids, are in their infancy.

Cyclodextrin carboxylate improves the stability and activity of nisin in a wider range of application conditions

Nisin is a natural bacteriocin that exhibits good antibacterial activity against Gram-positive bacteria. It has good solubility, stability, and activity under acidic conditions, but it becomes less soluble, stable, and active when the solution pH exceeds 6.0, which severely restricted the industrial application range of nisin as antibacterial agent. In this study, we investigated the potential of complexing nisin with a cyclodextrin carboxylate, succinic acid-β-cyclodextrin (SACD), to overcome the disadvantages. Strong hydrogen bonding was shown between the nisin and SACD, promoting the formation of nisin-SACD complexes. These complexes exhibited good solubility under neutral and alkaline conditions, and good stability after being held at high pH values during processing with high-steam sterilization. Moreover, the nisin-SACD complexes displayed significantly improved antibacterial activity against model Gram-positive bacteria (S. aureus). This study shows that complexation can improve the efficacy of nisin under neutral and alkaline situations, which may greatly broaden its application range in food, medical, and other industries.

Development of alkaline-stable nanoformulation of nisin: special insights through cytotoxic and antibacterial studies.

Nisin, a thermostable, approved food preservative, has limited therapeutic applications because of its high pH and proteolytic enzyme instability. The unavailability of a rapid, simple method of detection also restricts the research of nisin. The objective of this study was to adapt the simple, rapid protein estimation method of detection for nisin formulation and to formulate and evaluate site-specific nanoformulation for therapeutic applications, viz. colon cancer, and anti-bacterial action. Three nanoformulations of nisin with chitosan, gellan gum, and dextran (ECN, EGN, and EDN) were prepared and characterized in vitro. Among three, EGN was selected as a good formulation based on its size surface charge, morphology, drug loading, and release characteristics. FT-IR and DSC revealed the interaction pattern and stability nature. The stability of nisin in an alkaline environment was confirmed by CD. Its therapeutic applications were proved by efficiency against colon cancer cells evaluated by MTT assay and AO/EB staining using Caco-2 cell lines. The in situ sol-gel mechanism imparted by gellan gum was proved the sole reason for the stability and activity of nisin in EGN at lower GIT. This was confirmed (using rheometer) by shear-thickening characteristics of formulation EGN in simulated colon fluid. The antibacterial activity against Staphylococcus aureus by disk diffusion method was also performed to confirm the retention of antimicrobial activity of nisin in EGN. Hence, gellan gum-nisin colloidal nanoparticles are found good candidates for drug delivery at lower GIT and stabilizing alkaline food materials.

Ready-to-Eat Egg Products Formulated with Nisin and Organic Acids to Control Listeria monocytogenes

Formulating ready-to-eat (RTE) products with growth inhibitors minimizes the risk of listeriosis. In part I, RTE egg products formulated with 6.25 ppm nisin were evaluated to control Listeria monocytogenes. Individual experimental units were surface inoculated with 2.5-log CFU/g of L. monocytogenes, packaged in pouches with a headspace gas of 20:80 CO2:NO2, and stored at 4.4°C for 8 weeks. Formulations with finished product pH of 6.29 ± 0.07 limited growth to <2-log for 4 weeks. Products at pH values of 7.42 ± 0.12 and 7.84 ± 0.11 were not different (p > 0.05) from the control without nisin at pH 7.34 ± 0.13, all supported 4-log growth by 4 weeks. In part II, a nisin bioassay test was performed to evaluate the stability of nisin in eggs as affected by the product pH (6.00 ± 0.03, 7.00 ± 0.00, 7.50 ± 0.03, and 8.00 ± 0.02) and cooking to an internal temperature of 73.9 or 85°C for 90 s. The nisin activity loss increased as the product pH or the cooking temperature increased (p < 0.05). Part III evaluated the effectiveness of 6.25 ppm nisin in combination with either an acetate-based antimicrobial used at 1.0% (w/w) in egg formulation (A1.0), propionate at 0.3% (P0.3), acetate-diacetate at 1.0% (AD1.0), acetate-diacetate at 0.6% (AD0.6), and lactate at 2.0% (L2.0) as a positive control. These formulations had a finished product pH, moisture, and salt contents of 5.97 ± 0.21, 72.4 ± 0.9%, and 0.67 ± 0.05%, respectively. L. monocytogenes did not grow in formulations A1.0 and AD1.0, whereas L2.0 and P0.3 supported 2-log growth by weeks 6 and 15, respectively and AD0.6 supported <1-log growth over 20 weeks at 4.4°C. Evaluation of uninoculated control units in parts I and III showed no changes (p > 0.05) in the CO2 and O2 headspace gas composition, generally no detection or growth of background microbes, and no changes (p > 0.05) in the pH of the formulations during storage, all assuring absence of uncontrolled interferences for the growth of L. monocytogenes.