Production of plantaricin NC8 by Lactobacillus plantarum NC8 is induced in the presence of different types of gram-positive bacteria

Lactobacillus plantarum NC8 was shown to produce plantaricin NC8 (PLNC8), a recently purified and genetically characterized inducible class IIb bacteriocin, only when it was co-cultured with other gram-positive bacteria. Among 82 strains belonging to the genera Bacillus, Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Listeria, Pediococcus, Staphylococcus, and Streptococcus, 41 were shown to induce PLNC8 production in L. plantarum NC8. There was apparently no relationship between the sensitivity of the strains and their ability to induce the bacteriocin, indicating that the inducer and sensitive phenotypes may not be linked. In some instances, induction was promoted by both living and heat-killed cells of the inducing bacteria. However, no PLNC8-inducing activity was found in the respective cell-free, pure culture supernatants. Inducer strains also promoted the production of a PLNC8-autoinducing activity by L. plantarum NC8, which was found only in the cell-free co-culture supernatants showing inhibitory activity. This PLNC8-autoinducing activity was diffusible, heat resistant, and of a proteinaceous nature, and was different from the bacteriocin itself. Taken together, the results suggest that the presence of specific gram-positive bacteria acts as an environmental stimulus activating both PLNC8 production by L. plantarum NC8 and a PLNC8-autoinducing activity, which in turn triggers or maintains bacteriocin production in the absence of inducing cells.     

Purification and genetic characterization of plantaricin NC8, a novel coculture-inducible two-peptide bacteriocin from Lactobacillus plantarum NC8

A new, coculture-inducible two-peptide bacteriocin named plantaricin NC8 (PLNC8) was isolated from Lactobacillus plantarum NC8 cultures which had been induced with Lactococcus lactis MG1363 or Pediococcus pentosaceus FBB63. This bacteriocin consists of two distinct peptides, named alpha and beta, which were separated by C(2)-C(18) reverse-phase chromatography and whose complementary action is necessary for full plantaricin NC8 activity. N-terminal sequencing of both purified peptides showed 28 and 34 amino acids residues for PLNC8 alpha and PLNC8 beta, respectively, which showed no sequence similarity to other known bacteriocins. Mass spectrometry analysis showed molecular masses of 3,587 Da (alpha) and 4,000 Da (beta). The corresponding genes, designated plNC8A and plNC8B, were sequenced, and their nucleotide sequences revealed that both peptides are produced as bacteriocin precursors of 47 and 55 amino acids, respectively, which include N-terminal leader sequences of the double-glycine type. The mature alpha and beta peptides contain 29 and 34 amino acids, respectively. An open reading frame, orfC, which encodes a putative immunity protein was found downstream of plNC8B and overlapping plNC8A. Upstream of the putative -35 region of plNC8B, two direct repeats of 9 bp were identified, which agrees with the consensus sequence and structure of promoters of class II bacteriocin operons whose expression is dependent on an autoinduction mechanism.     

Factors affecting the production of an antimicrobial agent, plantaricin F, by Lactobacillus plantarum BF001

Lactobacillus plantarum BF001 produced plantaricin F in MRS broth but it was detected only after ca a 50-fold concentration. Growth on MRS broth and appearance of plantaricin F were similar under aerobic and anaerobic conditions. No growth occurred at pH 3 or at 4°C. Plantaricin F appeared first at early stationary growth phase (24 h) and was stable thereafter (pH 2). Amounts found in liquid cultures were ca 2–3 times higher than those from solidified MRS medium, and specific activities were ca 6 times higher in liquid culture (48 h). Maximal amounts of plantaricin F were found (48 h) when medium had an initial pH of 4 and growth was at 30°C. Under these conditions, cell growth and fermentation were partially uncoupled. Plantaricin F was not produced endogenously, organic nutrients were necessary. A molecular weight range of 500–3500 Da was indicated. Plantaricin F appears to be a secondary metabolite.     

Strain- and matrix-dependent adhesion of Lactobacillus plantarum is mediated by proteinaceous bacterial compounds

AIMS: The ability of 31 Lactobacillus plantarum strains to adhere to biological matrixes was evaluated, and the molecules involved in adherence were studied. METHODS AND RESULTS: Mucin, basement membrane proteins and Caco-2 cells were used in adhesion tests. These in vitro assays, together with a yeast agglutination test, were found to be discriminative for screening Lact. plantarum strains for adhesion. Some strains, such as 299v, CBE, BMCM12, Col4S and T25, were shown to possess interesting adhesion properties in at least two models. The adhesion of these strains was strongly inhibited when the bacterial cells were pretreated with trypsin. Lithium chloride and methyl-alpha-D-mannoside also inhibited adhesion to a lower extent. CONCLUSIONS: The adhesion of Lact. plantarum depends on both the model and the strain used. The chemical and enzymatic pretreatments applied to the bacterial cells suggested that lectin-like adhesins and other proteinaceous cell-surface structures are involved in adhesion of these strains. SIGNIFICANCE AND IMPACT OF THE STUDY: We found a great diversity in the adhesion properties between Lact. plantarum strains. Based upon the adhesive property of these strains interesting candidates were identified, that will undergo further study as potential probiotics.     

Adsorption of plantaricin Q7 on montmorillonite and application in feedback regulation of plantaricin Q7 synthesis by Lactobacillus plantarum Q7

Kieselguhr, bentonite, and montmorillonite were investigated as potential carriers of plantaricin Q7. Highest level of adsorption of plantaricin Q7 was obtained with montmorillonite. Meanwhile, visible inhibition zones were observed against Listeria monocytogenes for montmorillonite adsorbed with plantaricin Q7. Adsorption kinetics showed that the adsorption behaviour followed the pseudo‐first‐order and Weber's intra‐particle diffusion models, suggesting two steps had taken place during the adsorption process. X‐ray diffraction assays revealed that plantaricin Q7 was intercalated into the interlayer space of montmorillonites. Electrostatic, hydrogen bonding and hydrophobic interactions proved to play important roles in the mechanisms of interaction between montmorillonite and plantaricin Q7, as shown by infrared spectroscopy analysis. In addition, plantaricin Q7 production was inhibited by feedback regulation with its high concentrations. In order to remove product feedback inhibition in plantaricin Q7 production, a strategy was implemented for its adsorption onto montmorillonite during fermentation. The final plantaricin Q7 output reached 3713.40 IU/mL during fermentation using montmorillonite to adsorb plantaricin Q7, 41.61% higher than that of non‐ montmorillonite. These results indicate that montmorillonites are suitable carriers for plantaricin Q7 adsorption, and the adsorption of plantaricin Q7 onto montmorillonite during fermentation could be a good method to increase final plantaricin Q7 production.     

Recognition of gram-positive intestinal bacteria by hybridoma- and colostrum-derived secretory immunoglobulin A is mediated by carbohydrates

Humans live in symbiosis with 10(14) commensal bacteria among which >99% resides in their gastrointestinal tract. The molecular bases pertaining to the interaction between mucosal secretory IgA (SIgA) and bacteria residing in the intestine are not known. Previous studies have demonstrated that commensals are naturally coated by SIgA in the gut lumen. Thus, understanding how natural SIgA interacts with commensal bacteria can provide new clues on its multiple functions at mucosal surfaces. Using fluorescently labeled, nonspecific SIgA or secretory component (SC), we visualized by confocal microscopy the interaction with various commensal bacteria, including Lactobacillus, Bifidobacteria, Escherichia coli, and Bacteroides strains. These experiments revealed that the interaction between SIgA and commensal bacteria involves Fab- and Fc-independent structural motifs, featuring SC as a crucial partner. Removal of glycans present on free SC or bound in SIgA resulted in a drastic drop in the interaction with gram-positive bacteria, indicating the essential role of carbohydrates in the process. In contrast, poor binding of gram-positive bacteria by control IgG was observed. The interaction with gram-negative bacteria was preserved whatever the molecular form of protein partner used, suggesting the involvement of different binding motifs. Purified SIgA and SC from either mouse hybridoma cells or human colostrum exhibited identical patterns of recognition for gram-positive bacteria, emphasizing conserved plasticity between species. Thus, sugar-mediated binding of commensals by SIgA highlights the currently underappreciated role of glycans in mediating the interaction between a highly diverse microbiota and the mucosal immune system.     

Lactonase-expressing Lactobacillus plantarum NC8 attenuates the virulence factors of multiple drug resistant Pseudomonas aeruginosa in co-culturing environment

Pseudomonas aeruginosa possesses an arcade of both cell-associated and extracellular cytotoxic virulence factors which are regulated by a multi-component quorum sensing system. Many research studies report success of lactonase in combating the pathogenicity of P. aeruginosa but delivery of lactonase remains a challenge. The present study aims at developing a delivery vehicle for lactonase. Lactobacillus plantarum NC8 was used as host for aiiA (Bacillus thuringiensis 4A3 lactonase gene) using pSIP409 expression vector. pSIP409: aiiA construct was stably maintained in L. plantarum NC8. Co-culturing of multi-drug resistant (MDR) clinical isolates of P. aeruginosa and PAO1 with recombinant L. plantarum NC8 led to significant reduction (p < 0.001) in extracellular virulence factors like pyocyanin, protease, elastase and rhamnolipids in P. aeruginosa and also showed significant reduction in adhesion of P. aeruginosa strains to uroepithelial cells in vitro. This study shows the heterologous expression of AiiA lactonase in L. plantarum NC8. Co-culturing of lactonase expressing L. plantarum NC8 with MDR P. aeruginosa strains led to attenuation of their virulence significantly. These results underscore the potential application of recombinant L. plantarum NC8 with anti-quorum sensing properties to control infections caused by multidrug resistant P. aeruginosa.     

Antimicrobial activity of lactic acid bacteria isolated from Tenerife cheese: initial characterization of plantaricin TF711, a bacteriocin-like substance produced by Lactobacillus plantarum TF711

AIMS: The screening and initial characterization of bacteriocins produced by lactic acid bacteria (LAB) from raw Tenerife goats' cheese with possible application as biopreservatives or ripening accelerators for Tenerife cheese. METHODS AND RESULTS: One hundred and eighty LAB of the genera Lactobacillus (95), Leuconostoc (64) and Lactococcus (21) isolated from raw Tenerife goats' cheese were screened for the production of antimicrobial substances. Lactobacillus plantarum TF711, which had the broadest spectrum of antimicrobial activity, was selected for further characterization. The antimicrobial compound was determined as a proteinaceous substance, as it was sensitive to proteases. The bacteriocin-like substance, which we called plantaricin TF711, was active against the Gram-positive bacteria Bacillus cereus, Clostridium sporogenes and Staphylococcus aureus; and against the Enterobacteriaceae Shigella sonnei and Klebsiella pneumoniae. It was stable to heat and to treatment with surfactants and organic solvents. Highest antimicrobial activity was found between pH 1 and 9. Plantaricin TF711 exhibited primary metabolite kinetics, a bacteriostatic mode of action and a molecular mass of c. 2.5 kDa as determined by tricine SDS-PAGE. CONCLUSIONS: Lact. plantarum TF711 produces a low molecular mass bacteriocin-like compound with a wide spectrum of activity and interesting technological properties (thermostability, good pH stability and stability against surfactants and organic solvents). SIGNIFICANCE AND IMPACT OF THE STUDY: Plantaricin TF711 was found to have potential for use as a biopreservative in the food industry.     

Differences in Toll-like receptor expression and cytokine production after stimulation with heat-killed gram-positive and gram-negative bacteria

Innate immune surveillance in the blood is executed mostly by circulating monocytes, which recognize conserved bacterial molecules such as peptidoglycan and lipopolysaccharide. Toll-like receptors (TLR) play a central role in microbe-associated molecular pattern detection. The aim of this study was to compare the differences in TLR expression and cytokine production after stimulation of peripheral blood cells with heat-killed gram-negative and gram-positive human pathogens: Neisseria meningitidis, Escherichia coli, Staphylococcus aureus, and Streptococcus pneumoniae. We found that TLR2 expression is up-regulated on monocytes after stimulation with S. aureus, S. pneumoniae, E. coli, and N. meningitidis. Moreover, TLR2 up-regulation was positively associated with increasing concentrations of gram-positive bacteria, whereas higher concentrations of gram-negative bacteria, especially E. coli, caused a milder TLR2 expression increase when compared to low doses. Cytokines were produced in similar dose-dependent profiles regardless of the stimulatory pathogen; however, gram-negative pathogens induced higher cytokine levels when compared to gram-positive bacteria at the same density. These results indicate that gram-positive and gram-negative bacteria differ in their dose-dependent patterns of induction of TLR2 and TLR4, but not cytokine expression.     

Adhesion of Lactobacillus plantarum 423 and Lactobacillus salivarius 241 to the intestinal tract of piglets, as recorded with fluorescent in situ hybridization (FISH), and production of plantaricin 423 by cells colonized to the ileum

AIMS: To determine which intestinal section of pre and postweaned piglets are colonized by Lactobacillus plantarum 423 and Lactobacillus salivarius 241, and follow production of plantaricin 423 in a gastro-intestinal model. METHODS AND RESULTS: Lactobacillus plantarum 423 and Lact. salivarius 241, single or in combination, were administered to 1-, 14- and 28-day-old (postweaned) piglets. According to results obtained by fluorescent in situ hybridization (FISH), Lact. plantarum 423 adhered strongly to the ileum and posterior colon and Lact. salivarius 241 to the duodenum in preweaned piglets. High numbers of strain 241 were recorded in the duodenum and posterior colon of postweaned piglets, whereas strain 423 remained localized to the ileum. Lowering in Enterococcus faecalis cell numbers were recorded when preweaned piglets were challenged with strain 241. Plantaricin 423 was produced for 96 h in the ileum section of a gastro-intestinal model. CONCLUSIONS: Lactobacillus plantarum 423 and Lact. salivarius 241 adhere to different sections of the intestinal tract, depending on the piglet's age. Ent. faecalis were inhibited in vivo, probably by plantaricin 423. SIGNIFICANCE AND IMPACT OF THE STUDY: Fluorescent in situ hybridization proved valuable in the detection of probiotic bacteria adhered to the intestine. This is the first report of bacteriocin production in a model simulating the porcine gastro-intestinal tract.     

Structure and mechanism of action of an indolicidin peptide derivative with improved activity against gram-positive bacteria

Indolicidin, an antimicrobial peptide with a unique amino acid sequence (ILPWKWPWWPWRR-NH(2)) is found in bovine neutrophils. A derivative of indolicidin, CP10A, has alanine residues substituted for proline residues and has improved activity against Gram-positive organisms. Transmission electron microscopy of Staphylococcus aureus and Staphylococcus epidermidis treated with CP10A showed mesosome-like structures in the cytoplasm. The peptide at 2-fold the minimal inhibitory concentration did not show significant killing of S. aureus ISP67 (a histidine, uridine, and thymidine auxotroph) but did show an early effect on histidine and uridine incorporation and, later, an effect on thymidine incorporation. Upon interaction with liposomes, detergents, and lipoteichoic acid, CP10A was shown by circular dichroism spectroscopy to undergo a change in secondary structure. Fluorescence spectroscopy indicated that the tryptophan residues were located at the hydrophobic/hydrophilic interface of liposomes and detergent micelles and were inaccessible to the aqueous quencher KI. The three-dimensional structure of CP10A in the lipid mimetic dodecylphosphocholine was determined using two-dimensional NMR methods and was characterized as a short, amphipathic helical structure, whereas indolicidin was previously shown to have an extended structure. These studies have introduced a cationic peptide with a unique structure and an ability to interact with membranes and to affect intracellular synthesis of proteins, RNA, and DNA.     

Conversion of Phenylalanine to Benzaldehyde Initiated by an Aminotransferase in Lactobacillus plantarum

The production of benzaldehyde from phenylalanine has been studied in various microorganisms, and several metabolic pathways have been proposed in the literature for the formation of this aromatic flavor compound. In this study, we describe benzaldehyde formation from phenylalanine by using a cell extract of Lactobacillus plantarum. Phenylalanine was initially converted to phenylpyruvic acid by an aminotransferase in the cell extract, and the keto acid was further transformed to benzaldehyde. However, control experiments with boiled cell extract revealed that the subsequent conversion of phenylpyruvic acid was a chemical oxidation step. It was observed that several cations could replace the extract in the conversion of phenylpyruvic acid to benzaldehyde. Addition of Cu(II) ions to phenylpyruvic acid resulted not only in the formation of benzaldehyde, but also in the generation of phenylacetic acid, mandelic acid, and phenylglyoxylic acid. These compounds have been considered intermediates in the biological conversion of phenylalanine. The chemical conversion step of phenylpyruvic acid was dependent on temperature, pH, the availability of cations, and the presence of oxygen.     

Enhancement of tannase production by Lactobacillus plantarum CIR1: validation in gas-lift bioreactor

The optimization of tannase production by Lactobacillus plantarum CIR1 was carried out following the Taguchi methodology. The orthogonal array employed was L18 (2¹ × 3⁵) considering six important factors (pH and temperature, also phosphate, nitrogen, magnesium, and carbon sources) for tannase biosynthesis. The experimental results obtained from 18 trials were processed using the software Statistical version 7.1 using the character higher the better. Optimal culture conditions were pH, 6; temperature, 40 °C; tannic acid, 15.0 g/L; KH₂PO₄, 1.5 g/L; NH₄Cl, 7.0 g/L; and MgSO₄, 1.5 g/L which were obtained and further validated resulting in an enhance tannase yield of 2.52-fold compared with unoptimized conditions. Tannase production was further carried out in a 1-L gas-lift bioreactor where two nitrogen flows (0.5 and 1.0 vvm) were used to provide anaerobic conditions. Taguchi methodology allowed obtaining the optimal culture conditions for the production of tannase by L. plantarum CIR1. At the gas-lift bioreactor the tannase productivity yields increase 5.17 and 8.08-fold for the flow rates of 0.5 and 1.0 vvm, respectively. Lactobacillus plantarum CIR1 has the capability to produce tannase at laboratory-scale. This is the first report for bacterial tannase production using a gas-lift bioreactor.     

Optimization of probiotic and lactic acid production by Lactobacillus plantarum in submerged bioreactor systems

Biomass and lactic acid production by a Lactobacillus plantarum strain isolated from Serrano cheese, a microorganism traditionally used in foods and recognized as a potent probiotic, was optimized. Optimization procedures were carried out in submerged batch bioreactors using cheese whey as the main carbon source. Sequential experimental Plackett–Burman designs followed by central composite design (CCD) were used to assess the influence of temperature, pH, stirring, aeration rate, and concentrations of lactose, peptone, and yeast extract on biomass and lactic acid production. Results showed that temperature, pH, aeration rate, lactose, and peptone were the most influential variables for biomass formation. Under optimized conditions, the CCD for temperature and aeration rate showed that the model predicted maximal biomass production of 14.30 g l⁻¹ (dw) of L. plantarum. At the central point of the CCD, a biomass of 10.2 g l⁻¹ (dw), with conversion rates of 0.10 g of cell g⁻¹ lactose and 1.08 g lactic acid g⁻¹ lactose (w/w), was obtained. These results provide useful information about the optimal cultivation conditions for growing L. plantarum in batch bioreactors in order to boost biomass to be used as industrial probiotic and to obtain high yields of conversion of lactose to lactic acid.     

Efflux of bile acids in Lactobacillus reuteri is mediated by ATP

Purpose of work  

To study whether an active bile acid (BA) efflux occurs in Lactobacillus reuteri CRL 1098 as well as the nature (ATP or proton motive force [PMF] mediated primary transport) of the BA extrusion mechanism.     

Induction of bacteriocin production in Lactobacillus sake by a secreted peptide.

Lactobacillus sake LTH673 is known to produce a bacteriocin called sakacin P. Production of and immunity to sakacin P were found to depend on the presence of a protease-sensitive component that is produced by L. sake LTH673 itself. This component (called inducing factor [IF]) was purified from culture supernatants and shown to be a basic, nonbacteriocin peptide consisting of 19 amino acids, which in principle is capable of forming a highly amphiphilic helical structure. Circular dichroism studies showed that IF indeed could adopt a helical structure, but only in membrane-mimicking environments. Both purified IF and chemically synthesized IF induced expression of the structural gene for sakacin P and concomitant secretion of the gene product. In addition, IF induced its own production and immunity to sakacin P and related bacteriocins. These results indicate that bacteriocin production by L. sake LTH673 is controlled by an autoinduction pathway in which IF may function as a cell density signal.     

Coexistence of antibiotic-producing and antibiotic-sensitive bacteria in biofilms is mediated by resistant bacteria

Antibiotic-sensitive bacteria have been found to coexist with antibiotic-producing bacteria in biofilms, but little is known about how the former develop in such an environment. Here we isolated pyocyanin-sensitive bacteria belonging to the genus Brevibacillus from a biofilm derived from soil extract and based on the preestablished biofilm of a pyocyanin producer, Pseudomonas aeruginosa strain P1. In addition, pyocyanin-resistant strains belonging to the genus Raoultella were isolated from the same biofilm. Microbial relationships within biofilms were examined by using three strains, strain P1, Brevibacillus strain S1, and Raoultella strain R1, each of which individually formed a biofilm within 2 days in a flow cell. Strain S1 did not fully develop on the preestablished biofilm of strain P1 during 4 days of cultivation, whereas a mutant of strain P1 which was deficient in pyocyanin production allowed strain S1 to cocolonize within a biofilm. On the other hand, strain R1 developed on the biofilm of strain P1 regardless of pyocyanin production. When mixed 1:1 inocula of strains S1 and R1 were introduced into the strain P1 biofilm, all three species were found in the 4-day biofilm. In the mixed biofilm, strain S1 was surrounded by the layer of strain R1 and seemed to be separated from strain P1 and the outflow solution. However, strain S1 did not survive in a three-species mixed culture under planktonic conditions. These results indicate that the survival of sensitive bacteria in biofilm with a pyocyanin producer is achieved by covering them with a layer of resistant bacteria. We also evaluated the influence of antibiotic production on the producer.