Antibiotic Nisin Research Articles

Improvement of meat storage technology as a factor in increasing the efficiency of the development of the agro-industrial complex

The formula for a membrane solution with antioxidant and antimicrobial spectrum has been developed. 4% of agar agar, and 2% of glycerol were used as the main ingredients of the membrane solution, 1% of food additive arabinogalactan (E 409) was used as an antioxidant, and 0.02% of antibiotic nisin was used as an antimicrobial agent. These ingredients were diluted with distilled water to 100%. Addition to the basic formula of the membrane arabinogalactan makes it possible to increase strength and elongation. The biodegradability of the membrane when placed in a 0.5% solution of hydrochloric acid, containing all the enzymes of gastric juice was 25.4 minutes. It was found that samples of chilled meat packed in the developed membrane after 10 days of refrigerated storage corresponded to fresh organoleptic, physicochemical and microbiological indicators. Acid and peroxide numbers of fat samples were within normal limits. As a result, it was revealed that the packaging of chilled meat in the developed membranes helps prevent microbial and oxidative damage to the product, which allows positioning it for implementation in the agro-industrial complex.

Phylogenetic signature of lateral exchange of genes for antibiotic production and resistance among bacteria highlights a pattern of global transmission of pathogens between humans and livestock

The exchange of bacterial virulence factors driven by lateral gene transfer (LGT) can help indicate possible bacterial transmission among different hosts. Specifically, overlaying the phylogenetic signal of LGT among bacteria onto the distribution of respective isolation sources (hosts) can indicate patterns of transmission among these hosts. Here, we apply this approach towards a better understanding of patterns of bacterial transmission between humans and livestock. We utilize comparative genomics to trace patterns of LGT for an 11-gene operon responsible for the production of the antibiotic nisin and infer transmission of bacteria among respective host species. A total of 147 bacterial genomes obtained from NCBI were determined to contain the complete operon. Isolated from human, porcine and bovine hosts, these genomes represented six Streptococcus and one Staphylococcus species. Phylogenetic analyses of the operon sequences revealed a signature of frequent and recent lateral gene transfer that indicated extensive bacterial transmission between humans and pigs. For 11 isolates, we detected a Tn916-like transposon inserted into the operon. The transposon contained the tetM gene (tetracycline resistance) and additional phylogenetic analyses indicated transmission among human and animal hosts. The bacteria possessing the nisin operon and transposon were isolated from hosts distributed globally. These findings possibly reflect both the globalization of the food industry and an increasingly mobile and expanding human population. In addition to concerns regarding zoonosis, these findings also highlight the potential threat to livestock worldwide due to reverse zoonosis.

Tackling Salmonella Persister Cells by Antibiotic-Nisin Combination via Mannitol.

Bacterial persisters (defined as dormant, non-dividing cells with globally reduced metabolism) are the major cause of recurrent infections. As they neither grow nor die in presence of antibiotics, it is difficult to eradicate these cells using antibiotics, even at higher concentrations. Reports of metabolites (which help in waking up of these inactive cells) enabled eradication of bacterial persistence by aminoglycosides, suggest the new potential strategy to improve antibiotic therapy. Here we propose, mannitol enabled elimination of Salmonella persister cells by the nisin-antibiotic combination. For this, persister cells were developed and characterized for their typical properties such as non-replicative state and metabolic dormancy. Different carbon sources viz. glucose, glycerol, and mannitol were used, each as an adjunct to ampicillin for the eradication of persister cells. The maximum (but not complete) killing was observed with mannitol-ampicillin, out of all the combinations used. However, significant elimination (about 78%) could be observed, when nisin (an antimicrobial peptide) was used with ampicillin in presence of mannitol, which might have mediated the transfer of antibiotic-nisin combination at the same time when the cells tried to grab the carbon molecule. Further, the effectiveness of the trio was confirmed by flow cytometry. Overall, our findings highlight the potential of this trio-combination for developing it as an option for tackling Salmonella persister cells.

The Lantibiotic NAI-107 Binds to Bactoprenol-bound Cell Wall Precursors and Impairs Membrane Functions

The lantibiotic NAI-107 is active against Gram-positive bacteria including vancomycin-resistant enterococci and methicillin-resistant Staphylococcus aureus. To identify the molecular basis of its potency, we studied the mode of action in a series of whole cell and in vitro assays and analyzed structural features by nuclear magnetic resonance (NMR). The lantibiotic efficiently interfered with late stages of cell wall biosynthesis and induced accumulation of the soluble peptidoglycan precursor UDP-N-acetylmuramic acid-pentapeptide (UDP-MurNAc-pentapeptide) in the cytoplasm. Using membrane preparations and a complete cascade of purified, recombinant late stage peptidoglycan biosynthetic enzymes (MraY, MurG, FemX, PBP2) and their respective purified substrates, we showed that NAI-107 forms complexes with bactoprenol-pyrophosphate-coupled precursors of the bacterial cell wall. Titration experiments indicate that first a 1:1 stoichiometric complex occurs, which then transforms into a 2:1 (peptide: lipid II) complex, when excess peptide is added. Furthermore, lipid II and related molecules obviously could not serve as anchor molecules for the formation of defined and stable nisin-like pores, however, slow membrane depolarization was observed after NAI-107 treatment, which could contribute to killing of the bacterial cell.

Activity and Export of Engineered Nisin-(1-22) Analogs

The pentacyclic peptide antibiotic nisin, produced by Lactococcus lactis is ubiquitously applied as a food preservative. We previously demonstrated that the truncated nisin-(1-22) has only 10-fold lower activity than nisin. Here we aimed at further developing this tricyclic nisin analog to reach activity comparable to that of nisin. Our data demonstrate that: (1) ring A has a large mutational freedom; (2) the composition of residues 20–22 strongly affects production levels of nisin-(1-22); (3) a positively charged C-terminus of nisin-(1-22) significantly enhances its antimicrobial activity; (4) nisin-(1-22) inhibits in vitro growth of a target strain using different dynamics than nisin.

Synthetic Approach to the B-Ring of the Peptide Antibiotic Nisin

Synthetic Approach to the B-Ring of the Peptide Antibiotic Nisin

Loss of IrpT Function in Lactococcus lactis subsp. lactis N8 Results in Increased Nisin Resistance

The antibiotic nisin, produced by Lactococcus lactis subsp. lactis N8, offers an extensive commercial prospect as natural food preservatives. The nisin immunity of the L. lactis strains is regulated by a variety of mechanisms. In this study, we isolated a L. lactis L31 strain with increased nisin resistance from a mini-Mu transposon mutant pool of strain N8. The single Mu insertion in strain L31 was in the irpT gene with unknown function. By comparing the proteomic profiles of L. lactis L31 and its parental strain, we found that changes occurred in the synthesis of a protein involved in cell wall biosynthesis (RmlD). Strain L31 had 13.7% higher content of rhamnose in the cell wall than the N8 strain. Overexpression of RmlD involved in the synthesis of dTDP-L: -rhamnose in the nisin-sensitive MG1363 strain increased nisin resistance of the strain. The results indicate that these cellular proteins effected nisin resistance in L. lactis N8.

Oxidation of lanthionines renders the lantibiotic nisin inactive.

The peptide antibiotic nisin A belongs to the group of antibiotics called lantibiotics. They are classified as lantibiotics because they contain the structural group lanthionine. Lanthionines are composed of a single sulfur atom that is linked to the beta-carbons of two alanine moieties. These sulfur atoms are vulnerable to environmental oxidation. A mild oxidation reaction was performed on nisin A to determine the relative effects it would have on bioactivity. High-mass-accuracy Fourier transform ion cyclotron resonance mass spectrometry data revealed the addition of seven, eight, and nine oxygens. These additions correspond to the five lanthionines, two methionines, and two histidines that would be susceptible to oxidation. Subsequent bioassays revealed that the oxidized form of nisin A had a complete loss of bactericidal activity. In a competition study, the oxidized nisin did not appear to have an antagonistic affect on the bioactivity of nisin A, since the addition of an equal molar concentration of the oxidized variant did not have an influence on the bactericidal activity of the native antibiotic. Electron microscopy data revealed that the oxidized forms were still capable of assembling into large circular complexes, demonstrating that oxidation does not disrupt the lateral assembly mechanism of the antibiotic. Affinity thin-layer chromatography and fluorescence microscopy experiments suggested that the loss of activity is due to the inability of the oxidized form of nisin to bind to the cell wall precursor lipid II. Given the loss of bioactivity following oxidation, oxidation should be an important factor to consider in future production, purification, pharmacokinetic, and pharmacodynamic studies.

Rings, radicals, and regeneration: the early years of a bioorganic laboratory.

This Perspective provides an overview of the progress in two of the original programs in my research group focused on the biosynthesis of the antibiotics nisin, lacticin 481, fosfomycin, and bialaphos. The path from start-up funds to tenure and beyond offers insights into the opportunities realized and missed along the road.

Five Golden Rings

The antibiotic nisin bears five rings of different sizes in its structure. The enzyme that catalyzes the five distinct cyclization reactions is flexible, yet stringent, enough to form each ring in the correct order and conformation.

Ring-Closing Alkyne Metathesis Approach toward the Synthesis of Alkyne Mimics of Thioether A-, B-, C-, and DE-Ring Systems of the Lantibiotic Nisin Z

[reaction: see text] Ring-closing alkyne metathesis toward the synthesis of the alkyne-brigded A-, B-, C-, and (D)E-ring mimics of the peptide antibiotic nisin Z is described. We have successfully synthesized alkyne-bridged cyclic peptides containing 4-7 amino acid residues in yields ranging from 18 to 82%.

Mammalian defensins: structures and mechanism of antibiotic activity

Antibiotic peptides are important effector molecules in host-parasite interactions throughout the living world. In vertebrates, they function in first-line host defense by antagonizing a wide range of microbes including bacteria, fungi, and enveloped viruses. The antibiotic activity is thought to be based on their cationic, amphipathic nature, which enables the peptides to impair vital membrane functions. Molecular details for such activities have been elaborated with model membranes; however, there is increasing evidence that these models may not reflect the complex processes involved in the killing of microbes. For example, the overall killing activity of the bacterial peptide antibiotic nisin is composed of independent activities such as the formation of target-mediated pores, inhibition of cell-wall biosynthesis, formation of nontargeted pores, and induction of autolysis. We studied the molecular modes of action of human defense peptides and tried to determine whether they impair membrane functions primarily and whether additional antibiotic activities may be found. We compared killing kinetics, solute efflux kinetics, membrane-depolarization assays, and macromolecular biosynthesis assays and used several strains of Gram-positive cocci as test strains. We found that membrane depolarization contributes to rapid killing of a significant fraction of target cells within a bacterial culture. However, substantial subpopulations appear to survive the primary effects on the membrane. Depending on individual strains and species and peptide concentrations, such subpopulations may resume growth or be killed through additional activities of the peptides. Such activities can include the activation of cell-wall lytic enzymes, which appears of particular importance for killing of staphylococcal strains.

Pre-organization induced synthesis of a crossed alkene-bridged nisin Z DE-ring mimic by ring-closing metathesis

An alkene-bridged mimic of the complex DE-bisthioether-ring system of the antibiotic nisin was prepared in one step from the linear precursor.

Assembly and stability of nisin-lipid II pores.

The peptide antibiotic nisin was the first reported example of an antibiotic that kills bacteria via targeted pore formation. The specific target of nisin is Lipid II, an essential intermediate in the bacterial cell-wall synthesis. High-affinity binding of the antibiotic to Lipid II is followed by rapid permeabilization of the membrane. Here, we investigated the assembly and stability of nisin-Lipid II pore complexes by means of pyrene fluorescence and circular dichroism. We demonstrated that nisin uses all available Lipid II molecules in the membrane to form pore complexes. The pore complexes have a uniform structure and consist of 8 nisin and 4 Lipid II molecules. Moreover, the pores displayed a remarkable stability, because they were able to resist the solubilization of the membrane environment by mild detergents. Similar experiments with [N20P/M21P]nisin showed that the hinge region is essential for the assembly into stable pore complexes. The new insights were used to propose a refined model for nisin pore formation.

Lipid II-mediated pore formation by the peptide antibiotic nisin: a black lipid membrane study.

The antibiotic peptide nisin is the first known lantibiotic that uses a docking molecule within the bacterial cytoplasmic membrane for pore formation. Through specific interaction with the cell wall precursor lipid II, nisin forms defined pores which are stable for seconds and have pore diameters of 2 to 2.5 nm.

In vitro Inhibition of Soft-Rotting Bacteria by EDTA and Nisin and in vivo Response on Inoculated Fresh Cut Carrots.

EDTA and the antibiotic nisin, in combination with heat, were tested for inhibition of growth of six pectolytic, soft-rotting bacteria in 80% trypticase soy broth (TSB). Fifty percent reduction of growth by EDTA at 25°C in TSB occurred at 3.24 mM for Erwinia chrysanthemi, 2.57 mM for Pseudomonas fluorescens, 0.96 mM for E. carotovora (subsp. carotovora), 0.48 mM for P. viridiflava, 0.17 mM for Xanthomonas campestris (pv. campestris), and 0.16 mM for Cytophaga johnsonae. Nisin at 50 ug/ml was effective against X. campestris and C. johnsonae (over 90% inhibition of growth) but not against the other four bacteria (less than 20% inhibition), which are the more economically important soft-rotters. Combinations of EDTA and nisin were synergistic. A combination of 0.3 mM EDTA + nisin at 50 μg/ml inhibited growth of E. carotovora, E. chrysanthemi, and P. viridiflava by over 70%, and growth of P. fluorescens by 37%. Hot water treatments for 0.3 min at 37 or 49°C reduced survival of bacteria in the presence of EDTA + nisin, but not of EDTA, nisin, or water alone. EDTA + nisin at 37°C reduced CFU/ml of E. carotovora, E. chrysanthemi, P. fluorescens, and P. viridiflava by 2 log units, and at 49°C by 3 log units, compared with the 25°C treatment. Decay of carrot disks inoculated at two inoculum levels (103 and 104 CFU per disk) with E. carotovora, P. fluorescens, or P. viridiflava was reduced by a 1.5-min immersion in 45°C water, with or without EDTA and nisin additives. Immersion in 0.3 mM EDTA + nisin at 15 to 50 μg/ml at 45°C reduced decay due to E. carotovora and to P. fluorescens at the lower inoculum level by an average of about 50% compared with water alone at 45°C, but differences were statistically significant only at the 90% level of confidence and no different than a standard chlorine dip, current commercial practice for cut carrot slices.

Antibacterial efficacy of nisin against multidrug-resistant Gram-positive pathogens.

The rapid spread of multidrug-resistant bacterial pathogens necessitates the search for alternative antibacterial agents. We examined the efficacy of the antibiotic nisin against 56 multidrug-resistant isolates of Streptococcus pneumoniae, 33 Staphylococcus aureus and 29 vancomycin-resistant Enterococcus faecium and Enterococcus faecalis isolates. The test strains represented a large variety of clonal types (as determined by a combination of DNA fingerprints) isolated from a variety of geographic sources, and included some of the major internationally-spread multiresistant epidemic clones of S. pneumoniae and methicillin-resistant S. aureus (MRSA), MRSA strains resistant to over 16 generically distinct antibacterial agents, and enterococcal strains resistant to all currently available chemotherapeutic agents including glycopeptides. In the overwhelming majority of cases, treatment of growing cultures with nisin at 1 mg/L (S. pneumoniae) or 10-20 mg/L (in MRSA and enterococci) caused extensive (10(3)- to 10(4)-fold) loss of viable titre accompanied by various degrees of loss in the optical density of the cultures, which was most extensive in pneumococci (>90%) and least extensive (40-50%) in enterococci. Nevertheless, extensive variation in rates of nisin-induced autolysis was observed in each bacterial species. Serial exposure of a penicillin-susceptible strain of S. pneumoniae to nisin (1 mg/L) in liquid culture resulted in the rapid appearance of stable nisin-resistant mutants in which the MIC increased from 0.4 to 6.4 mg/L and the resistance trait was transferable by genetic transformation.

Influence of charge differences in the C-terminal part of nisin on antimicrobial activity and signaling capacity.

Three mutants of the antibiotic nisin Z, in which the Val32 residue was replaced by a Glu, Lys or Trp residue, were produced and characterized for the purpose of establishing the role of charge differences in the C-terminal part of nisin on antimicrobial activity and signaling properties. 1H-NMR analyses showed that all three mutants harbor an unmodified serine residue at position 33, instead of the usual dehydroalanine. Apparently, the nature of the residue preceding the serine to be dehydrated, strongly affects the efficiency of modification. Cleavage of [Glu32,Ser33]nisin Z by endoproteinase Glu-C yielded [Glu32]nisin Z(1-32)-peptide, which has a net charge difference of -2 relative to wild-type nisin Z. The activity of [Lys32,Ser33]nisin Z against Micrococcus flavus was similar to that of wild-type nisin, while [Trp32,Ser33]nisin Z, [Glu32,Ser33]nisin Z and [Glu32]nisin Z(1-32)-peptide exhibited 3-5-fold reduced activity, indicating that negative charges in the C-terminal part of nisin Z are detrimental for activity. All variants showed significant loss of activity against Streptococcus thermophilus. The potency of the nisin variants to act as signaling molecules for auto-induction of biosynthesis was significantly reduced. To obtain mutant production, extracellular addition of (mutant) nisin Z to the lactococcal expression strains was essential.

Structure-activity relationships in the peptide antibiotic nisin: role of dehydroalanine 5

A mutant of the peptide antibiotic nisin in which the dehydroalanine residue at position 5 has been replaced by an alanine has been produced and structurally characterized. It is shown to have activity very similar to that of wild-type nisin in inhibiting growth of Lactococcus lactis and Micrococcus luteus but is very much less active than nisin as an inhibitor of the outgrowth of spores of Bacillus subtilis. These observations, which parallel those of W. Liu and J. N. Hansen (Appl. Environ. Microbiol. 59:648-651, 1993) on the corresponding mutant of the related antibiotic subtilin, are discussed in terms of the mechanism(s) of action of these antibiotics.

Structure-activity relationships in the peptide antibiotic nisin: antibacterial activity of fragments of nisin

The post-translationally modified peptide antibiotic nisin has been cleaved by a number of proteases and the fragments produced purified, characterised chemically, and assayed for activity in inhibiting the growth of Lactococcus lactis MG1614 and Micrococcus luteus NCDO8166. These results provide information on the importance of different parts of the nisin molecule for its growth-inhibition activity. Removal of the C-terminal five residues leads to approximately a 10-fold decrease in potency, while removal of a further nine residues, encompassing two of the lanthionine rings, leads to a 100-fold decrease. There are some differences between analogous fragments of nisin and subtilin, suggesting possible subtle differences in mode of action. Cleavage within, or removal of, lanthionine ring C essentially abolishes the activity of nisin. The fragment nisin 1−12 is inactive itself, and specifically antagonises the growth-inhibitory action of nisin. These results are discussed in terms of current models for the mechanism of action of nisin.