Characterization of a <Emphasis Type="Italic">Lactobacillus brevis</Emphasis> strain with potential oral probiotic properties

Background

The microflora composition of the oral cavity affects oral health. Some strains of commensal bacteria confer probiotic benefits to the host. Lactobacillus is one of the main probiotic genera that has been used to treat oral infections. The objective of this study was to select lactobacilli with a spectrum of probiotic properties and investigate their potential roles in oral health.

Results

An oral isolate characterized as Lactobacillus brevis BBE-Y52 exhibited antimicrobial activities against Streptococcus mutans, a bacterial species that causes dental caries and tooth decay, and secreted antimicrobial compounds such as hydrogen peroxide and lactic acid. Compared to other bacteria, L. brevis BBE-Y52 was a weak acid producer. Further studies showed that this strain had the capacity to adhere to oral epithelial cells. Co-incubation of L. brevis BBE-Y52 with S. mutans ATCC 25175 increased the IL-10-to-IL-12p70 ratio in peripheral blood mononuclear cells, which indicated that L. brevis BBE-Y52 could alleviate inflammation and might confer benefits to host health by modulating the immune system.

Conclusions

L. brevis BBE-Y52 exhibited a spectrum of probiotic properties, which may facilitate its applications in oral care products.

     

Expression and characterization of glutamate decarboxylase from <Emphasis Type="Italic">Lactobacillus brevis</Emphasis> HYE1 isolated from <Emphasis Type="Italic">kimchi</Emphasis>

A putative gene (gadlbhye1) encoding glutamate decarboxylase (GAD) was cloned from Lactobacillus brevis HYE1 isolated from kimchi, a traditional Korean fermented vegetable. The amino acid sequences of GADLbHYE1 showed 48% homology with the GadA family and 99% identity with the GadB family from L. brevis. The cloned GADLbHYE1 was functionally expressed in Escherichia coli using inducible expression vectors. The expressed recombinant GADLbHYE1 was successfully purified by Ni–NTA affinity chromatography, and had a molecular mass of 54 kDa with optimal hydrolysis activity at 55 °C and pH 4.0. Its thermal stability was determined to be higher than that of other GADs from L. brevis, based on its melting temperature (75.18 °C). Kinetic parameters including K_m and V_max values for GADLbHYE1 were 4.99 mmol/L and 0.224 mmol/L/min, respectively. In addition, the production of gamma-aminobutyric acid in E. coli BL21 harboring gadlbhye1/pET28a was increased by adding pyridoxine as a cheaper coenzyme.     

Surface Display of the Receptor-Binding Region of the Lactobacillusbrevis S-Layer Protein in Lactococcuslactis Provides Nonadhesive Lactococci with the Ability To Adhere to Intestinal Epithelial Cells

Lactobacillus brevis is a promising lactic acid bacterium for use as a probiotic dietary adjunct and a vaccine vector. The N-terminal region of the S-layer protein (SlpA) of L. brevis ATCC 8287 was recently shown to mediate adhesion to various human cell lines in vitro. In this study, a surface display cassette was constructed on the basis of this SlpA receptor-binding domain, a proteinase spacer, and an autolysin anchor. The cassette was expressed under control of the nisA promoter in Lactococcus lactis NZ9000. Western blot assay of lactococcal cell wall extracts with anti-SlpA antibodies confirmed that the SlpA adhesion domain of the fusion protein was expressed and located within the cell wall layer. Whole-cell enzyme-linked immunosorbent assay and immunofluorescence microscopy verified that the SlpA adhesion-mediating region was accessible on the lactococcal cell surface. In vitro adhesion assays with the human intestinal epithelial cell line Intestine 407 indicated that the recombinant lactococcal cells had gained an ability to adhere to Intestine 407 cells significantly greater than that of wild-type L. lactis NZ9000. Serum inhibition assay further confirmed that adhesion of recombinant lactococci to Intestine 407 cells was indeed mediated by the N terminus-encoding part of the slpA gene. The ability of the receptor-binding region of SlpA to adhere to fibronectin was also confirmed with this lactococcal surface display system. These results show that, with the aid of the receptor-binding region of the L. brevis SlpA protein, the ability to adhere to gut epithelial cells can indeed be transferred to another, nonadhesive, lactic acid bacterium.     

A New Broad-Spectrum Peptide Antibiotic Produced by <Emphasis Type="Italic">Bacillus brevis</Emphasis> Strain MH9 Isolated from Margalla Hills of Islamabad,Pakistan

The antimicrobial peptide from a bacterial strain is isolated from soil sample of Margalla Hills of Islamabad, Pakistan. The peptide is found to significantly inhibit the growth of both Gram-positive (Staphylococcus aureus ATCC 6538 and Micrococcus luteus ATCC 10240) and Gram-negative (Escherichia coli ATCC 25922 and Salmonella typhi ATCC 14028) bacteria as compared to gramicidin as standard. The bacterium is identified as Bacillus brevis strain MH9 based on phenotype and phylogenetic analysis. The antibacterial polypeptide was produced optimally at 35 °C after 48 h of growth, precipitated by 50 % ammonium sulphate, and further purified using HPLC. The sequential steps of purification decrease the peptide contents with prominent antibacterial activity. The peptide composed of 11 amino acid was further characterized by FT-IR and NMR. Results suggested that the peptide molecule is a novel antibacterial agent that is effective against both Gram-positive and Gram-negative bacteria. This study may have important implications for new peptide antibiotic that could be a new addition to treat infections.     

The impact of heterologous catalase expression and superoxide dismutase overexpression on enhancing the oxidative resistance in <Emphasis Type="Italic">Lactobacillus casei</Emphasis>

Two heme-dependent catalase genes were amplified from genomic DNA of Lactobacillus plantarum WCFS1 (KatE1) and Lactobacillus brevis ATCC 367 (KatE2), respectively, and a manganese-containing superoxide dismutase from Lactobacillus casei MCJΔ1 (MnSOD) were cloned into plasmid pELX1, yielding pELX1-KatE1, pELX1-KatE2 and pELX1-MnSOD, then the recombinant plasmids were transferred into L. casei MCJΔ1. The strains of L. casei MCJΔ1/pELX1-KatE1 and L. casei MCJΔ1/pELX1-KatE2 were tolerant at 2 mM H₂O₂. The survival rates of L. casei MCJΔ1/pELX1-KatE1 and L. casei MCJΔ1/pELX1-KatE2 were 270-fold and 300-fold higher than that of the control strain on a short-term H₂O₂ exposure, and in aerated condition, the survival cells counts were 146- and 190-fold higher than that of the control strain after 96 h of incubation. Furthermore, L. casei MCJΔ1/pELX1-MnSOD was the best in three recombinants which was superior in the living cell viability during storage when co-storage with Lactobacillus delbrueckii subsp. lactis LBCH-1.     

Female Responses to Synthetic Pheromone and Plant Compounds in <Emphasis Type="Italic">Agriotes brevis</Emphasis> Candeze (Coleoptera: Elateridae)

Traps baited with synthetic pheromone components of Agriotes brevis [geranyl butanoate + (E,E)-farnesyl butanoate] captured significantly higher numbers of not only male, but also female beetles, compared to unbaited controls. Catches of both sexes showed a clear positive relationship with increasing doses. In electroantennogram tests, antennal responses of females and males to a number of known Agriotes pheromone components, identified from pheromone glands, showed a similar trend, with geranyl butanoate eliciting the strongest responses. This suggests that the female and male A. brevis antennae are similar with respect to the perception of pheromone compounds, and female beetles have the sensory capabilities to perceive the pheromone components which they produce. Addition of the plant-derived compounds (Z)-3-hexenyl acetate, methyl benzoate, (Z)-3-hexenol and methyl salicylate (identified earlier from foliage as attractive for A. brevis) to the synthetic pheromone significantly increased catches. All the above results suggest that geranyl butanoate and (E,E)-farnesyl butanoate are constituents of an aggregation pheromone of A. brevis, in contrast to the general view of click beetle pheromones being “classical” sex pheromones. These findings could be useful for more precise monitoring and forecasting of damage, based on female catches.     

A physiological comparative study of acid tolerance of <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> ZDY 2013 and <Emphasis Type="Italic">L. plantarum</Emphasis> ATCC 8014 at membrane and cytoplasm levels

This study aimed to disclose the acid tolerance mechanism of Lactobacillus plantarum by comparing L. plantarum ZDY 2013 with the type strain L. plantarum ATCC 8014 in terms of cell membrane, energy metabolism, and amino acid metabolism. L. plantarum ZDY 2013 had a superior growth performance under acidic condition with 100-fold higher survival rate than that of L. plantarum ATCC 8014 at pH 2.5. To determine the acid tolerance physiological mechanism, cell integrity was investigated through scanning electron microscopy. The study revealed that L. plantarum ZDY 2013 maintained cell morphology and integrity, which is much better than L. plantarum ATCC 8014 under acid stress. Analysis of energy metabolism showed that, at pH 5.0, L. plantarum ZDY 2013 enhanced the activity of Na⁺/K⁺-ATPase and decreased the ratio of NAD⁺/NADH in comparison with L. plantarum ATCC 8014. Similarly, amino acid metabolism of intracellular arginine, glutamate, and alanine was improved in L. plantarum ZDY 2013. Correspondingly, the activity of arginine deiminase and glutamate decarboxylase of L. plantarum ZDY 2013 increased by 1.2-fold and 1.3-fold compared with L. plantarum ATCC 8014 in acid stress. In summary, it is demonstrated that the special physiological behaviors (integrity of cell membrane, enhanced energy metabolism, increased amino acid and enzyme level) of L. plantarum ZDY 2013 can protect the cells from acid stress.     

Cloning and characterization of two distinct water-forming NADH oxidases from <Emphasis Type="Italic">Lactobacillus pentosus</Emphasis> for the regeneration of NAD

Two uncharacterized nicotinamide adenine dinucleotide (NADH) oxidases (named as LpNox1, LpNox2) from Lactobacillus pentosus ATCC 8041 were cloned and overexpressed in Escherichia coli BL21 (DE3). The sequence analysis revealed that the two enzymes are water-forming Noxs with 64 % and 52 % identity to LbNox from Lactobacillus brevis DSM 20054. The optimal pH and temperature of the purified LpNox1 and LpNox2 were 7.0 and 8.0 and 35 and 40 °C, respectively, with K _M of 99.0 μM (LpNox1) and 27.6 μM (LpNox2), and yielding catalytic efficiency k _cat/K _M of 1.0 and 0.2 μM^?1 s^?1, respectively. Heat inactivation studies revealed that the two enzymes are relatively instable. The application of LpNox1 for the regeneration of NAD⁺ was demonstrated by coupling with a glycerol dehydrogenase-catalyzed oxidation of glycerol to 1,3-dihydroxyacetone. The characteristics of the LpNox1 could prove to be of interest in industrial application such as NAD⁺ regeneration in dehydrogenase-catalyzed oxidations.     

Enhanced butyric acid tolerance and production by Class I heat shock protein-overproducing <Emphasis Type="Italic">Clostridium tyrobutyricum</Emphasis> ATCC 25755

The response of Clostridium tyrobutyricum to butyric acid stress involves various stress-related genes, and therefore overexpression of stress-related genes can improve butyric acid tolerance and yield. Class I heat shock proteins (HSPs) play an important role in the process of protecting bacteria from sudden changes of extracellular stress by assisting protein folding correctly. The results of quantitative real-time PCR indicated that the Class I HSGs grpE, dnaK, dnaJ, groEL, groES, and htpG were significantly upregulated under butyric acid stress, especially the dnaK and groE operons. Overexpression of groESL and htpG could significantly improve the tolerance of C. tyrobutyricum to butyric acid, while overexpression of dnaK and dnaJ showed negative effects on butyric acid tolerance. Acid production was also significantly promoted by increased GroESL expression levels; the final butyric acid and acetic acid concentrations were 28.2 and 38% higher for C. tyrobutyricum ATCC 25755/groESL than for the wild-type strain. In addition, when fed-batch fermentation was carried out using cell immobilization in a fibrous-bed bioreactor, the butyric acid yield produced by C. tyrobutyricum ATCC 25755/groESL reached 52.2 g/L, much higher than that for the control. The improved butyric acid yield is probably attributable to the high GroES and GroEL levels, which can stabilize the biosynthetic machinery of C. tyrobutyricum under extracellular butyric acid stress.     

<Emphasis Type="Italic">In situ</Emphasis> fermentation dynamics during production of <Emphasis Type="Italic">gundruk</Emphasis> and <Emphasis Type="Italic">khalpi</Emphasis>, ethnic fermented vegetable products of the Himalayas

Gundruk is a fermented leafy vegetable and khalpi is a fermented cucumber product, prepared and consumed in the Himalayas. In situ fermentation dynamics during production of gundruk and khalpi was studied. Significant increase in population of lactic acid bacteria (LAB) was found during first few days of gundruk and khlapi fermentation, respectively. Gundruk fermentation was initiated by Lactobacillus brevis, Pediococcus pentosaceus and finally dominated by Lb. plantarum. Similarly in khalpi fermentation, heterofermentative LAB such as Leuconostoc fallax, Lb. brevis and P. pentosaceus initiated the fermentation and finally completed by Lb. plantarum. Attempts were made to produce gundruk and khalpi using mixed starter culture of LAB previously isolated from respective products. Both the products prepared under lab condition had scored higher sensory-rankings comparable to market products.     

Analysis of antimicrobial and immunomodulatory substances produced by heterofermentative <Emphasis Type="Italic">Lactobacillus reuteri</Emphasis>

Antimicrobial and immunomodulatory potential of various Lactobacillus reuteri strains is closely connected to their metabolite production profile under given cultivation conditions. We determined the in vitro production of antimicrobial substances such as organic acids, ethanol, and reuterin by four strains of L. reuteri (L. reuteri E, L. reuteri KO5, L. reuteri CCM 3625, and L. reuteri ATCC 55730). All studied L. reuteri strains showed the ability to produce lactic acid, acetic acid, and ethanol with concominant consumption of glucose and together with phenyllactic acid—a potent antifungal compound—with concominant consumption of phenylalanine. The reuterin production from glycerol was confirmed for all analyzed lactobacilli strains except L. reuteri CCM 3625. Production of organic acids, ethanol, and reuterin is significantly involved in antimicrobial activity of lactobacilli which was determined using the dual-culture overlay diffusion method against six indicator bacteria and five indicator moulds. In comparison to the referential L. reuteri ATCC 55730, the highest inhibition potential was observed against Escherichia coli CCM 3988 and Pseudomonas aeruginosa CCM 3955. Among analyzed indicators of moulds, the growth of Alternaria alternata CCM F-128 was the most inhibited by all four analyzed L. reuteri strains. Finally, the immunomodulatory potential of analyzed lactobacilli were proven by the determination of the in vitro production of biogenic amines histamine and tyramine. L. reuteri CCM 3625 was able to produce tyramine, and L. reuteri E and L. reuteri KO5 were able to produce histamine under given cultivation conditions.     

Probiotic Characteristics of <Emphasis Type="Italic">Lactobacillus curvatus</Emphasis> DN317, a Strain Isolated from Chicken Ceca

The probiotic characteristics of Lactobacillus curvatus DN317, a strain isolated from chicken ceca, were evaluated. This strain was selected for study from the isolated Lactobacillus strains because it has specific anti-microbial activity against Campylobacter jejuni ATCC 33560, Camp. jejuni NCTC 11168, Listeria monocytogenes ATCC 7644, and Bacillus subtilis ATCC 8633. Lact. curvatus DN317 showed an auto-aggregation percentage of 72% and presented the highest co-aggregation with Lact. monocytogenes ATCC 7644 (68%) compared to B. subtilis ATCC 8633 (45%), Camp. jejuni ATCC 33560 (36%), and Camp. jejuni NCTC 11168 (35%). The data revealed that Lact. curvatus DN317 could survive at 0.25% bile, maintain viability at pH 2.5 for 30 min, produce biosurfactants, and adhere to Caco-2 cells. Quantification of IL-6, IL-8, IL-10, and β-defensin 2 levels shows that Lact. curvatus DN317 induces an increase in IL-8 and β-defensin 2 secretion, while the levels of IL-6 and IL-10 do not change. Lact. curvatus DN317 showed high levels of esterase and cysteine arylamidase activities (5); moderate levels of esterase lipase, β-galactosidase, and α-galactosidase activities (4, 3); and weak levels of leucine arylamidase, valine arylamidase, and acid phosphatase activity (1). Various activities were obtained of α-chymotrypsin, β-glucuronidase, β-glucosidase, and N-acetyl-β-glucosaminidase, which have been associated with intestinal diseases. Lact. curvatus DN317 lowered the cholesterol level in MRS with and without bile. Antibiotic susceptibility tests indicated that DN317 was sensitive to ampicillin, gentamicin, kanamycin, streptomycin, tetracycline, clindamycin, erythromycin, and vancomycin but was resistant to chloramphenicol and ciprofloxacin. These results suggest that Lact. curvatus DN317 could potentially function as a probiotic.     

Clinical strains of <Emphasis Type="Italic">Lactobacillus</Emphasis> reduce the filamentation of <Emphasis Type="Italic">Candida albicans</Emphasis> and protect <Emphasis Type="Italic">Galleria mellonella</Emphasis> against experimental candidiasis

Candida albicans is the most common human fungal pathogen and can grow as yeast or filaments, depending on the environmental conditions. The filamentous form is of particular interest because it can play a direct role in adherence and pathogenicity. Therefore, the purpose of this study was to evaluate the effects of three clinical strains of Lactobacillus on C. albicans filamentation as well as their probiotic potential in pathogen-host interactions via an experimental candidiasis model study in Galleria mellonella. We used the reference strain Candida albicans ATCC 18804 and three clinical strains of Lactobacillus: L. rhamnosus strain 5.2, L. paracasei strain 20.3, and L. fermentum strain 20.4. First, the capacity of C. albicans to form hyphae was tested in vitro through association with the Lactobacillus strains. After that, we verified the ability of these strains to attenuate experimental candidiasis in a Galleria mellonella model through a survival curve assay. Regarding the filamentation assay, a significant reduction in hyphae formation of up to 57% was observed when C. albicans was incubated in the presence of the Lactobacillus strains, compared to a control group composed of only C. albicans. In addition, when the larvae were pretreated with Lactobacillus spp. prior to C. albicans infection, the survival rate of G. mellonela increased in all experimental groups. We concluded that Lactobacillus influences the growth and expression C. albicans virulence factors, which may interfere with the pathogenicity of these microorganisms.     

Genome sequences and comparative genomics of two <Emphasis Type="Italic">Lactobacillus ruminis</Emphasis> strains from the bovine and human intestinal tracts

Background

The genus Lactobacillus is characterized by an extraordinary degree of phenotypic and genotypic diversity, which recent genomic analyses have further highlighted. However, the choice of species for sequencing has been non-random and unequal in distribution, with only a single representative genome from the L. salivarius clade available to date. Furthermore, there is no data to facilitate a functional genomic analysis of motility in the lactobacilli, a trait that is restricted to the L. salivarius clade.

Results

The 2.06 Mb genome of the bovine isolate Lactobacillus ruminis ATCC 27782 comprises a single circular chromosome, and has a G+C content of 44.4%. In silico analysis identified 1901 coding sequences, including genes for a pediocin-like bacteriocin, a single large exopolysaccharide-related cluster, two sortase enzymes, two CRISPR loci and numerous IS elements and pseudogenes. A cluster of genes related to a putative pilin was identified, and shown to be transcribed in vitro. A high quality draft assembly of the genome of a second L. ruminis strain, ATCC 25644 isolated from humans, suggested a slightly larger genome of 2.138 Mb, that exhibited a high degree of synteny with the ATCC 27782 genome. In contrast, comparative analysis of L. ruminis and L. salivarius identified a lack of long-range synteny between these closely related species. Comparison of the L. salivarius clade core proteins with those of nine other Lactobacillus species distributed across 4 major phylogenetic groups identified the set of shared proteins, and proteins unique to each group.

Conclusions

The genome of L. ruminis provides a comparative tool for directing functional analyses of other members of the L. salivarius clade, and it increases understanding of the divergence of this distinct Lactobacillus lineage from other commensal lactobacilli. The genome sequence provides a definitive resource to facilitate investigation of the genetics, biochemistry and host interactions of these motile intestinal lactobacilli.

     

Cellulase and Xylanase Production by the Mexican Strain <Emphasis Type="Italic">Talaromyces stollii</Emphasis> LV186 and Its Application in the Saccharification of Pretreated Corn and Sorghum Stover

A Mexican strain of Talaromyces stollii LV186 was isolated from decaying pretreated corn stover. The production of cellulase and xylanase enzyme cocktails was evaluated with corn and sorghum stover used as inducers in a mineral medium. The volumetric and specific activities of T. stollii LV186 were compared with the values produced by Trichoderma reesei ATCC 26921 in a time-course experiment. After the submerged culture and a posterior ultrafiltration stage, the enzyme complexes were evaluated over acid-pretreated corn or sorghum stover in baffled flasks under controlled temperature and agitation conditions, and hydrolysis levels of 30 and 39 % of the theoretical maximum were obtained after only 72-h reactions, for each substrate. A side-by-side comparison showed a better ratio of endoglucanase to cellobiohydrolase to β-glucosidase and of xylanase to β-xylosidase enzymes in T. stollii than in T. reesei ATCC 26921. Furthermore, the hydrolysis of pretreated corn and sorghum stover achieved by T. stollii is significantly higher compared with that of a commercial cocktail from T. reesei ATCC 26921 (Celluclast). Therefore, the T. stollii LV186 strain is a good candidate for the hydrolysis of complex lignocellulose substrates. To the authors’ knowledge, this study is the first to describe the cellulolytic and hemicellulolytic activities produced by a T. stollii strain.     

Sporulation of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> in binary cultures with ultramicrobacteria

The effect of ultramicrobacterial epibionts of the genera Kaistia (strain NF1), Chryseobacterium (strain NF4), and Stenotrophomonas (strain FM3) on the process of sporulation of Bacillus subtilis ATCC 6633 was studied. The investigated strains of ultramicrobacteria (UMB) were found to inhibit the sporulation process of B. subtilis ATCC 6633 in binary mixed cultures, exhibiting a 3-day delay of the onset of sporulation compared to the control one, an extended period of the prospore maturation, formation of the fraction of immature spores, and development of ultrastructural defects in many endospores. Thus, investigation of binary mixed cultures of B. subtilis and UMB revealed that, apart from suppression of reproduction and lysis of host vegetative cells, inhibition of spore formation and destruction of endospores was yet another feature of intermicrobial parasitism. The UMB parasites of the studied genera are assumed to participate in the regulation of development and reproduction of B. subtilis in natural habitats of this spore-forming bacterium.     

Evaluation of lytic bacteriophages for control of multidrug-resistant <Emphasis Type="Italic">Salmonella</Emphasis> Typhimurium

Background

The emergence of antibiotic-resistant bacteria can cause serious clinical and public health problems. This study describes the possibility of using bacteriophages as an alternative agent to control multidrug-resistant Salmonella Typhimurium.

Methods

The potential lytic bacteriophages (P22-B1, P22, PBST10, PBST13, PBST32, and PBST 35) were characterized by morphological property, heat and pH stability, optimum multiplicity of infection (MOI), and lytic activity against S. Typhimurium KCCM 40253, S. Typhimurium ATCC 19585, ciprofloxacin-induced antibiotic-resistant S. Typhimurium ATCC 19585, and S. Typhimurium CCARM 8009.

Results

P22-B1 and P22 belong to Podoviridae family and PBST10, PBST13, PBST32, and PBST 35 show a typical structure with polyhedral head and long tail, belonging to Siphoviridae family. Salmonella bacteriophages were highly stable at the temperatures (< 60 °C) and pHs (5.0–11.0). The reduction rates of host cells were increased at the MOI-dependent manner, showing the highest reduction rate at MOI of 10. The host cells were most effectively reduced by P22, while P22-B1 showed the least lytic activity. The ciprofloxacin-induced antibiotic-resistant S. Typhimurium ATCC 19585, and clinically isolated antibiotic-resistant S. Typhimurium CCARM 8009 were resistant to ciprofloxacin, levofloxacin, norfloxacin, and tetracycline. P22 showed the highest lytic activity against S. Typhimurium KCCM 40253 (> 5 log reduction), followed by S. Typhimurium ATCC 19585 (4 log reduction) and ciprofloxacin-induced antibiotic-resistant S. Typhimurium ATCC 19585 (4 log reduction).

Conclusion

The results would provide vital insights into the application of lytic bacteriophages as an alternative therapeutics for the control of multidrug-resistant pathogens.