Subinhibitory concentrations of nisin enhance virulence gene expression in Staphylococcus aureus and increase mortality in Galleria mellonella.

Alternative strategies for controlling Staphylococcus aureus and other pathogens have been continuously investigated, with nisin, a bacteriocin widely used in the food industry as a biopreservative, gaining increasing attention. In addition to its antimicrobial properties, bacteriocins have significant effects on genome functionality even at inhibitory concentrations. This study investigated the impact of subinhibitory concentrations of nisin on S. aureus. Culturing in the presence of 0.625 µmol l-1 nisin, led to the increased relative expression of hla, saeR, and sarA, genes associated with virulence while expression of the sea gene, encoding staphylococcal enterotoxin A (SEA), decreased. In an in vivo experiment, Galleria mellonella larvae inoculated with S. aureus cultured in the presence of nisin exhibited 97% mortality at 72 h post-infection, compared to over 40% of larvae mortality in larvae infected with S. aureus. A comprehensive understanding of the effect of nisin on the transcriptional response of virulence genes and the impact of these changes on the virulence of S. aureus can contribute to assessing the application of this bacteriocin in food and medical contexts.

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 (174 µM). 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.

Enhancing Antibiotic Efficacy in Regenerative Endodontics by Improving Biofilm Susceptibility.

INTRODUCTION: Various strategies have been researched to enhance the susceptibility of biofilms, given their tolerance to antibiotics. This study evaluated the effect of the anti-microbial peptide nisin in association with antibiotics used in regenerative endodontics, exploring different treatment times and biofilm growth conditions. METHODS: A mixture of 10 bacterial species was cultivated on dentin specimens anaerobically for 21 days. Biofilms were treated with 1 mL of high-purity nisin Z (nisin ZP, 200 µg/mL) and a triple antibiotic mixture (TAP: ciprofloxacin + metronidazole + minocycline, 5 mg/mL), alone or in combination. The effectiveness of antimicrobial agents was assessed after 1 and 7 days. During the 7-day period, biofilms were treated under 2 conditions: a single dose in a nutrient-depleted setting (ie, no replenishment of growth medium) and multiple doses in a nutrient-rich environment (ie, renewal of medium and antimicrobial agents every 48 h). After treatments, biofilm cells were dispersed, and total colony-forming units were counted. RESULTS: After 1 d-treatment, nisin ZP + TAP resulted in 2-log cell reduction compared to TAP alone (P < .05). After 7 d-treatment with a single dose, nisin ZP + TAP and TAP reduced bacteria to nonculturable levels (P < .05), whereas repeated antimicrobial doses did not eliminate bacteria in a nutrient-rich environment. No bacterial reduction was observed with nisin ZP alone in any treatment time. CONCLUSIONS: The additional use of nisin improved the TAP activity only after a short exposure time. Longer exposure to TAP or nisin + TAP in a nutrient-deprived environment effectively eliminated biofilms.

Effect of Nisin-based pretreatment solution on dentin bond strength, antibacterial property, and MMP activity of the adhesive interface.

OBJECTIVE: To evaluate the effect of a Nisin-based dentin pretreatment solution on microtensile bond strength, antibacterial activity, and matrix metalloproteinase (MMP) activity of the adhesive interface. MATERIALS AND METHODS: 100 human molars were sectioned to expose dentin. The teeth were assigned to five groups (n = 20), according to the dentin pretreatment: 0.5%, 1.0%, or 1.5% Nisin; 0.12% chlorhexidine (positive control), and no solution (negative control), and divided into 2 subgroups: no aging, and thermomechanical aging. Specimens were etched with 37% H3PO4 for 15 s and submitted to the dentin pretreatment. Then, they were bonded with an adhesive (Adper Single Bond 2) and a resin composite for microtensile bond strength (µTBS) evaluation. Antibacterial activity against Streptococcus mutans was qualitatively examined using an agar diffusion test. Anti-MMP activity within hybrid layers was examined using in-situ zymography. Data were analyzed with two-factor ANOVA and post-hoc Tukey's test (α = 0.050). RESULTS: For µTBS, significant differences were identified for the factors "solutions" (p = 0.002), "aging" (p = 0.017), and interaction of the two factors (p = 0.002). In the absence of aging, higher µTBS was observed for the group 0.5% Nisin. In the presence of aging, all groups showed similar µTBS values. All Nisin concentrations were effective in inhibiting the growth of S. mutans. Endogenous MMP activity was more significantly inhibited using 0.5% and 1.0% Nisin (p < 0.050). CONCLUSION: 0.5% and 1.0% Nisin solutions do not adversely affect resin-dentin bond strength and exhibit a potential bactericidal effect against S. mutans. Both concentrations effectively reduce endogenous gelatinolytic activity within the hybrid layer. CLINICAL RELEVANCE: The use of 0.5% and 1.0% Nisin solutions for dentin pretreatment potentially contributes to preserving the adhesive interface, increasing the longevity of composite restorations.

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.

Jabuticaba peel extract and nisin: A promising combination for reducing sodium nitrite in Bologna-type sausages.

This study investigated the effect of a 50% reduction in sodium nitrite and the addition of nisin (200 mg/kg) and different concentrations (0, 0.5%, 0.75%, and 1%) of jabuticaba peel extract (JPE) on the main attributes affected by this chemical additive in Bologna-type sausages. The modified treatments showed approximately 50% lower residual nitrite than the control throughout the storage (60 days at 4 °C). The proposed reformulation did not affect the color (L*, a*, and b*), and the ΔE values (< 2) demonstrated high color stability during storage. Physicochemical (TBARS and volatile compounds) and sensory analyses performed to evaluate oxidative stability indicated that JPE exhibited antioxidant activity comparable to sodium nitrite. The microbiological quality of the reformulated products was similar to the control, but further studies should be conducted to assess the effect of this reformulation strategy on the growth of pathogenic microorganisms impacted by nitrite.

Optimizing the Effects of Nisin and NaCl to Thermal Inactivate Listeria monocytogenes in Ground Beef with Chipotle Sauce During Sous-vide Processing.

Mild cooking thermal treatments, like sous-vide, can compromise ground meat entrees such as meatballs with chipotle sauce, especially when salt levels are reduced during its preparation. Listeria monocytogenes is a thermoresistant pathogen that can be in ready-to-eat food. On the other hand, nisin, due to its thermal stability, can be a good alternative to aid on the thermal inactivation of L. monocytogenes and ensure meat safety. The objective was to optimize the amount of nisin and salt concentrations to thermally inactivate L. monocytogenes during the sous-vide cooking of ground beef marinated in chipotle sauce, and to generate a predictive model. A four-strain cocktail was prepared and inoculated in ground beef in combination (3:2) with chipotle sauce added with nisin (0-150 IU) and salt (0-2%). After that, meat samples were sous-vide cooked at different temperatures, nisin, and salt concentrations, established by a central composite design. Depending on the levels of these factors, D-values ranged from 49.71 to 0.27 min. A predictive model (p < 0.05) was obtained by response surface, which described that D-values variation was explained by the linear effects of the three factors, the interaction between nisin and temperature, and the quadratic effects of salt and temperature. It was also observed that nisin presented a bactericidal effect while salt presented a protective effect during the thermal inactivation of L. monocytogenes. Adding 120 IU of nisin and 0.4% of salt to the meat product at 63°C temperature can help to ensure food safety by making L. monocytogenes cells more sensitive to the lethal effect of heat. The model developed in this study can be used by food processors for planning and designing effective levels of salt and nisin to thermally inactivate L. monocytogenes in ground beef products marinated with chipotle sauce to ensure their safety.

Headspace control and antimicrobials: Inhibition strategies to prevent the growth of Alicyclobacillus acidoterrestris in orange juice.

Alicyclobacillus acidoterrestris can cause spoilage in orange juice that leads to consumer rejection. Six different orange juices were physiochemically characterized (pH, total soluble solids, titratable acidity, total polyphenols and vitamin C). A bottle for each sampling point per juice was filled (headspace: 40% volume) and inoculated with 102 -103 CFU per ml of A. acidoterrestris ATCC® 49025™ (heat shocked before inoculation: 75°C, 20 min). Samples were stored for 21 days at 45 ± 1°C and plate counted periodically on acidified YSG agar (pH 3·7) incubated at 45 ± 1°C for 3 days. The effect of headspace (6% versus 40% volume) on A. acidoterrestris growth was also evaluated. The effect of nisin (0·006, 0·003, 0·0015, and 0·00075%), sodium benzoate (0·1%), potassium sorbate (0·1%) and a mix of benzoate and sorbate (0·05% each) on A. acidoterrestris was additionally addressed. Alicyclobacillus acidoterrestris reached up to 107 CFU per ml in five of the six juices in less than 1 week. Headspace significantly impacted (P < 0·05) A. acidoterrestris maximum population, which reached the critical value of 5 log CFU per ml at 40% headspace. All preservatives, regardless of concentration, showed a bacteriostatic effect during 22 days of storage with no significant differences amongst treatments (P > 0·05).

Listeria monocytogenes exposed to antimicrobial peptides displays differential regulation of lipids and proteins associated to stress response.

With the onset of Listeria monocytogenes resistance to the bacteriocin nisin, the search for alternative antimicrobial treatments is of fundamental importance. In this work, we set out to investigate proteins and lipids involved in the resistance mechanisms of L. monocytogenes against the antimicrobial peptides (AMPs) nisin and fengycin. The effect of sub-lethal concentrations of nisin and lipopeptide fengycin secreted by Bacillus velezensis P34 on L. monocytogenes was investigated by mass spectrometry-based lipidomics and proteomics. Both AMPs caused a differential regulation of biofilm formation, confirming the promotion of cell attachment and biofilm assembling after treatment with nisin, whereas growth inhibition was observed after fengycin treatment. Anteiso branched-chain fatty acids were detected in higher amounts in fengycin-treated samples (46.6%) as compared to nisin-treated and control samples (39.4% and 43.4%, respectively). In addition, a higher relative abundance of 30:0, 31:0 and 32:0 phosphatidylglycerol species was detected in fengycin-treated samples. The lipidomics data suggest the inhibition of biofilm formation by the fengycin treatment, while the proteomics data revealed downregulation of important cell wall proteins involved in the building of biofilms, such as the lipoteichoic acid backbone synthesis (Lmo0927) and the flagella-related (Lmo0718) proteins among others. Together, these results provide new insights into the modification of lipid and protein profiles and biofilm formation in L. monocytogenes upon exposure to antimicrobial peptides.

Biodegradable gelatin-based films with nisin and EDTA that inhibit Escherichia coli.

In this study, we developed gelatin-based films for active packaging with the ability to inhibit E. coli. We created these novel biodegradable gelatin-based films with a nisin-EDTA mix. FT-IR, TGA, and SEM analysis showed that nisin interacted with the gelatin by modifying its thermal stability and morphology. The use of nisin (2,500 IU/mL) with concentrations of Na-EDTA (1.052 M stock solution) distributed in the polymer matrix generated a significant decrease in the growth of E. coli when compared to the control. In freshly made films (t0), the growth of E. coli ATCC 25922 was reduced by approximately 3 logarithmic cycles. Two weeks after the films were made, a reduction in antimicrobial activity was observed in approximately 1, 1 and 3 logarithmic cycles of the films with 5%, 10% and 20% of the compound (nisin/Na-EDTA) distributed in the polymer matrix, respectively. This evidences an antimicrobial effect over time. Also, biodegradation tests showed that the films were completely degraded after 10 days. With all these results, an active and biodegradable packaging was successfully obtained to be potentially applied in perishable foods. These biodegradable, gelatin-based films are a versatile active packaging option. Further research on the barrier properties of these films is needed.

Bovicin HC5 and nisin reduce cell viability and the thermal resistance of Alicyclobacillus acidoterrestris endospores in fruit juices.

BACKGROUND: Alicyclobacillus acidoterrestris is an important thermoacidophilic spore-forming bacterium in fruit-juice deterioration, and alternative non-thermal methods have been investigated to control fruit juice spoilage. This work aimed to evaluate the capacity of bovicin HC5 and nisin to inhibit the growth of vegetative cells and reduce the thermal resistance of endospores of A. acidoterrestris inoculated (107 CFU mL-1 ) in different fruit juices. The number of viable cells was determined after 12 h incubation at 43 °C in the presence and absence of nisin or bovicin HC5 (10-100 AU mL-1 ). The exposure time (min) required to kill 90% of the initial population (reduction of one log factor) at 90 ºC (D90ºC ) was used to assess the thermal resistance of A. acidoterrestris endospores exposed (80 AU mL-1 ) or non-exposed to the bacteriocins. Additionally, the effect of bovicin and nisin on the morphology and cell structure of A. acidoterrestris was evaluated by atomic force microscopy (AFM). RESULTS: Bovicin HC5 and nisin were bactericidal against A. acidoterrestris inoculated in fruit juices and reduced the D90°C values up to 30-fold. AFM topographical images revealed substantial structural changes in the cellular framework of vegetative cells upon treatment with bovicin HC5 or nisin. CONCLUSIONS: These results emphasize the potential application of lantibiotics as additional hurdles in food processing to control thermoacidophilic spoilage bacteria in fruit juices. © 2022 Society of Chemical Industry.

Nisin Induces Cell-Cycle Arrest in Free-Living Amoebae Acanthamoeba castellanii.

PURPOSE: Acanthamoeba spp. are free-living amoebas with worldwide distribution and play an important role as disease-causing agents in humans. Drug inability to completely eradicate these parasites along with their toxic effects suggest urgent need for new antimicrobials. Nisin is a natural antimicrobial peptide produced by Lactococcus lactis. Nisin is also the only bacteriocin approved for use in food preservation. In this work, we analyzed the effect of nisin on the growth of Acanthamoeba castellanii trophozoites. METHODS: A total of 8 × 104 trophozoites were exposed to increasing concentrations of nisin to determine its activity. Changes in cell membrane and cellular cycle of trophozoites were investigated by flow cytometry, and nisin cytotoxicity in mammalian cells was evaluated in L929 cells by MTT method. RESULTS: After 24 h exposure to increasing nisin concentrations, an IC50 of 4493.2 IU mL-1 was obtained for A. castellanii trophozoites. However, after 72 h a recovery in amoebic growth was observed, and it was no longer possible to determine IC50. Flow cytometry analysis showed that nisin has no effect on the membrane integrity. Treatment with nisin induced cell-cycle arrest during G1 and S phases in A. castellanii trophozoites, which recovered their growth after 72 h. CONCLUSION: This is one of the first studies showing the effect of internationally approved nisin against A. castellanii trophozoites. Nisin caused cell-cycle arrest in trophozoites, momentarily interfering with the DNA replication process. The data highlight the amoebostatic activity of nisin, and suggest its use as an adjuvant for the treatment of infections caused by Acanthamoeba spp.

Citotoxicidad in vitro del polipéptido antimicrobiano nisina sobre células tumorales sanguíneas / In vitro cytotoxicity of the antimicrobial polypeptide nisin on blood tumor cells

RESUMEN: Las bacteriocinas son péptidos antimicrobianos de síntesis ribosomal secretadas por bacterias. Dentro de estas destaca nisina que posee potenciales usos en terapias antibióticas, como biopreservante de alimentos y probióticos. También se ha descrito que nisina posee citotoxicidad sobre líneas celulares neoplásicas, pero existe poca información de su efecto sobre células tumorales sanguíneas. Debido al potencial uso que presenta nisina, es relevante determinar la toxicidad que presenta sobre líneas celulares tumorales del tipo sanguíneo. Para esto, se realizaron ensayos de actividad hemolítica sobre eritrocitos humanos y de toxicidad sobre células mononucleares de sangre periférica humanas, determinándose que nisina no posee efecto citotóxico sobre este tipo de células normales humanas sanguíneas. Se realizaron también, ensayos de citotoxicidad con líneas celulares tumorales (K562 y U937), con el fin de determinar dosis, tiempo de exposición y selectividad en el efecto tóxico de nisina sobre las células tumorales humanas. Estos ensayos muestran que nisina presenta actividad citotóxica sobre líneas celulares K562 y U937 a las 72 h de exposición, a una concentración de 40 µg/mL, que corresponde a 100 veces la concentración mínima inhibitoria (MIC) usada para su acción sobre bacterias. Al comparar el efecto de nisina sobre células mononucleares de sangre periférica humanas con las líneas tumorales linfoides y mieloides (K562 y U937 respectivamente), se observa un efecto selectivo de nisina sobre las células tumorales sanguíneas.

SUMMARY: Bacteriocins are antimicrobial peptides of ribosomal synthesis secreted by bacteria. Among these, nisin stands out, which has potential uses in antibiotic therapies, as a food bio preservative and probiotics. Nisin has also been reported to have cytotoxicity on neoplastic cell lines, but there is little information on its effect on blood tumor cells. Due to the potential use that nisin presents, it is relevant to determine the toxicity it presents on tumor cell lines of the blood type. For this, hemolytic activity tests were carried out on human erythrocytes and toxicity on human peripheral blood mononuclear cells, determining that nisin does not have a toxic effect on this type of normal human blood cells. Cytotoxicity tests were also carried out with tumor cell lines (K562 and U937), to determine dose, exposure time and selectivity in the toxic effect of nisin on human tumor cells. These tests show that nisin shows cytotoxic activity on K562 and U937 cell lines at 72 h of exposure, at a concentration of 40 µg / mL, which corresponds to 100 times the minimum inhibitory concentration (MIC) used for its action on bacteria. When comparing the effect of nisin on human peripheral blood mononuclear cells with lymphoid and myeloid tumor lines (K562 and U937 respectively), a selective effect of nisin on blood tumor cells is observed.

Combined effect of carvacrol, thymol and nisin against Staphylococcus aureus and Salmonella Enteritidis.

The aim of this study was to investigate the combined effect of carvacrol, thymol and nisin against Staphylococcus aureus and the combined effect of carvacrol and thymol against Salmonella Enteritidis. Minimum inhibitory concentrations (MIC) of carvacrol, thymol, and nisin for S. aureus were 200, 150 and 30 µg/mL, respectively. MIC of carvacrol and thymol for Salmonella Enteritidis was 200 µg/mL. A factorial method of independent variables was then used to study the combined effect of antimicrobials. Results showed that combinations of carvacrol-thymol-nisin (reduction of 1.2 log CFU/mL for MIC and 4.98 log CFU/mL for 2MIC), carvacrol-thymol (reduction of 1.33 log CFU/mL for 2MIC), nisin-thymol (reduction of 3.52 log CFU/mL for 2MIC) and nisin-carvacrol (reduction of 3.41 log CFU/mL for 2MIC) attained a significant inhibition of S. aureus. Similarly, there was significant reduction of Salmonella Enteritidis due to combined effect of thymol-carvacrol (reduction of 4.5 log CFU/mL for MIC and inhibition below detection limit for 2MIC). Therefore, the combinations of natural antimicrobials described in this work showed potential to be used as an additional barrier for food safety.

Proteomic analysis reveals differential responses of Listeria monocytogenes to free and nanoencapsulated nisin.

The ability of Listeria monocytogenes grow on ready-to-eat food is a major concern in food safety. Natural antimicrobials, such as nisin, can be used to control this pathogen, but the increasing reports of nisin tolerance and resistance make necessary novel approaches to increase its effectiveness, such as encapsulation. The goal of this study was to investigate how L. monocytogenes ATCC7644 regulates and shapes its proteome in response to sublethal doses of nisin and nisin-loaded phosphatidylcholine liposomes (lipo-nisin), compared to untreated cells growing under optimal conditions. Total proteins were extracted from L. monocytogenes cells treated for 1 h with free and lipo-nisin. As result, of 803 proteins that were initially identified, 64 and 53 proteins were differentially upregulated and downregulated respectively, in the treatments with nisin and lipo-nisin. Changes of Listeria proteome in response to treatments containing nisin were mainly related to ATP-binding cassette (ABC) transporter systems, transmembrane proteins, RNA-binding proteins and diverse stress response proteins. Some of the proteins uniquely detected in samples treated with free nisin were the membrane proteins SecD, Lmo1539 and the YfhO enzyme, which are related to translocation of L. monocytogenes virulence factors, activation of the LiaR-mediated stress defense and glycosylation of wall teichoic acid, respectively. The L. monocytogenes treated with liposome encapsulated nisin showed no expression of some stress response factors as compared with the free nisin, suggesting a reduction of stress mediated response and production of nisin-resistance factors by exposure to encapsulated nisin.

Nisin resistance in Gram-positive bacteria and approaches to circumvent resistance for successful therapeutic use.

Antibiotic resistance among bacterial pathogens is one of the most worrying problems in health systems today. To solve this problem, bacteriocins from lactic acid bacteria, especially nisin, have been proposed as an alternative for controlling multidrug-resistant bacteria. Bacteriocins are antimicrobial peptides that have activity mainly against Gram-positive strains. Nisin is one of the most studied bacteriocins and is already approved for use in food preservation. Nisin is still not approved for human clinical use, but many in vitro studies have shown its therapeutic effectiveness, especially for the control of antibiotic-resistant strains. Results from in vitro studies show the emergence of nisin-resistant bacteria after exposure to nisin. Considering that nisin has shown promising results for clinical use, studies to elucidate nisin-resistant mechanisms and the development of approaches to circumvent nisin-resistance are important. Thus, the objectives of this review are to identify the Gram-positive bacterial strains that have shown resistance to nisin, describe their resistance mechanisms and propose ways to overcome the development of nisin-resistance for its successful clinical application.

Control of the growth of Alicyclobacillus acidoterrestris in industrialized orange juice using rosemary essential oil and nisin.

The use of rosemary essential oil (RO) and its combination with nisin (RO+N) in preventing the multiplication of Alicyclobacillus acidoterrestris in orange juice was evaluated. The minimum inhibitory and bactericidal concentrations (MIC and MBC) for RO were both 125 µg ml-1 while RO+N displayed a synergistic effect. The use of RO and RO+N at concentrations of 1, 4 and 8× MIC in orange juice for 96 h was evaluated in terms of their sporicidal effectiveness. With regard to the action against A. acidoterrestris spores, RO at 8× MIC was sporostatic, whereas RO+N at 1× MIC was sporicidal. Morphological changes in the structure of the micro-organism after treatment were also observed by microscopy. Furthermore, flow cytometric analysis showed that most cells were damaged or killed after treatment. In general, the antioxidant activity after addition of RO+N decreased with time. The results demonstrate that using the combination of RO and nisin can prevent the A. acidoterrestris growth in orange juice.

Metabolic extract of the endophytic fungus Flavodon flavus isolated from Justicia brandegeana in the control of Alicyclobacillus acidoterrestris in commercial orange juice.

In this work, the antibacterial activity of a crude extract of the endophytic fungus Flavodon flavus (JB257), isolated from leaves of Justicia brandegeana, was evaluated against both the vegetative and sporulated forms of Alicyclobacillus acidoterrestris. The microdilution technique was performed in order to determine the antibacterial activity of the crude extract alone as well as in combination with the bacteriocin, nisin. The minimum inhibitory concentration (MIC) of the crude extract and nisin alone against A. acidoterrestris vegetative forms were 250 µg/mL and 31.5 µg/mL, respectively, while the minimum bactericidal concentrations (MBC) were 1000 µg/mL and 62.5 µg/mL,respectively. For A. acidoterrestris spores, treatment with the crude extract at a concentration of 500 µg/mL caused a 47% reduction in growth, while nisin at 62.5 µg/mL could reduce 100% of the growth. The in vitro evaluation of the crude extract combined with nisin against A. acidoterrestris by the Checkerboard method showed a synergistic interaction between the two compounds. In addition, greater selectivity towards bacterial cells over host cells, a human hepatocyte cell line, was achieved when the crude extract was combined with nisin, Using scanning electron microscopy, interferences in the cell membrane of A. acidoterrestris could be observed after treatment with the crude extract. The results presented in this study indicate that the crude extract of the endophyte F. flavus has biotechnological potential in the food industry, especially for the treatment of orange juices through the control of A. acidoterrestris.

Nisin influence on the expression of Listeria monocytogenes surface proteins.

In this work, a comparative analysis of the peripheral cell component (PCC) proteins of Listeria monocytogenes was carried out. The study was conducted on two set of samples consisting of bacteria treated with sub-lethal concentration of nisin and untreated bacteria as control. PCC proteins were extracted by Tris-Urea-EDTA treatment and then subjected to trypsin digestion and mass spectrometry analysis. The whole cell proteome was analyzed through label-free quantitative proteomics approach. Proteomic analysis was carried out using OrbiTrap Mass Spectrometer coupled to nanoflow liquid chromatography. The treatment with sub-lethal nisin concentration resulted in 62 up regulated and 97 down regulated proteins compared to untreated samples. Using PSORTb 3.0, 19 and 18 surface proteins were detected among the up regulated and down regulated proteins, respectively. Proteins related with increased biofilm formation by L.monocytogenes, such as moonlight proteins of the pyruvate dehydrogenase complex and flagellin-related proteins, were identified as up regulated surface proteins. Proteins associated with virulence of L.monocytogenes, including listeriolysin O, internalin B and actin assembly-inducing protein, were detected among the down regulated proteins. To confirm proteomics data, increased production of biofilm was experimentally confirmed in nisin-treated cells through crystal violet method. BIOLOGICAL SIGNIFICANCE: Proteosurfaceomics can be defined as the "omics" science applied to the proteins of the peripheral cell component (PCC). The surface proteins of Listeria monocytogenes, an important foodborne pathogen were investigated after treatment with nisin, a bacteriocin approved as a natural food preservative by regulatory agencies. Recent cases of nisin tolerance by Listeria spp. were documented, and deeper studies on the molecular process behind the bacterial survival may help in both understanding the development of tolerance process and comparing nisin effect with other antimicrobial compounds.

Nisin and acid resistance in Salmonella is enhanced by N-dodecanoyl-homoserine lactone.

Salmonella is a foodborne pathogen that can develop resistance to different stresses, which is essential for successful infection of the host. Some genes directly related to acid resistance are also involved in cationic peptide resistance in Gram-negative bacteria and could be under the control of quorum sensing (QS) mediated by autoinducer 1, known as acyl-homoserine lactone. Here, we investigated the influence of autoinducer 1, N-dodecanoyl-homoserine lactone (C12-HSL) on the resistance of Salmonella enterica subspecies enterica serovar Enteritidis to nisin and acid stress. Salmonella cells growing in anaerobic tryptic soy agar (TSB) at a pH of 7.0 for 7 h were submitted to acid stress at a pH of 4.5 in the presence and absence of nisin and were either supplemented or not with C12-HSL. Viable cell counts, gene expression, membrane charge alterations, fatty acid composition, and intracellular content leakage were observed. The autoinducer C12-HSL increased nisin resistance and survival at a pH of 4.5 in Salmonella. Also, C12-HSL increased the expression of the genes, phoP, phoQ, pmrA, and pmrB, which are involved with antimicrobial and acid resistance. The positive charge on the cell surface and concentration of cyclopropane fatty acid of the cellular membrane were increased in the presence of C12-HSL under acidic conditions, whereas membrane fluidity decreased. The loss of K+ and NADPH, promoted by nisin, was reduced in the presence of C12-HSL at a pH of 4.5. Taken together, these findings suggest that quorum sensing plays an important role in enhanced nisin and acid resistance in Salmonella.