M13 DNA fingerprinting, a new tool for classification and identification of Lactobacillus spp.

The optimal conditions for the application of M13 DNA fingerprinting to the genus Lactobacillus were determined. Comparative fingerprint analysis of representative strains of Lactobacillus delbrueckii subsp. delbrueckii, Lact. delbrueckii subsp. lactis, Lact. delbrueckii subsp. bulgaricus, Lact. helveticus and Lact. casei permitted the differentiation of species, subspecies and individual strains and the quantitative determination of their genetic relatedness. The results confirm the high specificity of M13 DNA fingerprinting and indicate that it might be used in the classification of Lactobacillus spp.     

M13 DNA fingerprinting, a new tool for classification and identification of Lactobacillus spp.

The optimal conditions for the application of M13 DNA fingerprinting to the genus Lactobacillus were determined. Comparative fingerprint analysis of representative strains of Lactobacillus delbrueckii subsp. delbrueckii, Lact. delbrueckii subsp. lactis, Lact. delbrueckii subsp. bulgaricus, Lact. helveticus and Lact. casei permitted the differentiation of species, subspecies and individual strains and the quantitative determination of their genetic relatedness. The results confirm the high specificity of M13 DNA fingerprinting and indicate that it might be used in the classification of Lactobacillus spp.     

In vitro inhibition of Helicobacter pylori NCTC 11637 by organic acids and lactic acid bacteria

In this Study the effects of both pH and organic acids on Helicobacter pylori NCTC 11637 were tested. Lactobacillus acidophilus, Lact. casei, Lact. bulgaricus, Pediococcus pentosaceus and Bifidobacterium bifidus were assayed for their lactic acid production, pH and inhibition of H. pylori growth. A standard antimicrobial plate well diffusion assay was employed to examine inhibitory effects. Lactic, acetic and hydrochloric acids demonstrated inhibition of H. pylori growth in a concentration-dependent manner with the lactic acid demonstrating the greatest inhibition. This inhibition was due both to the pH of the solution and its concentration. Six strains of Lact. acidophilus and one strain of Lact. casei subsp. rhamnosus inhibited H. pylori growth where as Bifidobacterium bifidus, Ped. pentosaceus and Lact. bulgaricus did not. Concentrations of lactic acid produced by these strains ranged from 50 to 156 mmol 1⁻¹ and correlated with H. pylori inhibition. The role of probiotic organisms and their metabolic by-products in the eradication of H. pylori in vivo remains to be determined.     

Selenium and Lactobacillus species

Selenium (Se) is an essential element for man and animal. Supplementation of Se has been done with Se-enriched yeast and inorganic Se compounds. In the present study a quantitative and qualitative evaluation was made of whether lactobacilli are able to concentrate Se. A high correlation was found between the bacterial Se concentration and the concentration of Se in the medium. The Se concentration in biomass was respectively 253 ± 50, 375 ± 33 and 407 ± 108 μg g⁻¹ dry weight for Lactobacillus delbrueckii subsp. bulgaricus, Lact. plantarum and Lact. casei subsp. casei when 1 mg 1⁻¹ Se⁴⁺ was present in the medium. Manganese (Mn) was concentrated in Lact. plantarum and Lact. casei subsp. casei but not in Lact. delbrueckii subsp. bulgaricus. The Mn concentration in biomass was higher compared to the Se concentration but this difference decreased when the concentration of Mn/Se increased in the culture medium. Copper, zinc and iron were also concentrated in biomass of Lact. delbrueckii subsp. bulgaricus. Characterization of the bacterial selenocompound revealed that ⁷⁵Se was generally incorporated, as selenocysteine, into protein of Lact. delbrueckii subsp. bulgaricus. Addition of L-cysteine to the medium decreased the bacterial Se content. It was concluded that Lact. delbrueckii subsp. bulgaricus incorporated Se non-specifically into bacterial protein. The application of Se-enriched lactobacilli (Se-Lb) in Se supplementation would be an interesting approach since selenomethionine, which is the major selenocompound in commercialized Se-yeast, was not detected in Se-Lb and because lactobacilli are already widely used in human nutrition.     

Evaluation of numerical analyses of RAPD and API 50 CH patterns to differentiate Lactobacillus plantarum, Lact. fermentum, Lact. rhamnosus, Lact. sake, Lact. parabuchneri, Lact. gallinarum, Lact. casei, Weissella minor and related taxa isolated from kocho and tef

AIM: The study was carried out to assess the agreement of API 50 CH fermentation data of food lactobacilli with their RAPD profiles to determine whether the system could be used alone as a reliable taxonomic tool for this genus. METHODS AND RESULTS: API 50 CH, RAPD and DNA:DNA reassociation data for 42 lactobacilli from tef and kocho were compared with 30 type strains. Discrepancies were observed between the three methods in assigning strains of Lactobacillus plantarum, Lact. fermentum, Weissella minor and Lact. gallinarum, and Lact. fermentum, Lact. amylophilus, Lact. casei subsp. pseudoplantarum and Lact. rhamnosus. DNA reassociation data agreed well with RAPD results. CONCLUSIONS: API 50 CH profiles should be complemented with molecular genetic results for effective identification in Lactobacillus. SIGNIFICANCE AND IMPACT OF THE STUDY: The study suggested less dependability of metabolic data alone as an identification tool.     

A survey of lactic acid bacteria in Italian silage

G razia , L. & S uzzi , G. 1984. A survey of lactic acid bacteria in Italian silage. Journal of Applied Bacteriology 56 , 373–379.
Lactic acid bacteria, isolated from Italian ensiled products, were represented by strains of the genera Lactobacillus and Leuconostoc . The predominant strains were heterofermentative lactobacilli, with Lactobacillus buchneri being the most frequent. Among homofermentative lactic acid bacteria, strains of Lact. plantarum and Lact. casei were recovered. Almost all strains utilized malic acid and showed good acid-tolerance, but only some of them were able to metabolize malic acid at extremely low pH; these were five homofermentative lactobacilli (4 Lact. plantarum and 1 Lacr. casei var. casei ) and two heterofermentative lactobacilli ( Lact. cellobiosus and Lactobacillus sp.).     

Antagonistic Action of Lactobacilli and Bifidobacteria in Relation to Staphylococcus aureus and Their Influence on the Immune Response in Cases of Intravaginal Staphylococcosis in Mice

The antibacterial activity of Lactobacillus casei IMV B-7280, Lact. acidophilus IMV B-7279, Bifidobacterium longum VK1, and B. bifidum VK2 strains or their various compositions in relation to Staphylococcus aureus in vitro and on models of experimental intravaginal staphylococcosis of mice was determined. It was found that under the influence of these strains and their various compositions, the in vitro growth of Staph. aureus was inhibited, and the number of colonies of Staph. aureus plated from the vagina of infected mice was significantly reduced. The antibacterial activity of these strains separately and in compositions correlated with their ability to improve the performance of the immune response. These strains were the most effective in the following compositions: Lact. casei IMV B-7280-B. longum VK1-B. bifidum VK2. Strains of Lact. casei IMV B-7280, Lact. acidophilus IMV B-7279, B. bifidum VK2, and B. longum VK1 are prospective components of future probiotic drugs efficient in treating staphylococcosis and for immunity correction.     

Lactose metabolism and lactase gene sequence homologies amongst lactobacilli

A number of strains of Lactobacillus spp., including the thermophilic and mesophilic dairy species, were screened for the presence of β -galactosidase ( β -gal) and phospho- β -galactosidase (pbg) enzyme activities. The majority of lactose fermenting strains exhibited β -gal rather than pbg enzyme activity with the highest levels in the thermophilic dairy species.
Correlation between these enzymes and the presence of specific genetic determinants was sought using probes for β -gal and pbg genes from Lactobacillus casei ssp. casei strain 64H. Southern transfer and filter hybridization showed that the β-gal probe shared homology with one strain of Lact. casei ssp. casei only. Sequences homologous to the pbg gene were detected only in plasmid DNA from the same strain of Lact. casei ssp. casei and with plasmid DNA from an apparently unrelated strain of Lactobacillus which exhibited no pbg activity. Two other strains of Lact. casei ssp. casei appeared to show homology between their chromosomal DNA and the pbg gene probe. No other homologies were detected. Therefore, although lactase activity could be detected in many strains of Lactobacillus spp., the genetic determinants involved did not share extensive homology.     

Antibacterial polypeptides of Lactobacillus species

Twelve of 79 strains of the genus Lactobacillus , mainly isolated from plants or fermenting material, were found to inhibit at least one of the nine indicator strains of the species Lact. brevis, Pediococcus damnosus and Leucanostoc oenos . The antimicrobial activities from Lact. brevis B 37 and Lact. casei B 80 were caused by polypeptides detectable in the culture liquids. They are bacteriocins with a narrow antimicrobial spectrum. Brevicin 37 from Lact. brevis B 37 was active against many lactic acid bacteria and Nocardia corallina , whereas caseicin 80 from Lact. casei B 80 inhibits only one other strain of Lact. casei . Brevicin 37 is stable at 121C, caseicin 80 is inactivated above 60C, and both are inactivated under alkaline conditions.     

Antibacterial activity of Lactobacillus strains isolated from dry fermented sausages

One hundred strains of lactic acid bacteria isolated from dry cured sausages were tested for antagonistic activity against a set of test strains. Nine of 52 strains of Lactobacilus casei and three of 48 strains of Lact. plantarun produced inhibition zones against the indicator species. The substance excreted by Lact. casei CRL 705 was active against Lact. plantarum, Listeria monocytogenes, Staphylococcus aureus and a wide range of Gram-negative bacteria. The activity of the antibacterial compound from Lact. casei CRL 705 was destroyed by papain, trypsin and pepsin, but was resistant to heat (100°C for 20 min), lysozyme and catalase. The agent was produced during the growth cycle and when the concentrated and neutralized supernatant fluid was added to a fresh culture of sensitive cells it produced a rapid inactivation. A decrease in optical density (O.D.) over time, indicative of cell lysis, was also observed. These characteristics allowed us to identify the inhibitory compound as a bacteriocin which we termed Lactocin 705.     

Fermentation of fructans by epiphytic lactic acid bacteria

A total of 712 strains of lactic acid bacteria isolated from forage grasses were studied for their ability to ferment fructans of phlein- as well as inulin-type. Only 16 strains utilized phlein and eight of these also fermented inulin. They were identified as Lactobacillus paracasei subsp. paracasei, Lact. plantarum, Lact. brevis and Pediococcus pentosaceus . In the species Lact. paracasei subsp. paracasei , all strains gave positive results, whereas the other positive strains possessed unique properties within their own species. In all but two cases (strains of the species Lact. plantarum ), the phlein was more intensively fermented than the inulin, as indicated by a lower pH and a higher lactic acid concentration. On the basis of the outcome of this study it seems worthwhile to inoculate grasses of low sugar content before ensiling with an active strain that can ferment fructans.     

Ribotyping ofLactobacillus casei group strains isolated from dairy products

A series of lactobacilli isolated from dairy products were characterized using biotyping and ribotyping with EcoRI and HindIII restriction enzymes. Biotyping assigned 14 strains as Lactobacillus casei, 6 strains as Lactobacillus paracasei subsp. paracasei and 12 as Lactobacillus rhamnosus. The obtained ribotype patterns separated all analyzed strains into two clearly distinguished groups corresponding to L. rhamnosus and L. casei/L. paracasei subsp. paracasei. The HindIII ribotypes of individual strains representing these two groups were visually very similar. In contrast, EcoRI ribotyping revealed high intraspecies variability. All ribotypes of L. casei and L. paracasei subsp. paracasei dairy strains were very close and some strains even shared identical ribotype profiles. The type strains L. casei CCM 7088T (= ATCC 393T) and Lactobacillus zeae CCM 7069T revealing similar ribopatterns formed a separate subcluster using both restriction enzymes. In contrast, the ribotype profile of L. casei CCM 7089 (= ATCC 334) was very close to ribopatterns obtained from the dairy strains. These results support synonymy of L. casei and L. paracasei species revealed by other studies as well as reclassification of the type strain L. casei CCM 7088T as L. zeae and designation of L. casei CCM 7089 as the neotype strain.     

Lactobacillus casei and Lactobacillus plantarum initiate catabolism of methionine by transamination

AIMS: To study the ability of Lactobacillus casei and Lact. plantarum strains to convert methonine to cheese flavour compounds. METHODS AND RESULTS: Strains were assayed for methionine aminotransferase and lyase activities, and amino acid decarboxylase activity. About 25% of the strains assayed showed methionine aminotransferase activity. The presence of glucose in the reaction mixture increased conversion of methionine to 4-methylthio-2-ketobutanoate (KMBA) and 4-methylthio-2-hydroxybutanoate (HMBA) in all strains. The methionine aminotransferase activity in Lact. plantarum and Lact. casei showed variable specificity for the amino group acceptors glyoxylate, ketoglutarate, oxaloacetate and pyruvate. None of the strains showed methionine lyase or glutamate and methionine decarboxylase activities. CONCLUSION: The presence of amino acid converting enzymes in lactobacilli is strain specific. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings of this work suggest that lactobacilli can be used as adjuncts for flavour formation in cheese manufacture.     

Comparative study on cell envelope-associated proteinases in natural isolates of mesophilic lactobacilli

Lactobacilli isolated from different natural sources were screened for the presence of cell envelope-associated proteinases (Prt⁺ strains). Among them 17 of 75 tested isolates were Prt⁺. All Prt⁺ strains were producers of a serine-type proteinase, since their proteolytic activity was inhibited by phenylmethylsulfonyl fluoride. Most of the natural isolates of mesophilic lactobacilli degraded only β-casein such as Lactobacillus paracasei subsp. paracasei strains BGLI17 and BGLI18 and Lact. rhamnosus BGEN1. Only Lact. divergens BG742 cleaved all three, α-, β- and κ-caseins, even in the presence of Ca²⁺ ions. Total DNA isolated from Lact. paracasei subsp. paracasei strains BGLI17 and BGLI18 hybridized to the lactococcal proteinase gene probes originated from Lactococcus lactis subsp. cremoris Wg2. Hybridization could not be linked to the plasmid DNA, and pulse-field gel electrophoresis analysis suggested that the proteinase genes of these two strains are most probably chromosomally located.     

Taxonomic characterization of Lactobacillus delbrueckii subsp. bulgaricus isolates from a Cameroonian zebu's fermented raw milk

Atypical thermophilic homofermentative lactobacilli were isolated as one of the major microflora from Pindidam, an indigenous Cameroonian zebu's fermented raw milk. On the basis of their physiological characteristics, obtained isolates constituted a homogeneous group belonging to the species Lactobacillus delbrueckii. Nevertheless, their carbohydrate fermentation pattern was unusual and their identification to one of two subspecies Lact. delbrueckii subsp. bulgaricus (Lact. d. bulgaricus ) or Lact. delbrueckii subsp. lactis (Lact. d. lactis ) was rendered difficult. DNA-DNA hybridization confirmed that they belonged to the species Lact. delbrueckii and the electrophoretic mobility of the D-(—)-lactate dehydrogenase (LDH) furthermore precisely assigned them to Lact. d. bulgaricus. Whole-cell protein profiles were only able to weakly discriminate between these wild isolates and type strains of the species Lact. delbrueckii. The isolates from Pindidam were well adapted to their specific environmental conditions and demonstrated both high growth rates and ability to support elevated acidic levels in fermented milk.     

Differentiation of Lactobacillus isolates from infant faeces by SDS-PAGE and rRNA-targeted oligonucleotide probes

Isolates of lactobacilli from infant faeces phenotypically characterized as Lactobacillus paracasei subsp. paracasei (six strains), Lact. rhamnosus (six strains), Lact. gasseri (three strains), Lact. acidophilus (one strain) and Lact. fermentum/reuteri (three strains) according to recent classification systems were subjected to SDS-PAGE of whole cell proteins and rRNA-targeted oligonucleotide probe hybridization, in order to confirm the phenotypic characterization and elucidate the exact taxonomic position of the three strains that had properties between fermentum and reuteri. Results suggested a good agreement between the phenotypic characterization, SDS-PAGE and rRNA-targeted oligonucleotide probe hybridization for strains of all species except for the Lact. fermentum/reuteri strains. Results obtained by rRNA probes suggested a possible phylogenetic relatedness of the strains to Lact. reuteri. Isolates from infant faeces with interesting probiotic properties could be used as components of fermented milk products.     

Role of viability of probiotic strains in their persistence in the gut and in mucosal immune stimulation

AIMS: To determine how probiotic bacteria contact with intestinal epithelial and immune cells and the conditions to induce a good mucosal immune stimulation. METHODS AND RESULTS: Lactobacillus casei was studied by transmission electron microscopy (TEM) to determine its interaction with the gut. We compared the influence of viable and nonviable lactic acid bacteria on the intestinal mucosal immune system (IMIS) and their persistence in the gut of mice. TEM showed whole Lact. casei adhered to the villi; the bacterial antigen was found in the cytoplasm of the enterocytes. Viable bacteria stimulated the IMIS to a greater extent than nonviable bacteria with the exception of Lact. delbrueckii subsp. bulgaricus. For all the strains assayed at 72 h no antigenic particles were found in the intestine. CONCLUSION: Antigenic particles but not the whole bacteria can enter to epithelial cells and contact with the immune cells. Bacterial viability is a condition for a better stimulation of the IMIS. SIGNIFICANCE AND IMPACT OF THE STUDY: We demonstrated that only antigenic particle interact with the immune cells and their fast clearance from the gut agrees with those described for the particulate antigens. The regular consumption of probiotics should not adversely affect the host.     

Differentiation of Lactobacillus delbrueckii subspecies by ribotyping and amplified ribosomal DNA restriction analysis (ARDRA)

AIMS: To differentiate the subspecies of Lactobacillus delbrueckii, subsp. delbrueckii, subsp. lactis and subsp. bulgaricus. METHODS AND RESULTS: Amplified ribosomal DNA restriction analysis (ARDRA) and ribotyping were applied to over 30 strains. Both methods analyse the ribosomal genes which carry useful information about the evolutionary and taxonomic relationship among bacteria. The methods proved to be reliable and highly reproducible. ARDRA was applied to 16S rDNA, 23S rDNA and the IGS region, thus covering the whole rrn operon with eight restriction enzymes. Only EcoRI differentiated Lact. delbrueckii subsp. bulgaricus from Lact. delbrueckii subsp. delbrueckii/Lact. delbrueckii subsp. lactis, which confirmed the finding of other authors. Ribotyping with different enzymes under precisely optimized conditions revealed a high level of strain polymorphism. Only ribotyping with EcoRI allowed differentiation of the three subspecies on the basis of typical hybridization patterns. CONCLUSION: The successful differentiation of the three subspecies of Lact. delbrueckii by EcoRI ribotyping offers a new possibility for precise identification and differentiation of strains and new isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: Both methods could be used for differentiation of Lact. delbrueckii subspecies.     

Detection of permanent Lactobacillus casei subsp. casei strains in weaned infants' gut

The Lactobacillus microflora in the faeces of two infants was followed in the days after weaning. All colonies isolated at the highest dilution were Lactobacillus casei subsp. casei . They were identified to strain level by plasmid profile, total soluble cell protein and surface cell protein pattern. Two dominant strains of L. casei subsp. casei were identified on successive days, which suggests that these were permanent members of the infant bowel microflora.     

Expression of Lactobacillus casei ATCC 393 β-galactosidase encoded by plasmid pLZ15 in Lactococcus lactis CNRZ 1123

Lactococcus lactis subsp. lactis CNRZ 1123, a Lac^- derivative of CNRZ 1122 was transformed by electroporation with the Lactobacillus casei ATCC 393 plasmid pLZ15, which bears a β-galactosidase gene. The transformants expressed a constitutive β-galactosidase activity at a higher level than in Lact. casei , and in the cell-free extract two additional protein bands were detected by SDS-PAGE which could correspond to lactose metabolism enzymes. Both plasmid and β-gal activity were stable in Lactococcus after 100 generations in glucose-containing medium.