The species-specific mode of action of the antimicrobial peptide subtilosin against Listeria monocytogenes Scott A

Aims: To elucidate the molecular mechanism of action of the antimicrobial peptide subtilosin against the foodborne pathogen Listeria monocytogenes Scott A. Methods and Results: Subtilosin was purified from a culture of Bacillus amyloliquefaciens. The minimal inhibitory concentration of subtilosin against L. monocytogenes Scott A was determined by broth microdilution method. The effect of subtilosin on the transmembrane electrical potential (ΔΨ) and pH gradient (ΔpH), and its ability to induce efflux of intracellular ATP, was investigated. Subtilosin fully inhibited L. monocytogenes growth at a concentration of 19 μg ml^?1. Subtilosin caused a partial depletion of the ΔΨ and had a similar minor effect on the ΔpH. There was no significant efflux of intracellular ATP. Conclusion: Subtilosin likely acts upon L. monocytogenes Scott A by perturbing the lipid bilayer of the cellular membrane and causing intracellular damage, leading to eventual cell death. Subtilosin’s mode of action against L. monocytogenes Scott A differs from the one previously described for another human pathogen, Gardnerella vaginalis. Significance and Impact of the Study: This is the first report on the specific mode of action of subtilosin against L. monocytogenes and the first report of a bacteriocin with a species‐specific mode of action.     

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.     

Mechanism of Nisin, Pediocin 34, and Enterocin FH99 Resistance in Listeria monocytogenes

Nisin-, pediocin 34-, and enterocin FH99-resistant variants of Listeria monocytogenes ATCC 53135 were developed. In an attempt to clarify the possible mechanisms underlying bacteriocin resistance in L. monocytogenes ATCC 53135, sensitivity of the resistant strains of L. monocytogenes ATCC 53135 to nisin, pediocin 34, and enterocin FH99 in the absence and presence of different divalent cations was assessed, and the results showed that the addition of divalent cations significantly reduced the inhibitory activity of nisin, pediocin 34, and enterocin FH99 against resistant variants of L. monocytogenes ATCC 53135. The addition of EDTA, however, restored this activity suggesting that the divalent cations seem to affect the initial electrostatic interaction between the positively charged bacteriocin and the negatively charged phospholipids of the membrane. Nisin-, pediocin 34-, and enterocin-resistant variants of L. monocytogenes ATCC 53135 were more resistant to lysozyme as compared to the wild-type strain both in the presence as well as absence of nisin, pediocin 34, and enterocin FH99. Ultra structural profiles of bacteriocin-sensitive L. monocytogenes and its bacteriocin-resistant counterparts revealed that the cells of wild-type strain of L. monocytogenes were maximally in pairs or short chains, whereas, its nisin-, pediocin 34-, and enterocin FH99-resistant variants tend to form aggregates. Results indicated that without a cell wall, the acquired nisin, pediocin 34, and enterocin FH99 resistance of the variants was lost. Although the bacteriocin-resistant variants appeared to lose their acquired resistance toward nisin, pediocin 34, and enterocin FH99, the protoplasts of the resistant variants appeared to be more resistant to bacteriocins than the protoplasts of their wild-type counterparts.     

Use of bacteriocinogenic lactic acid bacteria to inhibit spontaneous nisin-resistant mutants of Listeria monocytogenes Scott A

Nisin is a bacteriocin with a broad antibacterial spectrum including strains of Listeria monocytogenes . Populations of L. monocytogenes , however, frequently contain spontaneous nisin-resistant mutants. When a culture of L. monocytogenes Scott A was exposed to nisin concentrations between 10 and 500 IU ml⁻¹, the initial decrease in viable numbers was followed by regrowth of survivors to nisin. Nisin-resistant mutants of L. monocytogenes Scott A were isolated after a single exposure to nisin at 100 IU ml⁻¹ and were shown to be sensitive to the non-nisin bacteriocins, sakacin A and enterocin B, produced by Lactobacillus sake Lb 706 and Enterococcus faecium BFE 900, respectively. The regrowth of L. monocytogenes Scott A following the initial decrease due to exposure to nisin was prevented by nisin-resistant Lact. sake Lb 706–1a and to a somewhat lesser extent, by Ent. faecium BFE 900–6a. Listerial cells surviving nisin action were thus inhibited by the bacteriocin-producing strains that might be used as starter or protective cultures in foods. Growth of a nisin-resistant mutant of L. monocytogenes Scott A (Li3) was also suppressed by the bacteriocinogenic cultures. Use of nisin in combination with a starter culture producing a non-nisin antilisterial bacteriocin may therefore prevent the emergence of nisin-resistant mutants of L. monocytogenes .     

Selection and Identification of a Listeria monocytogenes Target Strain for Pulsed Electric Field Process Optimization

Nine Listeria monocytogenes strains were treated individually with a continuous pulsed electric field (PEF) apparatus, and their sensitivities to the treatment were compared at 25 kV/cm. When cell suspensions of these strains in 0.1% NaCl (pH 7.0) were treated at 23°C for 144 μs, inactivation ranged from 0.7 to 3.7 log₁₀ CFU/ml. Inactivation by 72-μs PEF treatments at 37°C ranged from 0.3 to 2.5 log₁₀ CFU/ml. L. monocytogenes OSY-8578 was substantially more resistant than other strains when cells were PEF treated in 0.1% NaCl, whereas Scott A was one of the most sensitive strains. The superiority of OSY-8578's resistance to that of Scott A was confirmed in 50% diluted acid whey (pH 4.2). Changes in sensitivity to PEF during phases of growth were minimal in OSY-8578 and substantial in Scott A. Use of L. monocytogenes OSY-8578, therefore, is recommended in studies to optimize PEF processes that target L. monocytogenes. The nine L. monocytogenes strains were genotyped with pulsed-field gel electrophoresis (PFGE) and arbitrarily primed PCR (AP-PCR) techniques. These strains were better differentiated with PFGE than with AP-PCR. The target strain (OSY-8578) was characterized by both molecular typing techniques, but resistance to PEF, in general, was not associated with a particular genotype group.     

Enterocin B3A-B3B produced by LAB collected from infant faeces: potential utilization in the food industry for <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> biofilm management

Enterococcus faecalis B3A-B3B produces the bacteriocin B3A-B3B with activity against Listeria monocytogenes, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA) and Clostridium perfringens, but apparently not against fungi or Gram-negative bacteria, except for Salmonella Newport. B3A-B3B enterocin has two different nucleotides but similar amino acid composition to the class IIb MR10A-MR10B enterocin. B3A-B3B consists of two peptides of predicted molecular mass of 5176.31 Da (B3A) and 5182.21 Da (B3B). Importantly, B3A-B3B impeded biofilm formation of the foodborne pathogen L. monocytogenes 162 grown on stainless steel. The antimicrobial treatment of stainless steel with nisin (1 or 16 mg ml^?1) decreased the cell numbers by about 2 log CFU ml^?1, thereby impeding the biofilm formation by L. monocytogenes 162 or its nisin-resistant derivative strain L. monocytogenes 162R. Furthermore, the combination of nisin and B3A-B3B enterocin reduced the MIC required to inhibit this pathogen grown in planktonic or biofilm cultures.     

Nisin‐induced expression of pediocin in dairy lactic acid bacteria

Aims: To test whether a single vector, nisin‐controlled expression (NICE) system could be used to regulate expression of the pediocin operon in Streptococcus thermophilus, Lactococcus lactis subsp. lactis and Lactobacillus casei. Methods and Results: The intact pediocin operon was cloned immediately into pMSP3535 downstream of the nisA promoter (PnisA). The resulting vector, pRSNPed, was electrotransformed into Strep. thermophilus ST128, L. lactis subsp. lactis ML3 and Lact. casei C2. Presence of the intact vector was confirmed by PCR, resulting in the amplification of a 0·8‐kb DNA fragment, and inhibition zones were observed for all lactic acid bacteria (LAB) transformants following induction with 50 ng ml^?1 nisin, when Listeria monocytogenes Scott A was used as the target bacterium. Using L. monocytogenes NR30 as target, the L. lactis transformants produced hazy zones of inhibition, while the Lact. casei transformants produced clear zones of inhibition. Zones of inhibition were not observed when the Strep. thermophilus transformants were tested against NR30. Conclusions: The LAB hosts were able to produce enough pediocin to inhibit the growth of L. monocytogenes Scott A; the growth of L. monocytogenes NR30 was effectively inhibited only by the Lact. casei transformants. Significance and Impact of the Study: This is the first time that the NICE system has been used to express the intact pediocin operon in these LAB hosts. This system could allow for the in situ production of pediocin in fermented dairy foods supplemented with nisin to prevent listeria contamination.     

The Acid Tolerance Response Alters Membrane Fluidity and Induces Nisin Resistance in Listeria monocytogenes

The ability of L. monocytogenes cells to adapt to a variety of stressors contributes to its growth in a wide range of foods. The present study examines the effect of acid and of the acid tolerance response (ATR) on membrane fluidity and on the organism’s resistance to acid and to the bacteriocin nisin. When ATR was induced in wild-type cells, these cells also became resistant to nisin. ATR(+) cells also had lower membrane rigidities than control ATR(?) cells that had not been subjected to the acid tolerance response. However, cells that were genetically resistant to nisin did not show any significant (P < 0.05) change in rigidity when grown in the presence of nisin. These studies suggest that the use of acid and nisin for L. monocytogenes control in ready-to-eat foods may be compromised if cross-resistance emerges.     

Suppression of Listeria monocytogenes Scott A in Fluid Milk by Free and Liposome-Entrapped Nisin

Nisin is an antimicrobial polypeptide inhibitory toward Gram-positive bacterial pathogens, including Listeria monocytogenes. Encapsulating nisin in lipid nanocapsules (i.e., liposomes) has been shown to protect antimicrobial functionality in complex food matrices. The capacity of liposomes to encapsulate a fluorescent reporter was determined via spectroscopy. Survival and growth of L. monocytogenes incubated in fluid milk containing 50 IU/ml free or liposome-entrapped nisin was assayed via periodic enumeration of survivors. Liposomes were formulated from phosphatidylcholine (PC) and phosphatidyl-DL-glycerol (PG) and prepared as PC, PC/PG 7/3 or PC/PG 6/4 (mol. fraction). Antilisterial activity of nisin-loaded liposomes was determined in ultra-high temperature processed fluid milk containing approximately 4.0 log₁₀ CFU/ml L. monocytogenes Scott A plus liposomal or free nisin at 50 IU/mL. Samples were aerobically held at 5 or 20°C; L. monocytogenes were enumerated via plating after 0, 1, 3, 6, 12, 24, 48, and 72 incubation hours. Liposome entrapment did not enhance pathogen inhibition when compared to free nisin as a function of storage temperature or incubation duration.     

PrfA activation in Listeria monocytogenes increases the sensitivity to class IIa bacteriocins despite impaired expression of the bacteriocin receptor

BackgroundThe scope of the present work was to characterize the activity of class IIa bacteriocins in Listeria (L.) monocytogenes cells that constitutively express an activated form of PrfA, the virulence master regulator, since bacteriocin sensitivity was only characterized in saprophytic cells so far. The mannose phosphotransferase system (Man-PTS) has been shown to be the class IIa bacteriocin receptor in Listeria; hence, special attention was paid to its expression in virulent bacteria.MethodsL. monocytogenes FBprfA* cells were obtained by transconjugation. Bacterial growth was studied in TSB and glucose containing-minimal medium. Sensitivity to antimicrobial peptides was assessed by killing curves. Membranes of L. monocytogenes FBprfA* cells were characterized using proteomic and lipidomic approaches.ResultsThe mannose phosphotransferase system (Man-PTS) was downregulated upon expression of PrfA*, and these cells turned out to be more sensitive to enterocin CRL35 and pediocin PA-1, while not to nisin. Proteomic and lipidomic analysis showed differences between wild type (WT) and PrfA* strains. For instance, phosphatidic acid was only detected in PrfA* cells, whereas, there was a significant decline of plasmalogen-phosphatidylglycerol in the same strain.ConclusionsOur results support a model in which Man-PTS acts just as a docking molecule that brings class IIa bacteriocins to the plasma membrane. Furthermore, our results suggest that lipids play a crucial role in the mechanism of action of bacteriocins.General significanceThis is the first demonstration of the link between L. monocytogenes virulence and the bacterial sensitivity toward pediocin-like peptides.     

Combined Effect of Bacteriocins on the Survival of Various Listeria Species in Broth and Meat System

The antilisterial efficiency of three bacteriocins from lactic acid bacteria, lactocin 705 (produced by L. casei CRL705, 17000 AU/ml), enterocin CRL35 (produced by E. faecium CRL35, 17000 AU/ml), and nisin (2000 IU/ml), was tested in broth, individually and in combination against Listeria monocytogenes and Listeria innocua. Both Listeria species showed an initial decrease in viable counts followed by the regrowth of the survivors after 1 h in the presence of each bacteriocin. A greater antilisterial effect was observed when the bacteriocins were combined in pairs, maximal inhibition being reached when nisin was involved. When a mix of the three bacteriocins was used, no survivors were observed after 24 h of incubation. Similar results were obtained when the bacteriocin combinations were tested in a meat system, indicating that the use of more than one LAB bacteriocin in combination may be effective in preventing the spontaneous emergence of a bacteriocin-resistant Listeria population. Received: 17 March 2000 / Accepted: 26 June 2000     

Spontaneous bacteriocin resistance in Listeria monocytogenes as a susceptibility screen for identifying different mechanisms of resistance and modes of action by bacteriocins of lactic acid bacteria

A practical system was devised for grouping bacteriocins of lactic acid bacteria (LAB) based on mode of action as determined by changes in inhibitory activity to spontaneously-acquired bacteriocin resistance (Bac^R). Wild type Listeria monocytogenes 39-2 was sensitive to five bacteriocins produced by 3 genera of LAB: pediocin PA-1 and pediocin Bac3 (Pediococcus), lacticin FS97 and lacticin FS56 (Lactococcus), and curvaticin FS47 (Lactobacillus). A spontaneous Bac^R derivative of L. monocytogenes 39-2 obtained by selective recovery against lacticin FS56 provided complete resistance to the bacteriocin made by Lactococcus lactis FS56. The lacticin FS56-resistant strain of L. monocyotgenes 39-2 was also cross-resistant to curvaticin FS47 and pediocin PA-1, but not to lacticin FS97 or pediocin Bac3. The same pattern of cross-resistance was also observed with Bac^R isolates obtained with L. monocytogenes Scott A-2. A spontaneous mutation that renders a strain cross-resistant to different bacteriocins indicates that they share a common mechanism of resistance due to similar modes of action of the bacteriocins. Spontaneous resistance was acquired to other bacteriocins (in aggregate) by following the same procedure against which the Bac^R strain was still sensitive. In subsequent challenge assays, mixtures of bacteriocins of different modes of action provided greater inhibition than mixtures of bacteriocins of the same mode of action (as determined by our screening method). This study identifies a methodical approach to classify bacteriocins into functional groups based on mechanism of resistance (i.e., mode of action) that could be used for identifying the best mixture of bacteriocins for use as biopreservatives.     

Impairment of the class IIa bacteriocin receptor function and membrane structural changes are associated to enterocin CRL35 high resistance in Listeria monocytogenes

BackgroundEnterocin CRL35 is a class IIa bacteriocin with anti-Listeria activity. Resistance to these peptides has been associated with either the downregulation of the receptor expression or changes in the membrane and cell walls. The scope of the present work was to characterize enterocin CRL35 resistant Listeria strains with MICs more than 10,000 times higher than the MIC of the WT sensitive strain.MethodsListeria monocytogenes INS7 resistant isolates R2 and R3 were characterized by 16S RNA gene sequencing and rep-PCR. Bacterial growth kinetic was studied in different culture media. Plasma membranes of sensitive and resistant bacteria were characterized by FTIR and Langmuir monolayer techniques.ResultsThe growth kinetic of the resistant isolates was slower as compared to the parental strain in TSB medium. Moreover, the resistant isolates barely grew in a glucose-based synthetic medium, suggesting that these cells had a major alteration in glucose transport. Resistant bacteria also had alterations in their cell wall and, most importantly, membrane lipids. In fact, even though enterocin CRL35 was able to bind to the membrane-water interface of both resistant and parental sensitive strains, this peptide was only able to get inserted into the latter membranes.ConclusionsThese results indicate that bacteriocin receptor is altered in combination with membrane structural modifications in enterocin CRL35-resistant L. monocytogenes strains.General significanceHighly enterocin CRL35-resistant isolates derived from Listeria monocytogenes INS7 have not only an impaired glucose transport but also display structural changes in the hydrophobic core of their plasma membranes.     

Ability of the Listeria monocytogenes Strain Scott A To Cause Systemic Infection in Mice Infected by the Intragastric Route

Listeriosis is an important food-borne disease that causes high rates of morbidity and mortality. For reasons that are not clear, most large outbreaks of human listeriosis involve Listeria monocytogenes serotype 4b. Relatively little is known about the pathogenesis of listeriosis following gastrointestinal exposure to food-borne disease isolates of L. monocytogenes. In the present study, we investigated the pathogenesis of systemic infection by the food-borne isolate Scott A in an intragastric (i.g.) mouse challenge model. We found that the severity of infection with L. monocytogenes Scott A was increased in mice made neutropenic by administration of monoclonal antibody RB6-8C5. This observation was similar to a previous report on a study with the laboratory strain L. monocytogenes EGD. Prior administration of sodium bicarbonate did not enhance the virulence of L. monocytogenes strain Scott A for i.g. inoculated mice. Following i.g. inoculation of mice, two serotype 4b strains of L. monocytogenes (Scott A and 101M) achieved a greater bacterial burden in the spleen and liver and elicited more severe histopathological damage to those organs than did a serotype 1/2a strain (EGD) and a serotype 1/2b stain (CM). Of the four strains tested, only strain CM exhibited poor survival in synthetic gastric fluid in vitro. The other three strains exhibited similar patterns of survival at pHs of greater than 5 and relatively rapid (<30 min) loss of viability at pHs of less than 5.0. Growth of L. monocytogenes Scott A at temperatures of 12.5 to 37°C did not affect its ability to cause systemic infection in i.g. inoculated mice. These observations suggest that the serotype 4b L. monocytogenes strains Scott A and 101M possess one or more virulence determinants that make them better able to cause systemic infection following inoculation via the g.i. tract than do the serotype 1/2 strains EGD and CM.     

Biocontrol of Listeria monocytogenes on Fresh-Cut Produce by Treatment with Lytic Bacteriophages and a Bacteriocin

The fresh-cut produce industry has been the fastest-growing portion of the food retail market during the past 10 years, providing consumers with convenient and nutritious food. However, fresh-cut fruits and vegetables raise food safety concerns, because exposed tissue may be colonized more easily by pathogenic bacteria than intact produce. This is due to the higher availability of nutrients on cut surfaces and the greater potential for contamination because of the increased amount of handling. We found that applied Listeria monocytogenes populations survived and increased only slightly on fresh-cut Red Delicious apples stored at 10°C but increased significantly on fresh-cut honeydew melons stored at 10°C over 7 days. In addition, we examined the effect of lytic, L. monocytogenes-specific phages via two phage application methods, spraying and pipetting, on L. monocytogenes populations in artificially contaminated fresh-cut melons and apples. The phage mixture reduced L. monocytogenes populations by 2.0 to 4.6 log units over the control on honeydew melons. On apples, the reduction was below 0.4 log units. In combination with nisin (a bacteriocin), the phage mixture reduced L. monocytogenes populations by up to 5.7 log units on honeydew melon slices and by up to 2.3 log units on apple slices compared to the control. Nisin alone reduced L. monocytogenes populations by up to 3.2 log units on honeydew melon slices and by up to 2.0 log units on apple slices compared to the control. The phage titer was stable on melon slices, but declined rapidly on apple slices. The spray application of the phage and phage plus nisin reduced the bacterial numbers at least as much as the pipette application. The effectiveness of the phage treatment also depended on the initial concentration of L. monocytogenes.     

Bioengineered Nisin A Derivatives with Enhanced Activity against Both Gram Positive and Gram Negative Pathogens

Nisin is a bacteriocin widely utilized in more than 50 countries as a safe and natural antibacterial food preservative. It is the most extensively studied bacteriocin, having undergone decades of bioengineering with a view to improving function and physicochemical properties. The discovery of novel nisin variants with enhanced activity against clinical and foodborne pathogens has recently been described. We screened a randomized bank of nisin A producers and identified a variant with a serine to glycine change at position 29 (S29G), with enhanced efficacy against S. aureus SA113. Using a site-saturation mutagenesis approach we generated three more derivatives (S29A, S29D and S29E) with enhanced activity against a range of Gram positive drug resistant clinical, veterinary and food pathogens. In addition, a number of the nisin S29 derivatives displayed superior antimicrobial activity to nisin A when assessed against a range of Gram negative food-associated pathogens, including E. coli, Salmonella enterica serovar Typhimurium and Cronobacter sakazakii. This is the first report of derivatives of nisin, or indeed any lantibiotic, with enhanced antimicrobial activity against both Gram positive and Gram negative bacteria.     

Antimicrobial activity and occurrence of bacteriocin structural genes in Enterococcus spp. of human and animal origin isolated in Portugal

The main objective of this study was to detect the antimicrobial activity and the presence of bacteriocin structural genes in 224 enterococcal isolates from fecal origin obtained from humans, pets, wild animals and birds. Direct antimicrobial activity against Listeria monocytogenes CECT4032 was detected in 102 (45.6%) of the tested isolates. From these, only 22 displayed bacteriocin activity against this indicator. The bacteriocinogenic strains contained one or more of the bacteriocin structural genes tested in this study, with those of enterocins P, A and L50 (L50A and L50B) being the most abundant. Our results show a high occurrence of the combination of different bacteriocin structural genes in the enterococcal isolates analyzed, indicating an elevated genetic potential of these strains to produce various bacteriocins.     

Nisin Z produced by <Emphasis Type="Italic">Lactococcus lactis</Emphasis> from bullfrog hatchery is active against <Emphasis Type="Italic">Citrobacter freundii</Emphasis>, a red-leg syndrome related pathogen

Lactococcus lactis subsp. lactis CRL 1584 isolated from a bullfrog hatchery produces a bacteriocin that inhibits both indigenous Citrobacter freundii (a Red-Leg Syndrome related pathogen) and Lactobacillus plantarum, and Listeria monocytogenes as well. Considering that probiotics requires high cell densities and/or bacteriocin concentrations, the effect of the temperature on L. lactis growth and bacteriocin production was evaluated to find the optimal conditions. Thus, the growth rate was maximal at 36 °C, whereas the highest biomass and bacteriocin activity was achieved between 20 and 30 °C and 20–25 °C, respectively. The bacteriocin synthesis was closely growth associated reaching the maximal values at the end of the exponential phase. Since bacteriocins co-production has been evidenced in bacterial genera, a purification of the bacteriocin/s from L. lactis culture supernatants was carried out. The active fraction was purified by cationic-exchange chromatography and then, a RP-HPLC was carried out. The purified sample was a peptide with a 3353.05 Da, a molecular mass that matches nisin Z, which turned out to be the only bacteriocin produced by L. lactis CRL 1584. Nisin Z showed bactericidal effect on C. freundii and L. monocytogenes, which increased in the presence l-lactic acid?+?H₂O₂. This is the first report on nisin Z production by L. lactis from a bullfrog hatchery that resulted active on a Gram-negative pathogen. This peptide has potential probiotic for raniculture and as food biopreservative for bullfrog meat.