A Bioengineered Nisin Derivative To Control Streptococcus uberis Biofilms.

Antimicrobial peptides are evolving as novel therapeutic options against the increasing problem of multidrug-resistant microorganisms, and nisin is one such avenue. However, some bacteria possess a specific nisin resistance system (NSR), which cleaves the peptide reducing its bactericidal efficacy. NSR-based resistance was identified in strains of Streptococcus uberis, a ubiquitous pathogen that causes mastitis in dairy cattle. Previous studies have demonstrated that a nisin A derivative termed nisin PV, featuring S29P and I30V, exhibits enhanced resistance to proteolytic cleavage by NSR. Our objective was to investigate the ability of this nisin derivative to eradicate and inhibit biofilms of S. uberis DPC 5344 and S. uberis ATCC 700407 (nsr+) using crystal violet (biomass), 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) (viability) assays, and confocal microscopy (viability and architecture). When preestablished biofilms were assessed, both peptides reduced biofilm biomass by over 60% compared to that of the untreated controls. However, a 42% higher reduction in viability was observed following treatment with nisin PV compared to that of nisin A. Accordingly, confocal microscopy analysis revealed significantly more dead cells on the biofilm upper surface and a reduced thickness following treatment with nisin PV. When biofilm inhibition was assessed, nisin PV inhibited biofilm formation and decreased viability up to 56% and 85% more than nisin A, respectively. Confocal microscopy analysis revealed a lack of biofilm for S. uberis ATCC 700407 and only dead cells for S. uberis DPC 5344. These results suggest that nisin PV is a promising alternative to effectively reduce the biofilm formation of S. uberis strains carrying NSR. IMPORTANCE One of the four most prevalent species of bovine mastitis-causing pathogens is S. uberis. Its ability to form biofilms confers on the bacteria greater resistance to antibiotics, requiring higher doses to be more effective. In a bid to limit antibiotic resistance development, the need for alternative antimicrobials is paramount. Bacteriocins such as nisin represent one such alternative that could alleviate the impact of mastitis caused by S. uberis. However, many strains of S. uberis have been shown to possess nisin resistance determinants, such as the nisin resistance protein (NSR). In this study, we demonstrate the ability of nisin and a nisin derivative termed PV that is insensitive to NSR to prevent and remove biofilms of NSR-producing S. uberis strains. These findings will add new information to the antimicrobial bacteriocins and control of S. uberis research fields specifically in relation to biofilms and nsr+ mastitis-associated strains.

Inhibition of Listeria monocytogenes by the Staphylococcus capitis - derived bacteriocin capidermicin.

Natural methods to control food pathogens are required and bacteriocins have received much interest in this regard. The aim of this study was to investigate the ability of the novel bacteriocin capidermicin to inhibit Listeria monocytogenes. Agar-based deferred antagonism assays were carried out with the capidermicin producer against 17 L. monocytogenes strains and large zones of inhibition were observed for 12 strains. Minimal inhibitory concentration assays performed with purified capidermicin peptide revealed MIC values between 680 nM and 11 µM. Biofilm assays were performed with five L. monocytogenes strains. Addition of capidermicin prevented biofilm formation by one strain and could remove pre-established biofilms of all five strains. Broth based growth experiments demonstrated that addition of capidermicin resulted in an extended lag phase of both L. monocytogenes strains tested. Kill-curve experiments showed that capidermicin was able to potentiate the anti-Listeria effects of the lantibiotic nisin. This enhanced killing by the combination of both peptides was also observed in model food systems (cottage cheese and chocolate milk). In summary, we show that capidermicin can inhibit L. monocytogenes and warrants further investigation as a potential natural agent for the control of this pathogen.

Investigation of combinations of rationally selected bioengineered nisin derivatives for their ability to inhibit Listeria in broth and model food systems.

In this study, we examined the ability of nisin A and a rationally assembled bank of 36 nisin derivative producing Lactococcus lactis strains to inhibit Listeria. A broth-based bioluminescence assay for screening single and combinations of bioengineered nisin derivatives using cell-free supernatants (CFS) from nisin derivative producing strains was developed. In this way, we screened 630 combinations of nisin derivative producing strains, identifying two (CFS from M17Q + N20P and M17Q + S29E) which exhibited enhanced anti-listerial activity when used together compared to when used alone, or to the nisin A producing strain. Minimal inhibitory concentration assays performed with purified peptides revealed than when used singly, the specific activities of M17Q, N20P and S29E (3.75-7.5 µM) against L. innocua were equal to, or less than that of nisin A (MIC of 3.75 µM). Broth-based growth curve assays using purified peptides demonstrated that use of the double peptide combinations and a triple peptide combination (M17Q + N20P + S29E) resulted in an extended lag phase of L. innocua, while kill curve assays confirmed the enhanced bactericidal activity of the combinations in comparison to the single derivative peptides or nisin A. Furthermore, the enhanced activity of the M17Q + N20P combination was maintained in a model food system (frankfurter homogenate) at both chill (4 °C) and abusive (20 °C) temperature conditions, with final cell numbers significantly less (1-2 log10 CFU/ml) than those observed with the derivative peptides alone, or nisin A. To our knowledge, this study is the first investigation that combines bioengineered bacteriocins with the aim of discovering a combination with enhanced antimicrobial activity.

Assessing the ability of nisin A and derivatives thereof to inhibit gram-negative bacteria from the genus Thermus.

Nisin is a bacteriocin that is globally employed as a biopreservative in food systems to control gram-positive, and some gram-negative, bacteria. Here we tested the bioactivity of nisin A-producing Lactococcus lactis NZ9700 and producers of bioengineered variants thereof against representatives of the gram-negative genus Thermus, which has been associated with the pink discoloration defect in cheese. Starting with a total of 73 nisin variant-producing Lactococcus lactis, bioactivity against Thermus was assessed via agar diffusion assays, and 22 variants were found to have bioactivity greater than or equal to that of the nisin A-producing control. To determine to what extent this enhanced bioactivity was attributable to an increase in specific activity, minimum inhibitory concentrations were determined using the corresponding purified form of these 22 nisin A derivatives. From these experiments, nisin M17Q and M21F were identified as peptides with enhanced antimicrobial activity against the majority of Thermus target strains tested. In addition, several other peptide variants were found to exhibit enhanced specific activity against a subset of strains.

Bio-Engineered Nisin with Increased Anti-Staphylococcus and Selectively Reduced Anti-Lactococcus Activity for Treatment of Bovine Mastitis.

Bovine mastitis is a significant economic burden for dairy enterprises, responsible for premature culling, prophylactic and therapeutic antibiotic use, reduced milk production and the withholding (and thus wastage) of milk. There is a desire to identify novel antimicrobials that are expressly directed to veterinary applications, do not require a lengthy milk withholding period and that will not have a negative impact on the growth of lactic acid bacteria involved in downstream dairy fermentations. Nisin is the prototypical lantibiotic, a family of highly modified antimicrobial peptides that exhibit potent antimicrobial activity against many Gram-positive microbes, including human and animal pathogens including species of Staphylococcus and Streptococcus. Although not yet utilized in the area of human medicine, nisin is currently applied as the active agent in products designed to prevent bovine mastitis. Over the last decade, we have harnessed bioengineering strategies to boost the specific activity and target spectrum of nisin against several problematic microorganisms. Here, we screen a large bank of engineered nisin derivatives to identify novel derivatives that exhibit improved specific activity against a selection of staphylococci, including mastitis-associated strains, but have unchanged or reduced activity against dairy lactococci. Three such peptides were identified; nisin A M17Q, nisin A T2L and nisin A HTK.

Nisin J, a Novel Natural Nisin Variant, Is Produced by Staphylococcus capitis Sourced from the Human Skin Microbiota.

The skin microbiota is thought to play a key role in host protection from infection. Nisin J is a novel nisin variant produced by Staphylococcus capitis APC 2923, a strain isolated from the toe web space area in a screening study performed on the human skin microbiota. Whole-genome sequencing and mass spectrometry of the purified peptide confirmed that S. capitis APC 2923 produces a 3,458-Da bacteriocin, designated nisin J, which exhibited antimicrobial activity against a range of Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus (MRSA) and Cutibacterium acnes The gene order in the nisin J gene cluster (nsjFEGBTCJP) differs from that of other nisin variants in that it is lacking the nisin regulatory genes, nisRK, as well as the nisin immunity gene nisI Nisin J has 9 amino acid changes compared to prototypical nisin A, with 8 amino acid substitutions, 6 of which are not present in other nisin variants (Ile4Lys, Met17Gln, Gly18Thr, Asn20Phe, Met21Ala, Ile30Gly, Val33His, and Lys34Thr), and an extra amino acid close to the C terminus, rendering nisin J the only nisin variant to contain 35 amino acids. This is the first report of a nisin variant produced by a Staphylococcus species and the first nisin producer isolated from human skin.IMPORTANCE This study describes the characterization of nisin J, the first example of a natural nisin variant, produced by a human skin isolate of staphylococcal origin. Nisin J displays inhibitory activity against a wide range of bacterial targets, including MRSA. This work demonstrates the potential of human commensals as a source for novel antimicrobials that could form part of the solution to antibiotic resistance across a broad range of bacterial pathogens.

Bioengineering nisin to overcome the nisin resistance protein.

The emergence and dissemination of antibiotic resistant bacteria is a major medical challenge. Lantibiotics are highly modified bacterially produced antimicrobial peptides that have attracted considerable interest as alternatives or adjuncts to existing antibiotics. Nisin, the most widely studied and commercially exploited lantibiotic, exhibits high efficacy against many pathogens. However, some clinically relevant bacteria express highly specific membrane-associated nisin resistance proteins. One notable example is the nisin resistance protein that acts by cleaving the peptide bond between ring E and the adjacent serine 29, resulting in a truncated peptide with significantly less activity. We utilised a complete bank of bioengineered nisin (nisin A) producers in which the serine 29 residue has been replaced with every alternative amino acid. The nisin A S29P derivative was found to be as active as nisin A against a variety of bacterial targets but, crucially, exhibited a 20-fold increase in specific activity against a strain expressing the nisin resistance protein. Another derivative, nisin PV, exhibited similar properties but was much less prone to oxidation. This version of nisin with enhanced resistance to specific resistance mechanisms could prove useful in the fight against antibiotic resistant pathogens.

Nisin M: a Bioengineered Nisin A Variant That Retains Full Induction Capacity but Has Significantly Reduced Antimicrobial Activity.

Nisin A is a potent antimicrobial with potential as an alternative to traditional antibiotics, and a number of genetically modified variants have been created that target clinically relevant pathogens. In addition to antimicrobial activity, nisin autoregulates its own production via a signal transduction pathway, a property that has been exploited in a protein expression system termed the nisin-controlled gene expression (NICE) system. Although NICE has become one of the most popular protein expression systems, one drawback is that the inducer peptide, nisin A, also has inhibitory activity. It has already been demonstrated that the N-terminal region of nisin A contributes to antimicrobial activity and signal transduction properties; therefore, we conducted bioengineering of nisin at positions Pro9 and Gly10 within ring B to produce a bank of variants that could potentially be used as alternative induction peptides. One variant, designated nisin M, has threonines at positions 9 and 10 and retains induction capacity comparable to that of wild-type nisin A, while most of the antimicrobial activity is abolished. Further analysis confirmed that nisin M produces a mix of peptides as a result of different degrees of dehydration of the two threonines. We show that nisin M exhibits potential as a more suitable alternative to nisin A for the expression of proteins that may be difficult to express or for production of proteins in strains that are sensitive to wild-type nisin. Moreover, it may address the increasing demand by industry for optimization of peptide fermentations to increase yields or production rates.IMPORTANCE This study describes the generation of a nisin variant with superior characteristics for use in the NICE protein expression system. The variant, termed nisin M, retains an induction capacity comparable to that of wild-type nisin A but exhibits significantly reduced antimicrobial activity and can therefore be used at concentrations that are normally toxic to the expression host.

The microbiology and treatment of human mastitis.

Mastitis, which is generally described as an inflammation of breast tissue, is a common and debilitating disease which frequently results in the cessation of exclusive breastfeeding and affects up to 33% of lactating women. The condition is a primary cause of decreased milk production and results in organoleptic and nutritional alterations in milk quality. Recent studies employing culture-independent techniques, including metagenomic sequencing, have revealed a loss of bacterial diversity in the microbiome of mastitic milk samples compared to healthy milk samples. In those infected, the pathogens Staphylococcus aureus, Staphylococcus epidermidis and members of corynebacteria have been identified as the predominant etiological agents in acute, subacute and granulomatous mastitis, respectively. The increased incidence of antibiotic resistance in the causative species is also a key cause of concern for treatment of the disease, thus leading to the need to develop novel therapies. In this respect, probiotics and bacteriocins have revealed potential as alternative treatments.

Use of enhanced nisin derivatives in combination with food-grade oils or citric acid to control Cronobacter sakazakii and Escherichia coli O157:H7.

Cronobacter sakazakii and Escherichia coli O157:H7 are well known food-borne pathogens that can cause severe disease. The identification of new alternatives to heating to control these pathogens in foods, while reducing the impact on organoleptic properties and nutritional value, is highly desirable. In this study, nisin and its bioengineered variants, nisin V and nisin S29A, are used alone, or in combination with plant essential oils (thymol, carvacrol and trans-cinnamaldehyde) or citric acid, with a view to controlling C. sakazakii and E. coli O157:H7 in laboratory-based assays and model food systems. The use of nisin variants (30 µM) with low concentrations of thymol (0.015%), carvacrol (0.03%) and trans-cinnamaldehyde (0.035%) resulted in extended lag phases of growth compared to those for corresponding nisin A-essential oil combinations. Furthermore, nisin variants (60 µM) used in combination with carvacrol (0.03%) significantly reduced viable counts of E. coli O157:H7 (3-log) and C. sakazakii (4-log) compared to nisin A-carvacrol treatment. Importantly, this increased effectiveness translated into food. More specifically, sub-inhibitory concentrations of nisin variants and carvacrol caused complete inactivation of E. coli O157:H7 in apple juice within 3 h at room temperature compared to that of the equivalent nisin A combination. Furthermore, combinations of commercial Nisaplin and the food additive citric acid reduced C. sakazakii numbers markedly in infant formula within the same 3 h period. These results highlight the potential benefits of combining nisin and variants thereof with carvacrol and/or citric acid for the inhibition of Gram negative food-borne pathogens.

Efficacies of nisin A and nisin V semipurified preparations alone and in combination with plant essential oils for controlling Listeria monocytogenes.

The food-borne pathogenic bacterium Listeria is known for relatively low morbidity and high mortality rates, reaching up to 25 to 30%. Listeria is a hardy organism, and its control in foods represents a significant challenge. Many naturally occurring compounds, including the bacteriocin nisin and a number of plant essential oils, have been widely studied and are reported to be effective as antimicrobial agents against spoilage and pathogenic microorganisms. The aim of this study was to investigate the ability of semipurified preparations (SPP) containing either nisin A or an enhanced bioengineered derivative, nisin V, alone and in combination with low concentrations of the essential oils thymol, carvacrol, and trans-cinnamaldehyde, to control Listeria monocytogenes in both laboratory media and model food systems. Combinations of nisin V-containing SPP (25 µg/ml) with thymol (0.02%), carvacrol (0.02%), or cinnamaldehyde (0.02%) produced a significantly longer lag phase than any of the essential oil-nisin A combinations. In addition, the log reduction in cell counts achieved by the nisin V-carvacrol or nisin V-cinnamaldehyde combinations was twice that of the equivalent nisin A-essential oil treatment. Significantly, this enhanced activity was validated in model food systems against L. monocytogenes strains of food origin. We conclude that the fermentate form of nisin V in combination with carvacrol and cinnamaldehyde offers significant advantages as a novel, natural, and effective means to enhance food safety by inhibiting food-borne pathogens such as L. monocytogenes.

Inhibition of Clinical MRSA Isolates by Coagulase Negative Staphylococci of Human Origin.

Staphylococcus aureus is frequently highlighted as a priority for novel drug research due to its pathogenicity and ability to develop antibiotic resistance. Coagulase-negative staphylococci (CoNS) are resident flora of the skin and nares. Previous studies have confirmed their ability to kill and prevent colonization by S. aureus through the production of bioactive substances. This study screened a bank of 37 CoNS for their ability to inhibit the growth of methicillin-resistant S. aureus (MRSA). Deferred antagonism assays, growth curves, and antibiofilm testing performed with the cell-free supernatant derived from overnight CoNS cultures indicated antimicrobial and antibiofilm effects against MRSA indicators. Whole genome sequencing and BAGEL4 analysis of 11 CoNS isolates shortlisted for the inhibitory effects they displayed against MRSA led to the identification of two strains possessing complete putative bacteriocin operons. The operons were predicted to encode a nukacin variant and a novel epilancin variant. From this point, strains Staphylococcus hominis C14 and Staphylococcus epidermidis C33 became the focus of the investigation. Through HPLC, a peptide identical to previously characterized nukacin KQU-131 and a novel epilancin variant were isolated from cultures of C14 and C33, respectively. Mass spectrometry confirmed the presence of each peptide in the active fractions. Spot-on-lawn assays demonstrated both bacteriocins could inhibit the growth of an MRSA indicator. The identification of natural products with clinically relevant activity is important in today's climate of escalating antimicrobial resistance and a depleting antibiotic pipeline. These findings also highlight the prospective role CoNS may play as a source of bioactive substances with activity against critical pathogens.

In vivo activity of nisin A and nisin V against Listeria monocytogenes in mice.

BACKGROUND: Lantibiotics are post-translationally modified antimicrobial peptides, of which nisin A is the most extensively studied example. Bioengineering of nisin A has resulted in the generation of derivatives with increased in vitro potency against Gram-positive bacteria. Of these, nisin V (containing a Met21Val change) is noteworthy by virtue of exhibiting enhanced antimicrobial efficacy against a wide range of clinical and food-borne pathogens, including Listeria monocytogenes. However, this increased potency has not been tested in vivo. RESULTS: Here we address this issue by assessing the ability of nisin A and nisin V to control a bioluminescent strain of Listeria monocytogenes EGDe in a murine infection model.More specifically, Balb/c mice were infected via the intraperitoneal route at a dose of 1 × 10(5) cfu/animal and subsequently treated intraperitoneally with either nisin V, nisin A or a PBS control. Bioimaging of the mice was carried out on day 3 of the trial. Animals were then sacrificed and levels of infection were quantified in the liver and spleen. CONCLUSION: This analysis revealed that nisin V was more effective than Nisin A with respect to controlling infection and therefore merits further investigation with a view to potential chemotherapeutic applications.

After a century of nisin research - where are we now?

It is almost a century since nisin was discovered in fermented milk cultures, coincidentally in the same year that penicillin was first described. Over the last 100 years this small, highly modified pentacyclic peptide has not only found success in the food industry as a preservative but has also served as the paradigm for our understanding of the genetic organization, expression, and regulation of genes involved in lantibiotic biosynthesis-one of the few cases of extensive post-translation modification in prokaryotes. Recent developments in understanding the complex biosynthesis of nisin have shed light on the cellular location of the modification and transport machinery and the co-ordinated series of spatio-temporal events required to produce active nisin and provide resistance and immunity. The continued unearthing of new natural variants from within human and animal gastrointestinal tracts has sparked interest in the potential application of nisin to influence the microbiome, given the growing recognition of the role the gastrointestinal microbiota plays in health and disease. Moreover, interdisciplinary approaches have taken advantage of biotechnological advancements to bioengineer nisin to produce novel variants and expand nisin functionality for applications in the biomedical field. This review will discuss the latest progress in these aspects of nisin research.

Impact of bacteriocin-producing strains on bacterial community composition in a simplified human intestinal microbiota.

Bacteriocins are antimicrobial peptides that have been studied for decades as food bio-preservatives or as alternatives to antibiotics. They also have potential as modulators of the gut microbiome, which has been linked to human health. However, it is difficult to predict a priori how bacteriocins will impact complex microbial communities through direct and indirect effects. Here we assess the effect of different bacteriocin-producing strains on a Simplified Human Intestinal Microbiota (SIHUMI) model, using a set of bacteriocin-producing strains (Bac+) and otherwise isogenic non-producers (Bac-). Bacteriocins from different classes and with different activity spectra were selected, including lantibiotics such as lacticin 3147 and nisin A, and pediocin-like bacteriocins such as pediocin PA-1 among other peptides. SIHUMI is a bacterial consortium of seven diverse human gut species that assembles to a predictable final composition in a particular growth medium. Each member can be individually tracked by qPCR. Bac+ and Bac- strains were superimposed on the SIHUMI system, and samples were taken at intervals up to 48 h. The genome copy number of each SIHUMI member was evaluated using specific primers. We establish that the composition of the community changes in response to the presence of either broad- or narrow-spectrum bacteriocin producers and confirm that there are significant off-target effects. These effects were analyzed considering antagonistic inter-species interactions within the SIHUMI community, providing a comprehensive insight into the possible mechanisms by which complex communities can be shaped by bacteriocins.

Bioengineered Nisin A Derivatives Display Enhanced Activity against Clinical Neonatal Pathogens.

Neonatal infection is a significant cause of mortality and morbidity in infants. The global incidence of multi-drug resistance continues to rise among neonatal pathogens, indicating a need for alternative treatment strategies. Nisin is an antimicrobial peptide that exhibits broad-spectrum activity against a wide variety of clinical pathogens and can be used in combination with antibiotics to improve their effectiveness. This study examined the activity of nisin and bioengineered derivatives against multi-drug resistant Streptococcus agalactiae and Staphylococcus capitis isolates and investigated the potential synergy between nisin peptides and selected antibiotics. Whole genome sequence analysis of the strains revealed the presence of multi-drug resistant determinants, e.g., macrolide, tetracycline, ß-lactam, aminoglycoside, while the S. agalactiae strains all possessed both nsr and nsrFP genes and the S. capitis strains were found to encode the nsr gene alone. Deferred antagonism assays demonstrated that nisin PV had improved antimicrobial activity against all strains tested (n = 10). The enhanced specific activity of this peptide was confirmed using minimum inhibitory concentrations (MIC) (0-4-fold lower MIC for nisin PV) and broth-based survival assays. Combinations of nisin peptides with antibiotics were assessed for enhanced antimicrobial activity using growth and time-kill assays and revealed a more effective nisin PV/ampicillin combination against one S. capitis strain while a nisin A/erythromycin combination displayed a synergistic effect against one S. agalactiae strain. The findings of this study suggest that nisin derivatives alone and in combination with antibiotics have potential as alternative antimicrobial strategies to target neonatal pathogens.

The dawning of a 'Golden era' in lantibiotic bioengineering.

There are many examples of highly modified antimicrobial peptides in nature, many of which are non-ribosomally synthesized. However, the bacterial lantibiotics are produced as gene-encoded pre-peptides that are subsequently modified by dedicated enzyme systems to form extraordinarily potent inhibitors. Consequently, they are much more amenable to bioengineering which could lead to the generation of a new arsenal of potent antimicrobials. However, although bioengineering of these compounds has been underway for at least two decades, significant progress has only been reported in recent years. This review charts these recent developments which suggest that we are entering a 'Golden era' of lantibiotic bioengineering.

Recipe for Success: Suggestions and Recommendations for the Isolation and Characterisation of Bacteriocins.

Bacteriocins are bacterially produced antimicrobial peptides. Although only two peptides have been approved for use as natural preservatives foods, current research is focusing on expanding their application as potential therapeutics against clinical pathogens. Our laboratory group has been working on bacteriocins for over 25 years, and during that time, we have isolated bacteriocin-producing microorganisms from a variety of sources including human skin, human faeces, and various foods. These bacteriocins were purified and characterised, and their potential applications were examined. We have also identified bioengineered derivatives of the prototype lantibiotic nisin which possess more desirable properties than the wild-type, such as enhanced antimicrobial activity. In the current communication, we discuss the main methods that were employed to identify such peptides. Furthermore, we provide a step-by-step guide to carrying out these methods that include accompanying diagrams. We hope that our recommendations and advice will be of use to others in their search for, and subsequent analysis of, novel bacteriocins, and derivatives thereof.

Simultaneous Production of Multiple Antimicrobial Compounds by Bacillus velezensis ML122-2 Isolated From Assam Tea Leaf [Camellia sinensis var. assamica (J.W.Mast.) Kitam.].

Bacillus velezensis ML122-2 is an antimicrobial-producing strain isolated from the leaf of Assam tea or Miang [Camellia sinensis var. assamica (J.W.Mast.) Kitam.]. The cell-free supernatant (CFS) of strain ML122-2 exhibits a broad-spectrum antimicrobial activity against various Gram-positive and Gram-negative bacteria as well as the mold Penicillium expansum. The genome of B. velezensis ML122-2 was sequenced and in silico analysis identified three potential bacteriocin-associated gene clusters, that is, those involved in the production of mersacidin, amylocyclicin, and LCI. Furthermore, six gene clusters exhibiting homology (75-100% DNA sequence identity) to those associated with the secondary metabolites bacilysin, bacillibactin, surfactin, macrolactin H, bacillaene, and plipastatin were identified. Individual antimicrobial activities produced by B. velezensis ML122-2 were purified and characterized by Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry analysis, revealing three antimicrobial peptides with molecular masses corresponding to surfactin, plipastatin, and amylocyclicin. Transcriptional analysis of specific genes associated with mersacidin (mrsA), amylocyclicin (acnA), plipastatin (ppsA), and surfactin (srfAA) production by B. velezensis ML122-2 showed that the first was not transcribed under the conditions tested, while the latter three were consistent with the presence of the associated peptides as determined by mass spectrometry analysis. These findings demonstrate that B. velezensis ML122-2 has the genetic capacity to produce a wide range of antimicrobial activities that may support a specific community structure and highlight the biotechnological properties of Assam tea.

Bioengineered Nisin Derivative M17Q Has Enhanced Activity against Staphylococcus epidermidis.

Staphylococcus epidermidis is frequently implicated in medical device-related infections. As a result of this, novel approaches for control of this opportunistic pathogen are required. We examined the ability of the natural peptide nisin A, produced by Lactococcus lactis, to inhibit S. epidermidis. In addition, a bank of 29 rationally selected bioengineered L. lactis strains were examined with the aim of identifying a nisin derivative with enhanced antimicrobial activity. Agar-based deferred antagonism assays revealed that wild type nisin A inhibited all 18 S. epidermidis strains tested. Larger zones of inhibition than those obtained from the nisin A producing L. lactis strain were observed for each derivative producer against at least one S. epidermidis strain tested. Six derivative producing strains, (VGA, VGT, SGK, M21A, M17Q, AAA), gave larger zones against all 18 strains compared to the wildtype producing strain. The enhanced bioactivity of M17Q was confirmed using well diffusion, minimum inhibitory concentration (MIC) and a broth-based survival assays. Biofilm assays were performed with plastic microtiter plates and medical device substrates (stainless-steel coupons and three catheter materials). The presence of nisin A significantly reduce the amount of biofilm formed on all surfaces. M17Q was significantly better at reducing biofilm production than nisin A on plastic and stainless-steel. Finally, M17Q was significantly better than nisin A at reducing bacterial numbers in a simulated wound fluid. The findings of this study suggest that nisin and bioengineered derivatives warrant further investigation as potential strategies for the control of S. epidermidis.