Production of a Class IIb Bacteriocin with Broad-spectrum Antimicrobial Activity in Lactiplantibacillus plantarum RUB1

Bacteriocins produced by lactic acid bacteria have potential use as natural food preservatives, which may alleviate current problems associated with the overuse of antibiotics and emerging multi-drug-resistant microbes. In this work, Lactiplantibacillus plantarum RUB1 was found to produce a class IIb bacteriocin with strong antibacterial activity. Except for plnXY encoding putative proteins, L. plantarum RUB1 contains most genes in five operons (plnABCD, plnGHSTUVW, plnMNOP, plnIEF, and plnRLJK) related to bacteriocin synthesis. Adding low (100 and 500 ng/mL) and medium (1 μg/mL) concentrations of PlnA to broth promoted bacteriocin production and upregulated bacteriocin gene plnA, while high concentrations (50 and 200 μg/mL) inhibited expression of these genes. Co-culturing L. plantarum RUB1 with Enterococcus hirae 1003, Enterococcus hirae LWS, Limosilactobacillus fermentum RC4, L. plantarum B6, and even Listeria monocytogenes ATCC 19111 and Staphylococcus aureus ATCC 6538 enhanced bacteriocin activity and expression of bacteriocin-related genes. This study verifies that PlnA can indeed upregulate the expression of bacteriocin genes, and also bacteriocin production can be induced by co-culture with some specific bacteria or their cell-free supernatants. Bacteriocin production by L. plantarum RUB1 is mediated by a quorum sensing mechanism, directly influenced by autoinducing peptide or specific strains. The findings provide new methods and insight into bacteriocin production mechanisms.

     

Inhibitory potential of lactobacilli against <Emphasis Type="Italic">Escherichia coli</Emphasis> internalization by HT 29 cells

A quantitative fluorescent in situ hybridization method was employed to evaluate the competitive inhibitory effect of three Lactobacillus strains (Lactobacillus reuteri, Lactobacillus gasseri, and Lactobacillus plantarum) against Escherichia coli internalization in a model system of HT 29 cells. Furthermore, aggregation and adhesion abilities of the Lactobacillus strains were examined. All lactobacilli were able to attach to the HT 29 cells and aggregate with pathogens; however, the adhesion and aggregation degree was strain-dependent. L. reuteri possessed a high capacity of adhesion (6.80 ± 0.63; log CFU ± SEM per well), whereas lower capacities were expressed by L. gasseri (4.52 ± 0.55) and L. plantarum (4.90 ± 0.98). Additionally, L. reuteri showed the rapid or normal ability to aggregate with selected E. coli in comparison with remaining two lactobacilli, which showed only slow or negative aggregative reaction. Internalization of E. coli into the cell lines was markedly suppressed by L. reuteri, while L. gasseri and L. plantarum caused only a minimum anti-invasion effect. The fact that L. reuteri in our experiments showed an outstanding potential for adhering to the colon epithelial cell line, compared with the rest strains, suggested that one of the possible mechanisms of preventing pathogen adhesion and invasion is simple competitions at certain receptors and capability to block receptor binding sites, or that an avid interaction between L. reuteri and the host cell might be modulating intracellular events responsible for the E. coli internalization. Moreover, L. reuteri exhibited a strong ability to aggregate with E. coli, which could be another limiting factor of pathogen invasion.     

Characterization of a new organization of the plantaricin locus in the inducible bacteriocin-producing<Emphasis Type="Italic"> Lactobacillus plantarum</Emphasis> J23 of grape must origin

Lactobacillus plantarum J23 was previously characterized as a bacteriocin-producer-strain when it was cocultured with other lactic acid bacteria. In this work, the genetic organization of the pln locus in the J23 strain was studied and compared with those of previously described L. plantarum C11, WCFS1 and NC8 strains. A new organization of the plantaricin locus was detected in the J23 strain. The sequenced fragment (20,266 bp) comprised plnJLR, plnMNOP, plnEFI, plnGHSTUVWXY, and plNC8IF-plNC8HK-plnD operons, as well as a new region that includes three new orfs (GenBank accession number DQ323671). When the J23 pln gene sequences were compared with those included in the GenBank database, the identity of the putative encoded proteins was in the range 67.1–100%. The regulatory system and the repertoire of putative bacteriocins of the J23 pln locus presented important differences with respect to the ones of C11, WCFS1 and NC8, such as the absence of plnK and the presence of a larger plnJ gene than the previously described for the other L. plantarum strains. The pln locus in L. plantarum strains seems to be a mosaic-like structure with different modules and reorganizations that presents highly conserved regions related to transport and bacteriocin maturation and variable regions related to regulation and bacteriocin production.     

Conjugal transfer of bacteriocin plasmids from different genera of lactic acid bacteria into Enterococcus faecalis JH2-2

The lactic acid bacteria (LAB) are of great interest because of their food grade quality and industrial importance. In the recent past, the pediocin PA-1 like bacteriocin was found to be synthesized in cross-species and cross-genera. Hence, the present work was carried out in order to determine the transfer of plasmid encoded pediocin PA-1 like bacteriocin among LAB. The objective of this study is to demonstrate the dissemination of bacteriocin-encoded plasmid from Pediococcus acidilactici NCIM 5424, Enterococcus faecium NCIM 5423 and Lactobacillus plantarum Acr2 to Enterococcus faecalis JH2-2 under in vitro (filter mating method) and in situ (soymilk model) conditions. The fermentation of the soymilk was determined by the selected pediocin producers. E. faecium NCIM 5423 was able to transfer the bacteriocin only under in situ conditions, whereas the native pediocin producer P. acidilactici NCIM 5424 transferred the bacteriocin under both the methods used. The in situ method gave more transfer frequency, ranging from 10^?7 to 10^?4 transconjugants per recipient cell. No conjugal transfer by L. plantarum Acr2 was observed. The physiological conditions like pH and temperature were found to influence the production of bacteriocin in the obtained transconjugants. The results suggest the horizontal gene transfer (HGT) and the natural spread of pediocin PA-1-like bacteriocin among LAB present in their close vicinity by means of conjugation. The dissemination of pediocin PA-1-like bacteriocin under in situ conditions is noteworthy, and such bacteriocin producers can be useful in the fermentation of dairy products and construction of novel cultures.     

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.     

Inhibition of Staphylococcus aureus adherence to Caco-2 cells by lactobacilli and cell surface properties that influence attachment

Staphylococcus aureus is an opportunistic pathogen that can colonize human and animal intestinal tracts, causing certain gastrointestinal diseases. The adherence of enteric pathogens to host intestinal epithelial cells is important for their pathogenesis. In the present study, Lactobacillus salivarius and Lactobacillus plantarum were investigated in vitro to examine their ability to competitively exclude S. aureus. Various factors involved in attachment, including bacterial status and cell concentration, growth phase, competition patterns, and surface-layer protein extracts, were also investigated. Live lactobacilli in the mid-log growth phase exhibited maximum inhibitory activity when lactobacilli were pre- or co-incubated with S. aureus. However, the inhibitory activity was significantly reduced when the lactobacilli were inactivated by heating or treated with LiCl. Furthermore, both lactobacilli possessed certain cell surface properties, such as hydrophobicity, autoaggregation, and coaggregation ability. L. salivarius and L. plantarum strongly inhibited S. aureus adherence to Caco-2 cells and their inhibition activity was significantly influenced by several factors that affect adhesion inhibition.     

Detection of Caries-Inducing Microorganisms in Hyposalivated Rats without Infection of Mutans Streptococci

Rampant dental caries was induced in hyposalivated rats fed a high sucrose diet without infection of mutans streptococci, in which increased numbers of lactobacilli and S. aureus were demonstrated in the oral flora. Administration of either penicillin or piperacillin, effective against all isolates of lactobacilli, markedly inhibited the caries induction in these rats, while severe dental caries was induced in hyposalivated rats given vancomycin that is inhibitory against S. aureus. These results suggested that certain lactobacilli might induce dental caries in hyposalivated rats fed a sucrose diet. Three strains of Lactobacillus species isolated from the hyposalivated rats were made resistant to erythromycin. The caries-inducing activity of these erythromycin-resistant lactobacilli was studied in hyposalivated rats giving erythromycin in the drinking water at a concentration of 500 μg/ml. After a 61-day experimental period, severe dental caries was induced in hyposalivated rats infected with L. fermentum TY1R. On the other hand, low caries incidence was found in hyposalivated rats infected with either L. acidophilus TY7R or L. plantarum TY3R. These results indicate that L. fermentum may be one of causative agents of dental caries in hyposalivated rats fed a sucrose diet.     

Evidence for in vitro anti-genotoxicity of cheese non-starter lactobacilli

The inhibition of direct acting DNA reactive agents by 63 non-starter lactobacilli isolated from raw ewes milk cheeses was examined by short-term assay (SOS-Chromotest) and compared with already characterized starter lactobacilli. The screening revealed strains active against the nitroarene 4-nitroquinoline-1-oxide (NQO) and the alkylating agent N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) in different species of the genus Lactobacillus (L. rhamnosus, L. casei, L. plantarum, L. brevis, Lactobacillus spp.). It was proved that the anti-genotoxicity was strain-dependent, and always associated with spectroscopic modification of genotoxins. The frequency of strains inhibiting nitroarene genotoxicity was comparable for non-starter and starter lactobacilli, whereas inhibition of the alkylating agent was largely predominant in non-starter isolates. Seventeen strains presented inhibitory activity against both genotoxins. DNA RAPD-PCR performed with M13, Pro-Up and RPO2 primers on the lactobacilli under examination showed genetic diversity in these strains. The non-starter isolates clustered in seven groups and the strains presenting a high degree of activity against 4-nitroquinoline-1-oxide clustered in a single group with a similarity around 75%. Interestingly, the strains with anti-genotoxic properties also showed acid-bile tolerance, indicating that the autochthonous lactobacilli which survive cheese ripening may also reach the gut as viable cells and could prevent genotoxin DNA damage to enterocytes, as is desirable for probiotic bacteria.     

In Vitro Evaluation of the Probiotic Potential of Halotolerant Lactobacilli Isolated from a Ripened Tropical Mexican Cheese

Three halotolerant lactobacilli (Lactobacillus plantarum, L. pentosus, and L. acidipiscis) isolated from a ripened Mexican tropical cheese (double cream Chiapas cheese) were evaluated as potential probiotics and compared with two commercial probiotic strains (L. casei Shirota and L. plantarum 299v) from human origin. All the strains survived the in vitro gastrointestinal simulation from the oral cavity to the ileum. During the stomach simulation, all the strains survived in satiety conditions (60 min, pH 3.0, 3 g/L pepsin, 150 rpm) and only L. pentosus could not survive under fasting conditions (60 min, pH 2.0, 3 g/L pepsin, 150 rpm). All the strains showed a strong hydrophilic character with low n-hexadecane and a variable chloroform affinity. L. plantarum showed a mucin adhesion rate similar to that of L. plantarum 299v and L. casei Shirota, while L. pentosus and L. acidipiscis had a lower mucin adhesion. The isolated halotolerant lactobacilli exhibited similar antimicrobial activity against some gram-positive and gram-negative pathogens in comparison with the two commercial strains. In addition, the proteinaceous character of the antimicrobial agents against the most pathogenic strains was demonstrated. The compounds showed a low molecular weight (less than 10 kDa). Besides, L. plantarum and L. acidipiscis were able to produce the enzyme β-galactosidase. Finally, L. pentosus was able to deconjugate taurocholic, taurodeoxycholic, glycocholic, and glycodeoxycholic acids better than the two commercial strains analyzed. All these results suggest that the halotolerant lactobacilli isolated from this ripened Mexican cheese could be potentially probiotic. This is the first time that halotolerant lactic acid bacteria have been shown to have probiotic properties.     

Characterization of the beneficial properties of lactobacilli isolated from bullfrog (<Emphasis Type="Italic">Rana catesbeiana</Emphasis>) hatchery

The present work addresses the isolation and partial identification of the microbial population of a R. catesbeiana hatchery in spring and summer as well as some beneficial properties of Lactobacillus strains isolated in different seasons and hatchery areas. The bacterial population was grouped into the following taxa: Lactobacillus spp., Pediococcus spp., Enterococcus faecalis and Ent. faecium, and Enterobacteriaceae (Enterobacter spp., Escherichia coli) while Pseudomonas aeruginosa and Staphylococcus epidermidis were isolated from frogs displaying red-leg syndrome. The Lactobacillus plantarum and L. curvatus strains isolated showed to inhibit the growth of red-leg syndrome associated pathogens and food-borne bacteria by organic acids. While L. plantarum CRL 1606 also inhibited red-leg syndrome related pathogens by hydrogen peroxide, meat spoilage bacteria were only inhibited by acidity. However, by using a MRS medium added with tetramethyl-benzidine and peroxidase, a high percentage of H₂O₂-producing lactobacilli were detected. The surface properties of Lactobacillus strains showed that a few strains were able to agglutinate ABO human erythrocytes, while the highest number of strains had a low to medium degree of hydrophobicity. This paper constitute the first study related to the beneficial properties of Lactobacillus isolated from a bullfrog hatchery, as well as the selection criteria applied to a group of strains, which could help to control or prevent bacterial infectious diseases in raniculture.     

Antibiotic resistance of potential probiotic bacteria of the genus <Emphasis Type="Italic">Lactobacillus</Emphasis> from human gastrointestinal microbiome

Thirteen Lactobacillus strains isolated from the gastrointestinal microbiome of people from the territory of the former Soviet Union have been studied for resistance to 15 antibiotics of different nature, namely, penicillins, aminoglycosides, macrolides, lincosamides, tetracyclines, chloramphenicol, and rifampicin. The strains included four strains of L. plantarum, four of L. helveticus, three of L. casei/paracasei, one of L. rhamnosus, and one of L. fermentum. All strains showed relative sensitivity to ampicillin, chloramphenicol, rifampicin, roxithromycin, erythromycin, and azithromycin, while none of them were sensitive to all tested antibiotics. L. plantarum strains had the broadest resistance spectra: one strain was resistant to tetracycline and three aminoglycosides and three strains were resistant to tetracycline and five aminoglycosides; one strain demonstrated high resistance to clindamycin and two strains to lincomycin. At the same time, two L. plantarum strains demonstrated resistance to benzylpenicillin coupled with sensitivity to ampicillin, another β-lactam antibiotic. Such resistance was clearly not related to the β-lactamase activity and could be explained by a specific mutation in one of the penicillin-binding proteins of the cell wall. Strains of L. helveticus, L. casei/paracasei, L. rhamnosus, and L. fermentum exhibited cross resistance to two to five different aminoglycosides. A PCR test of the resistance determinants for the widely clinically used antibiotics, tetracycline, chloramphenicol, and erythromycin revealed the presence of the tetM gene of conjugative transposon in L. casei/paracasei and two L. helveticus strains. Nucleotide sequence analysis of the amplified tetM fragments demonstrated their high homology with the tetM genes of Enterococcus faecalis and Streptococcus pneumoniae. The strains carrying tetM were tested for the genes of replication and conjugative transfer of plasmids in lactic acid bacteria. The results indicated that these strains contain genes identical or highly homologous to the rep and trsK genes of the plca36 plasmid and rep gene of the pLH1 and pLJ1 plasmids of lactic acid bacteria. The tetM gene is probably not expressed in strains sensitive to the corresponding antibiotic. However, the investigated lactobacilli cannot be directly used as probiotics, as they may serve as a source of genes for antibiotic resistance in the human microbiome.     

Effect of Two Probiotic Strains of Lactobacillus on In Vitro Adherence of Listeria monocytogenes,Streptococcus agalactiae,and Staphylococcus aureus to Vaginal Epithelial Cells

The lactobacilli probiotics maintain a normal vaginal biota and prevent disease recurrence. This microorganisms form a pellicle on the vaginal epithelium that acts as a biologic barrier against colonization by pathogenic bacteria. In this paper were realized assays of exclusion, competition, and displacement. For these test, vaginal epithelial cells, two strains of lactobacilli and pathogenic bacteria (Staphylococcus aureus, Streptococcus agalactiae and Listeria monocytogenes) were used. The lactobacilli strains showed a great capacity of adherence, with a mean of 83.5 ± 26.67 Lactobacillus fermentum cells and 56.2 ± 20.87 Lactobacillus rhamnosus cells per vaginal epithelial cells. L. fermentum and L. rhamnosus were able to reduce the adherence of S. aureus, S. agalactiae and L. monocytogenes in a significant level in this assay (P < 0.01). The lactobacilli used in this study protect the vaginal epithelium through a series of barriers and interference mechanisms. The aim of present study was to assess the ability of vaginal Lactobacillus strains, selected for their probiotic properties, to block the adherence of pathogenic microorganisms in vitro by displacement, competition, and exclusion mechanisms.     

Lactobacillus plantarum CS24.2 prevents Escherichia coli adhesion to HT‐29 cells and also down‐regulates enteropathogen‐induced tumor necrosis factor‐α and interleukin‐8 expression

The aim of the present study was to evaluate the potential of Lactobacillus plantarum CS24.2 to antagonize Escherichia coli adhesion and modulate expression of the responses by HT‐29 cells of inflammatory molecules to E. coli adhesion. Experiments were performed under different adhesion conditions and findings compared with the responses of Lactobacillus rhamnosus GG. Tests of competitive adhesion, adhesion inhibition and displacement assays were performed for lactobacilli (L. rhamnosus GG and L. plantarum CS24.2) and E. coli O26:H11 to HT‐29 cells. Both the lactobacilli significantly reduced E. coli adhesion to HT‐29 cells (P < 0.05). The ability of lactobacilli to modulate tumor necrosis factor‐α and interleukin‐8 expression was analyzed in HT‐29 cells stimulated with E. coli using qRT‐PCR. L. plantarum CS24.2 significantly down regulated expression of both the genes induced by E. coli in HT‐29 cells at 6 hr as well as 24 hr, which was more significant than the corresponding findings for L. rhamnosus GG. The present findings suggest that L. plantarum CS24.2 inhibits pathogen adhesion to a similar extent as does the established probiotic strain L. rhamnosus GG. It may also attenuate tumor necrosis factor‐α and interleukin‐8 expression in HT‐29 cells stimulated with E. coli.     

Identification and functional properties of dominant lactic acid bacteria isolated at different stages of solid state fermentation of cassava during traditional <Emphasis Type="Italic">gari</Emphasis> production

Culture-based technique was used to study the population dynamics of the bacteria and determine the dominant lactic acid bacteria (LAB) during cassava fermentation. LAB was consistently isolated from the fermented mash with an initial viable count of 6.00 log c.f.u. g⁻¹ observed at 12 h. The aerobic viable count of amylolytic lactic acid bacteria (ALAB) was higher than other group of LAB throughout the fermentation up to 96 h with the highest viable count of 8.08 log c.f.u. g⁻¹. Combination of phenotypic parameters and 16S rDNA gene sequencing identified the dominant group of LAB as Lactobacillus plantarum, L. fermentum and Leuconostoc mesenteroides while the pulse field gel electrophoresis determined that the strains were genotypically heterogeneous. The sugar fermentation profile of the isolates showed that indigestible sugars such as raffinose and stachyose can be fermented by the strains. Information was also generated about the functional properties of the strains. Only strain L. plantarum 9st0 isolate at 0 h of the fermentation produced bacteriocin with antagonism against closely related indicator strains. Quantitatively, the highest amylase activity was produced by strain L. plantarum 7st12, while appreciable amylase was also produced by L. fermentum 1st96. The result of this work showed that selection of mixed starter cultures of bacteriocin- and amylase-producing L. plantarum and L. fermentum will be highly relevant as starter cultures during the intermediate and large scale gari production.     

Characterization of wine Lactobacillus plantarum by PCR-DGGE and RAPD-PCR analysis and identification of Lactobacillus plantarum strains able to degrade arginine

Summary Screening of strains isolated from red wine undergoing malolactic fermentation allowed the identification of lactic acid bacteria able to degrade arginine. A denaturing gradient gel electrophoresis approach, using the rpoB gene as the molecular target, was developed in order to characterize the isolated strains. Several strains were identified as Lactobacillus plantarum and were typed by RAPD-PCR with several randomly designed primers. Almost all of the␣L. plantarum strains identified were able to produce citrulline and ammonia, suggesting that the ability of␣L.␣plantarum to degrade arginine is a common feature in wine. During the characterization of the newly identified L.␣plantarum strains, the presence of genes coding for the arginine deiminase (ADI) pathway was observed in the strains able to produce citrulline, while the lack of this genes was observed in strain unable to produce citrulline. These results suggest that the degradation of arginine in L. plantarum is probably strain-dependent.     

Characterization of a <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> Strain Able to Produce Tyramine and Partial Cloning of a Putative Tyrosine Decarboxylase Gene

The aim of this article was to analyze the ability of wine Lactobacillus plantarum strains to form tyramine. Preliminary identification of L. plantarum strains was performed by amplification of the recA gene. Primers pREV and PlanF, ParaF and PentF were used respectively as reverse and forward primers in the polymerase chain reaction tests as previously reported. Furthermore, the gene encoding for the tyrosine decarboxylase (TDC) was partially cloned from one strain identified as L. plantarum. The strain was further analyzed by 16S rDNA sequence and confirmed as belonging to L. plantarum species. The tyrosine decarboxylase activity was investigated and tyramine was determined by the high-performance liquid chromatography method. Moreover, a negative effect of sugars such as glucose and fructose and L-malic acid on tyrosine decarboxylase activity was observed. The results suggest that, occasionally, L. plantarum is able to produce tyramine in wine and this ability is apparently confined only to L. plantarum strains harboring the tdc gene.     

Use of Lactobacillus plantarum LPCO10, a Bacteriocin Producer, as a Starter Culture in Spanish-Style Green Olive Fermentations

Bacteriocin-producing Lactobacillus plantarum LPCO10 and its non-bacteriocin-producing, bacteriocinimmune derivative, L. plantarum 55-1, were evaluated separately for growth and persistence in natural Spanish-style green olive fermentations. Both strains were genetically marked and selectively enumerated using antibiotic-containing media. Plasmid profile and bacteriocin production (bac⁺) were used as additional markers. When olive brines were inoculated at 10⁵ CFU/ml, the parent strain, LPCO10, proliferated to dominate the epiphytic microflora, sharing high population levels with other spontaneously occurring lactobacilli and persisting throughout the fermentation (12 weeks). In contrast, the derivative strain could not be isolated after 7 weeks. Stability of both plasmid profile and bac⁺ (LPCO10 strain) or bac^- (55-1 strain) phenotype was shown by L. plantarum LPCO10 and L. plantarum 55-1 isolated throughout the fermentation. Bacteriocin activity could be found in the L. plantarum LPCO10-inoculated brines only after ammonium sulfate precipitation and concentration (20 times) of the final brine. Spontaneously occurring lactobacilli and lactic coccus populations, which were isolated from each of the fermenting brines studied during this investigation, were shown to be sensitive to the bacteriocins produced by L. plantarum LPCO10 when tested by the drop diffusion test. The declines in both pH and glucose levels throughout the fermentative process were similar in L. plantarum LPCO10- and in L. plantarum 55-1-inoculated brines and were comparable to the declines in the uninoculated brines. However, the final concentration of lactic acid in L. plantarum LPCO10-inoculated brines was higher than in the L. plantarum 55-1-inoculated brines and uninoculated brines. These results indicated that L. plantarum LPCO10 may be useful as a starter culture to control the lactic acid fermentation of Spanish-style green olives.     

Development of<Emphasis Type="Italic"> Lactobacillus plantarum</Emphasis> LL441 and its plasmid-cured derivatives in cheese

A wild Lactobacillus plantarum strain and two of its plasmid-cured derivatives were separately used as adjunct cultures in the manufacture of a Gouda-like traditional Spanish cheese. The wild strain, LL441, harbours seven plasmids and produces a lantibiotic-like bacteriocin. The LL441-B2 derivative has lost plasmids of 40 and 80 kb and the bacteriocin-producing capability. The LL441-B11 derivative has lost in addition a 70 kb plasmid encoding active α- and β-galactosidases. All three strains could be used as adjunct cultures as none of the technological and biochemical parameters of the cheeses was affected. Both the wild-type and the two derivatives were recovered from experimental cheeses up to 30 days after manufacture at similar rates of nearly 20%. Thus, the phenotypic traits under examination were not essential for L. plantarum to grow into the cheese matrix. Electronic Publication     

Probiotic Lactobacilli Interfere with <Emphasis Type="Italic">Streptococcus mutans</Emphasis> Biofilm Formation In Vitro

In clinical studies, probiotic bacteria have decreased the counts of salivary mutans streptococci (MS). We compared the effects of probiotic Lactobacillus strains on the biofilm formation of Streptococcus mutans. The bacterial strains used included four S. mutans strains (reference strains NCTC 10449 and Ingbritt and clinical isolates 2366 and 195) and probiotic strains Lactobacillus rhamnosus GG, L. plantarum 299v, and L. reuteri strains PTA 5289 and SD2112. The ability of MS to adhere and grow on a glass surface, reflecting biofilm formation, was studied in the presence of the lactobacilli (LB). The effect of LB culture supernatants on the viability of the MS was studied as well. All of the LB inhibited the biofilm formation of the clinical isolates of MS (P < 0.001). The biofilm formation of the reference strains of MS was also inhibited by the LB, but L. plantarum and L. reuteri PTA 5289 showed a weaker inhibition when compared to L. reuteri SD2112 and L. rhamnosus GG. Viable S. mutans cells could be detected in the biofilms and culture media only when the experiments were performed with the L. reuteri strains. The L. reuteri strains were less efficient in killing the MS also in the tests performed with the culture supernatants. The pHs of the supernatants of L. reuteri were higher compared to those of L. rhamnosus GG and L. plantarum; P < 0.001. In conclusion, our results demonstrated that four commonly used probiotics interfered with S. mutans biofilm formation in vitro, and that the antimicrobial activity against S. mutans was pH-dependent.     

Influence of lactobacilli on the adhesion ofStaphylococcus aureus andCandida albicans to fibers and epithelial cells

The ability of organisms to adhere to and form biofilms on fibrous materials is believed to be an important initiating step in the induction of several diseases, such as toxic shock syndrome. Using anin vitro assay, a moderately hydrophobic strain ofStaphylococcus aureus (water contact angle 35°) and a hydrophilicCandida albicans (shown by a hexadecane test) were highly adherent to commercial diaper fibers. The lumen side of the diaper was porous and the fibers were very hydrophobic (>140°), but the internal section was very hydrophilic (0°), presumably for adsorption purposes. There was evidence that adhesion of the pathogens was inhibited when one of fiveLactobacillus strains was present. Surfaces precoated with lactobacilli inhibited staphylococcal adhesion by 26–97%, and candida by 0–67%. When the lactobacilli were used to challenge adherent pathogens, there was 99% displacement of theS. aureus and up to 91% displacement ofC. albicans. HydrophobicL. acidophilus 76 (54°) and T-13 (80°) were the most effective of fiveLactobacillus isolates tested at interference by precoating. The moderately hydrophilicL. casei varrhamnosus GR-1 (33°) was the most effective at displacing the yeast. Experiments with uroepithelial cells also showed that the lactobacilli could significantly interfere with the adhesion of both pathogens to the cells. The results demonstrate the rapidity with which two pathogens adhered to fibers and epithelial cells, and raised the possibility that members of the normal female urogenital flora might interfere with infections caused by these organisms.