Rapid differentiation and in situ detection of 16 sourdough lactobacillus species by multiplex PCR

A two-step multiplex PCR-based method was designed for the rapid detection of 16 species of lactobacilli known to be commonly present in sourdough. The first step of multiplex PCR was developed with a mixture of group-specific primers, while the second step included three multiplex PCR assays with a mixture of species-specific primers. Primers were derived from sequences that specify the 16S rRNA, the 16S-23S rRNA intergenic spacer region, and part of the 23S rRNA gene. The primer pairs designed were shown to exclusively amplify the targeted rrn operon fragment of the corresponding species. Due to the reliability of simultaneously identifying Lactobacillus plantarum, Lactobacillus pentosus, and Lactobacillus paraplantarum, a previously described multiplex PCR method employing recA gene-derived primers was included in the multiplex PCR system. The combination of a newly developed, quick bacterial DNA extraction method from sourdough and this multiplex PCR assay allows the rapid in situ detection of several sourdough-associated lactobacilli, including the recently described species Lactobacillus rossii, and thus represents a very useful alternative to culture-based methodologies.     

Rapid Differentiation and In Situ Detection of 16 Sourdough Lactobacillus Species by Multiplex PCR

A two-step multiplex PCR-based method was designed for the rapid detection of 16 species of lactobacilli known to be commonly present in sourdough. The first step of multiplex PCR was developed with a mixture of group-specific primers, while the second step included three multiplex PCR assays with a mixture of species-specific primers. Primers were derived from sequences that specify the 16S rRNA, the 16S-23S rRNA intergenic spacer region, and part of the 23S rRNA gene. The primer pairs designed were shown to exclusively amplify the targeted rrn operon fragment of the corresponding species. Due to the reliability of simultaneously identifying Lactobacillus plantarum, Lactobacillus pentosus, and Lactobacillus paraplantarum, a previously described multiplex PCR method employing recA gene-derived primers was included in the multiplex PCR system. The combination of a newly developed, quick bacterial DNA extraction method from sourdough and this multiplex PCR assay allows the rapid in situ detection of several sourdough-associated lactobacilli, including the recently described species Lactobacillus rossii, and thus represents a very useful alternative to culture-based methodologies.     

Rapid identification of the species of the Bacteroides fragilis group by multiplex PCR assays using group- and species-specific primers

We report a rapid and reliable two-step multiplex polymerase chain reaction (PCR) assay to identify the 10 Bacteroides fragilis group species - Bacteroides caccae, B. distasonis, B. eggerthii, B. fragilis, B. merdae, B. ovatus, B. stercoris, B. thetaiotaomicron, B. uniformis and B. vulgatus. These 10 species were first divided into three subgroups by multiplex PCR-G, followed by three multiplex PCR assays with three species-specific primer mixtures for identification to the species level. The primers were designed from nucleotide sequences of the 16S rRNA, the 16S-23S rRNA intergenic spacer region and part of the 23S rRNA gene. The established two-step multiplex PCR identification scheme was applied to the identification of 155 clinical isolates of the B. fragilis group that were previously identified to the species level by phenotypic tests. The new scheme was more accurate than phenotypic identification, which was accurate only 84.5% of the time. The multiplex PCR scheme established in this study is a simple, rapid and reliable method for the identification of the B. fragilis group species. This will permit more accurate assessment of the role of various B. fragilis group members in infections and of the degree of antimicrobial resistance in each of the group members.     

Rapid species identification within two groups of closely related lactobacilli using PCR primers that target the 16S/23S rRNA spacer region

A rapid and reliable PCR-based method for distinguishing closely related species within two groups of lactobacilli is described. Primers complementary to species-specific sequences in the 16S/23S rDNA spacer regions were designed after sequencing and sequence comparison of the spacer regions of 32 strains. The strains belong to two groups of closely related Lactobacillus species; one composed of Lactobacillus curvatus, Lactobacillus graminis and Lactobacillus sake, the other of Lactobacillus paraplantarum, Lactobacillus pentosus and Lactobacillus plantarum. PCR assays with the designed primers and subsequent agarose gel analysis of the amplified fragments allowed the same species identification as the DNA/DNA hybridization procedure.     

Quantitative evaluation of adhesion of lactobacilli isolated from human intestinal tissues to human colonic mucin using surface plasmon resonance (BIACORE assay)

AIMS: To isolate lactobacilli from the mucus layer of the human intestine and evaluate their adhesion abilities using a BIACORE assay. METHODS AND RESULTS: Thirty strains of lactobacilli were isolated from the mucus layer of normal human intestinal tissues using conventional plate culture. The strains were identified using homology comparisons of the 16S rDNA sequence to databases as Lactobacillus salivarius (26%), Lactobacillus fermentum (13%), Lactobacillus gasseri (10%), Lactobacillus paracasei (7%), Lactobacillus casei (3%), Lactobacillus mucosae (3%) and Lactobacillus plantarum (3%). Lactobacillus plantarum LA 318 shows the highest adhesion to human colonic mucin (HCM) using the BIACORE assay at 115.30 +/- 12.37 resonance unit (RU). The adhesion of cell wall surface proteins from strain LA 318 was significantly higher to HCM than to bovine serum albumin (BSA; P < 0.05). CONCLUSIONS: We isolated 30 strains of lactobacilli. Lactobacillus salivarius was the predominant species of lactobacilli isolated in this study. The adhesion of strain LA 318 isolated from human transverse colon to its mucin was shown. The adhesion could be mediated by lectin-like components on the bacterial cell surface. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study where lactobacilli were isolated from human intestinal tissues and shown to adhere to HCM.     

Rapid identification of Lactobacillus and Bifidobacterium in probiotic products using multiplex PCR

Lactic acid bacteria (LAB) are beneficial for the gastrointestinal tract and reinforce immunity in human health. Recently, many functional products using the lactic acid bacteria have been developed. Among these LAB, Lactobacillus acidophilus, Lactobacillus rhamnosus, Bifidobacterium longum, and Bifidobacterium bifidum are frequently used for probiotic products. In order to monitor these LAB in commercial probiotic products, a multiplex PCR method was developed. We designed four species-specific primer pairs for multiplex PCR from the 16S rRNA, 16S-23S rRNA intergenic spacer region, and 23S rRNA genes in Lactobacillus acidophilus, Lactobacillus rhamnosus, Bifidobacterium longum, and Bifidobacterium bifidum. Using these primer pairs, 4 different LAB were detected with high specificity in functional foods. We suggest that the multiplex PCR method developed in this study would be an efficient tool for simple, rapid, and reliable identification of LAB used as probiotic strains.     

A one-step reaction for the rapid identification of Lactobacillus mindensis, Lactobacillus panis, Lactobacillus paralimentarius, Lactobacillus pontis and Lactobacillus frumenti using oligonucleotide primers designed from the 16S-23S rRNA intergenic sequences

Aims:  Species-specific primers targeting the 16S–23S ribosomal DNA (rDNA) intergenic spacer region (ISR) were designed to rapidly discriminate between Lactobacillus mindensis , Lactobacillus panis , Lactobacillus paralimentarius , Lactobacillus pontis and Lactobacillus frumenti species recently isolated from French sourdough.
Methods and Results:  The 16S–23S ISRs were amplified using primers 16S/p2 and 23S/p7, which anneal to positions 1388–1406 of the 16S rRNA gene and to positions 207–189 of the 23S rRNA gene respectively, Escherichia coli numbering (GenBank accession number V00331 ). Clone libraries of the resulting amplicons were constructed using a pCR2·1 TA cloning kit and sequenced. Species-specific primers were designed based on the sequences obtained and were used to amplify the 16S–23S ISR in the Lactobacillus species considered. For all of them, two PCR amplicons, designated as small ISR (S-ISR) and large ISR (L-ISR), were obtained. The L-ISR is composed of the corresponding S-ISR, interrupted by a sequence containing tRNA^Ile and tRNA^Ala genes. Based on these sequences, species-specific primers were designed and proved to identify accurately the species considered among 30 reference Lactobacillus species tested.
Conclusions:  Designed species-specific primers enable a rapid and accurate identification of L. mindensis , L. paralimentarius , L. panis , L. pontis and L. frumenti species among other lactobacilli.
Significance and Impact of the Study:  The proposed method provides a powerful and convenient means of rapidly identifying some sourdough lactobacilli, which could be of help in large starter culture surveys.     

Three Lactobacillus strains from healthy infant stool inhibit enterotoxigenic Escherichia coli grown in vitro

Enterotoxigenic Escherichia coli (ETEC) are a major cause of sporadic diarrhea disease in humans, affecting mainly infants in developing countries and travelers from industrialized countries visiting tropical or subtropical areas. In this study, we screen the antagonistic activity by inoculating wells among ETEC agar cultures to assess inhibition zones created by the lactobacilli spent culture supernatant (SCS) from healthy infant stool. Only three isolates possessed antagonistic activity, acid and bile tolerance and could adhere to the cultured human intestinal C2BBel (Caco-2) cell line. Isolate identification using API 50CHL strips showed that they belonged to different Lactobacillus species, i.e., Lactobacillus acidophilus RY2, Lactobacillus salivarius MM1 and Lactobacillus paracasei En4. The SCS still had an inhibitory effect on ETEC after heating (100 degrees C, 15 min). The lactate dehydrogenase treatment or the pH of SCS was adjusted to neutral (pH 7.2) to reduce the SCS inhibitory effect. Antimicrobial activity was performed by incubating the lactic acid bacteria (LAB)-SCS with ETEC suspension. After 4h of co-culture, ETEC growth was inhibited. This study suggests that L. acidophilus RY2, L. salivarius MM1 and L. paracasei En4 could be used as an effective control for ETEC.     

Genomic diversity of cultivable Lactobacillus populations residing in the neonatal and adult gastrointestinal tract

The objective of this study was to investigate the cultivable Lactobacillus population in adult and infant faecal material to identify strains shared across a number of individuals. A range of lactobacilli isolated on Lactobacillus-selective agar from faeces of 16 infants and 11 adults were genetically fingerprinted and further characterized by 16S rRNA gene sequencing. The relatedness of all the Lactobacillus strains isolated to known species was also determined both genetically and phenotypically. This study revealed that the human intestine is initially colonized by only a few (1-2) different cultivable strains whereas in adults the pattern becomes more complex with a higher diversity of strains. The adult samples contained three genetically distinct Lactobacillus strains in some cases, while infant samples generally harboured only one dominant Lactobacillus strain. Moreover, the species in general appeared to differ with Lactobacillus rhamnosus and Lactobacillus casei/paracasei found mainly in adults, whereas Lactobacillus gasseri and Lactobacillus salivarius were more commonly isolated in infant samples. The data reaffirm the differences in Lactobacillus populations both between individual subjects and between the infant and adult, with an overall change in the diversity and complexity from early stages of life to adulthood.     

Use of 16S-23S rRNA spacer-region (SR)-PCR for identification of intestinal clostridia

The suitability of a species identification technique based on PCR analysis of 16S-23S rRNA spacer region (SR) polymorphism for human intestinal Clostridium species was evaluated. This SR-PCR based technique is highly reproducible and successfully differentiated the strains tested, which included 17 ATCC type strains of Clostridium and 152 human stool Clostridium isolates, at the species or intraspecies level. Ninety-eight of 152 stool isolates, including C. bifermentans, C. butyricum, C. cadaveris, C. orbiscindens, C. paraputrificum, C. pefringens, C. ramosum, C. scindens, C. spiroforme, C. symbiosum and C. tertium, were identified to species level by SR-PCR patterns that were identical to those of their corresponding ATCC type strains. The other 54 stool isolates distributed among ten SR-PCR patterns that are unique and possibly represent ten novel Clostridium species or subspecies. The species identification obtained by SR-PCR pattern analysis completely agreed with that obtained by 16S rRNA sequencing, and led to identification that clearly differed from that obtained by cellular fatty acid analysis for 23/152 strains (15%). These results indicate that SR-PCR provides an accurate and rapid molecular method for the identification of human intestinal Clostridium species.     

Identification of lactobacilli and bifidobacteria

Selective culture media and phenotypic tests enable lactobacilli to be differentiated from morphologically similar bacteria. The accurate identification of Lactobacillus species can be accomplished by reference to 16S rRNA gene sequences. Species-specific, PCR primers that target the 16S-23S rRNA spacer region are available for a limited number of Lactobacillus species. Molecular methods for the comprehensive identification of Bifidobacterium species are not yet available. Only DNA-DNA reassociation provides a reliable means of species identification for this genus at present. Bifidobacteria can be differentiated from morphologically similar bacteria by the use of genus-specific, PCR primers or oligonucleotide probes.     

Strain typing with ISLpl1 in lactobacilli

Twenty-seven Lactobacillus plantarum ssp. plantarum, 11 Lactobacillus paraplantarum and five Lactobacillus casei-related strains, isolated from various autochthonous Serbian and Montenegro-fermented foods, were identified using phenotypical characterization and current PCR methods based on PCR of the recA gene or the 23S-5S rRNA gene intragenic spacer (IS) region. The strains were genotypically characterized by a new method based on the insertion sequence element ISLpl11 that grouped these lactobacilli into 10 IS-fingerprinting groups. Between six and 23 copies of the ISLpl1 were found in each strain and the ISLpl1-fingerprint groups correlated well with the origin of the strains. The method proved suitable for strain typing of lactic acid bacteria at the infraspecies level.     

Polyphasic approach to bacterial dynamics during the ripening of Spanish farmhouse cheese, using culture-dependent and -independent methods

We studied the dynamics of the microbial population during ripening of Cueva de la Magahá cheese using a combination of classical and molecular techniques. Samples taken during ripening of this Spanish goat's milk cheese in which Lactococcus lactis and Streptococcus thermophilus were used as starter cultures were analyzed. All bacterial isolates were clustered by using randomly amplified polymorphic DNA (RAPD) and identified by 16S rRNA gene sequencing, species-specific PCR, and multiplex PCR. Our results indicate that the majority of the 225 strains isolated and enumerated on solid media during the ripening period were nonstarter lactic acid bacteria, and Lactobacillus paracasei was the most abundant species. Other Lactobacillus species, such as Lactobacillus plantarum and Lactobacillus parabuchneri, were also detected at the beginning and end of ripening, respectively. Non-lactic-acid bacteria, mainly Kocuria and Staphylococcus strains, were also detected at the end of the ripening period. Microbial community dynamics determined by temporal temperature gradient gel electrophoresis provided a more precise estimate of the distribution of bacteria and enabled us to detect Lactobacillus curvatus and the starter bacteria S. thermophilus and L. lactis, which were not isolated. Surprisingly, the bacterium most frequently found using culture-dependent analysis, L. paracasei, was scarcely detected by this molecular approach. Finally, we studied the composition of the lactobacilli and their evolution by using length heterogeneity PCR.     

Application of a novel polyphasic approach to study the lactobacilli composition of sourdoughs from the Abruzzo region (central Italy)

AIMS: To characterize the lactobacilli community of 20 sourdoughs using a novel polyphasic approach. METHODS AND RESULTS: A polyphasic approach, consisting of a two-step multiplex polymerase chain reaction (PCR) system, 16S rRNA gene sequence analysis and physiological features, was applied to identify 127 isolates, representing about 37% of the presumptive lactobacilli collected from sourdough samples. Multiplex PCR successfully identified 111 isolates, while 16S rRNA gene sequencing was applied for the other 16 isolates, two of which could not be associated with any previously described lactic acid bacteria (LAB) species. Strain diversity was evaluated by phenotypic and random amplified polymorphic DNA-PCR analysis. Molecular detection of Lactobacillus group species was also performed on total DNA extracted from the doughs. CONCLUSIONS: Abruzzo region sourdough lactobacilli biodiversity, reflected in both Lactobacillus species composition and strain polymorphism, is similar to that of other Italian regions and is a source of novel LAB species. SIGNIFICANCE AND IMPACT OF THE STUDY: Within culture-independent methods, multiplex PCR is a rapid tool to study the lactobacilli population of sourdoughs.     

Multiplex PCR for the detection of Lactobacillus pontis and two related species in a sourdough fermentation

A specific multiplex PCR assay based on the amplification of parts of the 16S rRNA molecule was designed. Primers derived from variable regions of the 16S rRNA provided a means of easily differentiating the species Lactobacillus pontis and Lactobacillus panis. They could be clearly discriminated from the phylogenetically related species Lactobacillus vaginalis, Lactobacillus oris, and Lactobacillus reuteri and from other lactobacilli commonly known to be present in sourdough. Other strains isolated together with L. pontis from an industrial sourdough fermentation could be clearly separated from these species by comparative sequence analysis and construction of a specific PCR primer. For a fast identification a DNA isolation protocol based on the ultrasonic lysis of cells from single colonies was developed. To demonstrate the potential of such techniques for tracking these organisms in a laboratory-scale fermentation, we combined the specific PCR assay with direct DNA extraction from the organisms in the sourdough without previous cultivation.     

Antimicrobial potential of four Lactobacillus strains isolated from breast milk

AIMS: The antimicrobial potential of four lactobacilli (Lactobacillus salivarius CECT5713, Lactobacillus gasseri CECT5714, L. gasseri CECT5715 and Lactobacillus fermentum CECT5716), isolated from fresh human breast milk, was evaluated in this study and compared with Lactobacillus coryniformis CECT5711, a reuterin-producing strain isolated from an artisan goat's cheese. METHODS AND RESULTS: Agar diffusion tests, competitive adhesion assays and mucin expression assays were carried out in order to value the antibacterial properties of the lactobacilli strains. The antibacterial capability of the strains was tested in vivo by using a murine infection model with Salmonella choleraesuis. The results revealed that all the strains studied, displayed antibacterial properties against pathogenic bacteria. However, the antibacterial potential varied among the lactobacilli tested and, in fact, L. salivarius CECT5713 showed not only the best in vitro antibacterial activity, but also the highest protective effect against a Salmonella strain in the murine infection model. CONCLUSION: The four breast-milk lactobacilli, and particularly L. salivarius CECT5713, possess potent antibacterial activities that result in a higher protection against S. choleraesuis CECT4155 in a mouse infection model. SIGNIFICANCE AND IMPACT OF THE STUDY: These results suggest that lactobacilli from breast milk could contribute to an anti-infective protection in neonates and would be excellent candidates for the development of infant probiotic products.     

Genetic diversity of vaginal lactobacilli from women in different countries based on 16S rRNA gene sequences

AIMS: Lactobacilli are widely distributed in food and the environment, and some colonize the human body as commensal bacteria. The aim of this study was to determine the species of lactobacilli that colonize the vagina and compare them with those found in food and the environment. METHODS AND RESULTS: Thirty-five Lactobacillus strains from women from seven countries were isolated, and sequences from 16S rRNA genes were determined and compared with existing data in GenBank. A phylogenetic tree was achieved using the Neighbour-Joining method based on the analysis of 1465 nucleotides. The results showed that most vaginal isolates were L. crispatus, L. jensenii and L. gasseri. Some were L. vaginalis, L. fermentum, L. mucosae, L. paracasei and L. rhamnosus. Two isolates from a native American woman displayed distinct branches, indicating novel phylotypes. Few vaginal isolates matched food or environmental Lactobacillus species. CONCLUSIONS: Most women worldwide were colonized by three common Lactobacillus species: L. crispatus, L. jensenii and L. gasseri. SIGNIFICANCE AND IMPACT OF THE STUDY: Knowledge of vaginal Lactobacillus species richness and distribution in women worldwide may lead to the design of better probiotic products as bacterial replacement therapy.     

Differentiation of non-pylori Helicobacter species based on PCR–restriction fragment length polymorphism of the 23S rRNA gene

Phenotypic identification of non-pylori Helicobacter species has always been problematic and time-consuming in comparison with many other bacteria. We developed a rapid two-step identification assay based on PCR–restriction fragment length polymorphism (PCR–RFLP) analysis of the 23S rRNA gene for differentiating between non-pylori Helicobacter species. A new genus-specific primer pair based on all available complete and partial 23S rRNA sequences of Helicobacter species was designed. In silico restriction analysis of variable regions of the 23S rRNA gene suggested SmaI and HindIII endonucleases would provide a good level of differentiation. Analysis of the obtained 23S rRNA RFLP patterns divided all Helicobacter study strains into three species groups (groups A–C) and 12 unique restriction patterns. Wolinella succinogenes also gave a unique pattern. Our proposed PCR–RFLP method was found to be as a valuable tool for routine identification of non-pylori Helicobacter species from human or animal samples.     

Enzyme production by lactobacilli and the potential link with infective endocarditis

H.J. OAKEY, D.W.S. HARTY AND K.W. KNOX. 1995. Fifty-six strains of lactobacilli were examined for the production of glycosidases and proteases (arylamidases) that could be associated with the ability to grow in vivo and/or be a factor in the pathogenesis of endocarditis. The strains were from seven species, with an emphasis on Lactobacillus rhamnosus and Lact. paracasei subsp. paracasei , both of which have been associated with endocarditis and provided 12 of the 13 strains isolated from cases of the disease. Other species were Lact. acidophilus, Lact. plantarum, Lact. salivarius, Lact. fermentum and Lact. oris.
Commonly expressed glycosidase activities were α-D-galactosidase and β- N -acetyl-D-glucosaminidase followed by β-D-glucosidase and α-L-fucosidase. The combined production of β- N -acetyl-D-glucosaminidase and α-D-galactosidase was a feature of the endocarditis isolates. In contrast, β-D-galactosidase was produced by very few of the strains within species implicated in endocarditis but most of the strains of Lact. salivarius, Lact. fermentum and Lact. oris.
The most commonly produced arylamidases active against substrates employed for testing human blood clotting cascade were activated protein C(Ca)-like, activated factor X(Xa)-like and Hageman factor-like followed by kallikrein-like and chymotrypsin-like enzymes. Kallikrein-like enzyme activity was shown more frequently by strains from species commonly isolated from cases of endocarditis ( Lact. rhamnosus and Lact. paracasei subsp. paracasei ) than from other oral species ( Lact. plantarum, Lact. salivarius, Lact. fermentum and Lact. oris ).
The data indicate that some lactobacilli can produce enzymes that would enable the breakdown of human glycoproteins and the synthesis and lysis of human fibrin clots, characteristics which aid the colonization and survival of bacteria infecting an endocarditis vegetation.     

Rapid identification of potentially probiotic Bifidobacterium species by multiplex PCR using species-specific primers based on the region extending from 16S rRNA through 23S rRNA

This study aimed at developing a novel multiplex polymerase chain reaction (PCR) primer set for identification of the potentially probiotic Bifidobacterium species B. adolescentis, B. animalis subsp. animalis (B. animalis), B. bifidum, B. breve, B. longum biovar infantis (B. infantis), B. animalis subsp. lactis B. lactis, B. longum biovar longum (B. longum) and B. pseudolongum. The primer set comprised specific and conserved primers and was derived from the integrated sequences of 16S and 23S rRNA genes and the rRNA intergenic spacer region (ISR) of each species. It could detect and identify type strains and isolates from pharmaceuticals or dairy products corresponding to the eight Bifidobacterium species with high specificity. It was also useful for screening of the related strains from natural sources such as the gastro-intestinal tract and feces. We suggest that the assay system from this study is an efficient tool for simple, rapid and reliable identification of Bifidobacterium species for which probiotic strains are known.