Identification of Lactobacillus crispatus by polymerase chain reaction targeting S-layer protein gene

AIMS: This study aimed to develop a polymerase chain reaction (PCR) method to identify Lactobacillus crispatus. METHODS AND RESULTS: A primer set (CbsA2F-CbsA2R) for amplifying conserved regions of S-layer genes was designed to identify Lact. crispatus and the specificity of this set was compared with that of another primer set (Cri 16SI-Cri 16SII) which has been reported as a species-specific primer set targeting the 16S rRNA gene. Among species in the Lact. acidophilus A1-A4 groups, when KOD polymerase was used for amplification, the primer set CbsA2F-CbsA2R gave PCR products with Lact. crispatus strains only. However, when Taq polymerase was used, this primer set gave products with one Lact. amylovorus strain as well as with Lact. crispatus strains. The primer set Cri 16SI-Cri 16SII gave PCR products with Lact. crispatus strains and two Lact. acidophilus strains, regardless of whether the polymerase used was KOD or Taq. CONCLUSIONS: A PCR targeting the S-layer gene and amplified with KOD polymerase can identify Lact. crispatus accurately and rapidly. SIGNIFICANCE AND IMPACT OF THE STUDY: To the authors' knowledge, this is the first paper to provide a PCR method for the specific identification of Lact. crispatus.     

The use of multiplex PCR reactions to characterize populations of lactic acid bacteria associated with meat spoilage

A rapid, systematic and reliable approach for identifying lactic acid bacteria associated with meat was developed, allowing for detection of Carnobacterium spp., Lactobacillus curvatus, Lact. sakei and Leuconostoc spp. Polymerase chain reaction primers specific for Carnobacterium and Leuconostoc were created from 16S rRNA oligonucleotide probes and used in combination with species-specific primers for the 16S/23S rRNA spacer region of Lact. curvatus and Lact. sakei in multiplex PCR reactions. The method was used successfully to characterize lactic acid bacteria isolated from a vacuum-packaged pork loin stored at 2 degrees C. Seventy isolates were selected for identification and 52 were determined to be Lact. sakei, while the remaining 18 isolates were identified as Leuconostoc spp.     

Identification of Lactobacillus Isolates from the Gastrointestinal Tract, Silage, and Yoghurt by 16S-23S rRNA Gene Intergenic Spacer Region Sequence Comparisons

Lactobacillus isolates were identified by PCR amplification and sequencing of the region between the 16S and 23S rRNA genes (spacer region). The sequences obtained from the isolates were compared to those of reference strains held in GenBank. A similarity of 97.5% or greater was considered to provide identification. To check the reliability of the method, the V2-V3 region of the 16S rRNA gene was amplified and sequenced in the case of isolates whose spacer region sequences were less than 99% similar to that of a reference strain. Confirmation of identity was obtained in all instances. Spacer region sequencing provided rapid and accurate identification of Lactobacillus isolates obtained from gastrointestinal, yoghurt, and silage samples. It had an advantage over 16S V2-V3 sequence comparisons because it distinguished between isolates of Lactobacillus casei and Lactobacillus rhamnosus.     

Temporal temperature gradient gel electrophoresis (TTGE) as a tool for identification of Lactobacillus casei, Lactobacillus paracasei, Lactobacillus zeae and Lactobacillus rhamnosus

AIMS: To develop a tool for rapid and inexpensive identification of the Lactobacillus casei complex. METHODS AND RESULTS: Lactobacillus casei, Lactobacillus paracasei, Lactobacillus zeae and Lactobacillus rhamnosus were identified by PCR-amplification of the segment between the U1 and U2 regions of 16S rDNA (position 8-357, Escherichia coli numbering) and temporal temperature gradient gel electrophoresis (TTGE). Seven tested Lact. paracasei strains were divided into three TTGE-subgroups. CONCLUSION: TTGE successfully distinguished between the closely-related target species. TTGE is also a powerful method for revealing sequence heterogeneities in the 16S rRNA genes. SIGNIFICANCE AND IMPACT OF THE STUDY: Due to rapid and easy performance, TTGE of PCR-amplified 16S rDNA fragments will be useful for the identification of extended numbers of isolates.     

Differentiation of Lactobacillus casei, Lactobacillus paracasei and Lactobacillus rhamnosus by polymerase chain reaction

Lactobacillus casei, Lact. paracasei and Lact. rhamnosus form a closely related taxonomic group within the heterofermentative lactobacilli. These three species are difficult to differentiate using traditional fermentation profiles. We have developed polymerase chain reaction primers which are specific for each of these species based on differences in the V1 region of the 16S rRNA gene. Sixty-three Lactobacillus isolates from cheese were identified using these primers. The 12 Lact. rhamnosus and 51 Lact. paracasei identified in this way were also differentiated using a randomly amplified polymorphic DNA (RAPD) primer.     

Rapid identification of 11 human intestinal Lactobacillus species by multiplex PCR assays using group- and species-specific primers derived from the 16S-23S rRNA intergenic spacer region and its flanking 23S rRNA

Rapid and reliable two-step multiplex polymerase chain reaction (PCR) assays were established to identify human intestinal lactobacilli; a multiplex PCR was used for grouping of lactobacilli with a mixture of group-specific primers followed by four multiplex PCR assays with four sorts of species-specific primer mixtures for identification at the species level. Primers used were designed from nucleotide sequences of the 16S-23S rRNA intergenic spacer region and its flanking 23S rRNA gene of members of the genus Lactobacillus which are commonly isolated from human stool specimens: Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus delbrueckii (ssp. bulgaricus and ssp. lactis), Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus jensenii, Lactobacillus paracasei (ssp. paracasei and ssp. tolerans), Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus and Lactobacillus salivarius (ssp. salicinius and ssp. salivarius). The established two-step multiplex PCR assays were applied to the identification of 84 Lactobacillus strains isolated from human stool specimens and the PCR results were consistent with the results from the DNA-DNA hybridization assay. These results suggest that the multiplex PCR system established in this study is a simple, rapid and reliable method for the identification of common Lactobacillus isolates from human stool samples.     

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.     

Phylogeny of the defined murine microbiota: altered Schaedler flora.

The "altered Schaedler flora" (ASF) was developed for colonizing germfree rodents with a standardized microbiota. The purpose of this study was to identify each of the eight ASF strains by 16S rRNA sequence analysis. Three strains were previously identified as Lactobacillus acidophilus (strain ASF 360), Lactobacillus salivarius (strain ASF 361), and Bacteroides distasonis (strain ASF 519) based on phenotypic criteria. 16S rRNA analysis indicated that each of the strains differed from its presumptive identity. The 16S rRNA sequence of strain ASF 361 is essentially identical to the 16S rRNA sequences of the type strains of Lactobacillus murinis and Lactobacillus animalis (both isolated from mice), and all of these strains probably belong to a single species. Strain ASF 360 is a novel lactobacillus that clusters with L. acidophilus and Lactobacillus lactis. Strain ASF 519 falls into an unnamed genus containing [Bacteroides] distasonis, [Bacteroides] merdae, [Bacteroides] forsythus, and CDC group DF-3. This unnamed genus is in the Cytophaga-Flavobacterium-Bacteroides phylum and is most closely related to the genus Porphyromonas. The spiral-shaped strain, strain ASF 457, is in the Flexistipes phylum and exhibits sequence identity with rodent isolates of Robertson. The remaining four ASF strains, which are extremely oxygen-sensitive fusiform bacteria, group phylogenetically with the low-G+C-content gram-positive bacteria (Firmicutes, Bacillus-Clostridium group). ASF 356, ASF 492, and ASF 502 fall into Clostridium cluster XIV of Collins et al. Morphologically, ASF 492 resembles members of this cluster, Roseburia cecicola, and Eubacterium plexicaudatum. The 16S rRNA sequence of ASF 492 is identical to that of E. plexicaudatum. Since the type strain and other viable original isolates of E. plexicaudatum have been lost, strain ASF 492 is a candidate for a neotype strain. Strain ASF 500 branches deeply in the low-G+C-content gram-positive phylogenetic tree but is not closely related to any organisms whose 16S rRNA sequences are currently in the GenBank database. The 16S rRNA sequence information determined in the present study should allow rapid identification of ASF strains and should permit detailed analysis of the interactions of ASF organisms during development of intestinal disease in mice that are coinfected with a variety of pathogenic microorganisms.     

A real-time PCR assay for the rapid determination of 16S rRNA genotype in Vibrio vulnificus

In a terminal restriction fragment polymorphism (T-RFLP) study, we recently reported a significant association between the type B 16S rRNA gene and clinical strains of Vibrio vulnificus associated with the consumption of raw oysters. In the present study we describe a real-time PCR assay for the rapid determination of the 16S rRNA type of V. vulnificus isolates. This assay was used to reexamine the 16S rRNA gene type in the strains studied previously by T-RFLP and additional isolates from selected sources. Analyses revealed that 15 of the strains (10 environmental and 5 clinical) previously found to be 16S rRNA type A actually appear to possess both the type A and B genes. The presence of both alleles was confirmed by cloning and sequencing both gene types from one strain. To our knowledge, this is the first report of 16S rRNA sequence heterogeneity within individual strains of V. vulnificus. The findings confirm the T-RFLP data that 16S rRNA type may be a useful marker for determining the clinical significance of V. vulnificus in disease in humans and cultured eels. The real-time PCR assay is much more rapid and less resource-intensive than T-RFLP, and should facilitate further study of the occurrence and distribution of the 16S rRNA genotypes of V. vulnificus. These studies should provide more definitive estimates of the risks associated with this organism and may lead to a better understanding of its virulence mechanism(s).     

Detection and identification of gastrointestinal Lactobacillus species by using denaturing gradient gel electrophoresis and species-specific PCR primers

Denaturing gradient gel electrophoresis (DGGE) of DNA fragments obtained by PCR amplification of the V2-V3 region of the 16S rRNA gene was used to detect the presence of Lactobacillus species in the stomach contents of mice. Lactobacillus isolates cultured from human and porcine gastrointestinal samples were identified to the species level by using a combination of DGGE and species-specific PCR primers that targeted 16S-23S rRNA intergenic spacer region or 16S rRNA gene sequences. The identifications obtained by this approach were confirmed by sequencing the V2-V3 region of the 16S rRNA gene and by a BLAST search of the GenBank database.     

Characterization of cell envelope-associated proteinases of thermophilic lactobacilli

The proteolytic activities of two natural isolates of thermophilic lactobacilli, Lactobacillus acidophilus BGRA43 and Lact. delbrueckii BGPF1, and Lact. acidophilus CH2 (Chr. Hansen's strain) and Lact. acidophilus V74 (Visby's strain), were compared. Results revealed that optimal pH for all four proteinases is 6.5, whereas temperature optimum varied among proteinases. Determination of caseinolytic activity done under optimal conditions for each strain revealed that the CH2 and V74 proteinases completely hydrolysed both alphaS1-casein and beta-casein, showing very low activity towards kappa-casein. The BGPF1 proteinase completely hydrolysed only beta-casein. The BGRA43 proteinase completely hydrolysed all three casein fractions. The proteolytic activities of whole cells were inhibited by serine proteinase inhibitors, suggesting that all four strains produce serine proteinases. DNA-DNA hybridization and PCR analysis showed that BGPF1 contains the prtB-like proteinase gene. Characterized thermophilic strains BGPF1 and BGRA43 were successfully used as starter cultures for production of yoghurt and acidophilus milk, respectively.     

Characterization of cell envelope-associated proteinases of thermophilic lactobacilli

D. FIRA, M. KOJIC, A. BANINA, I. SPASOJEVIC, I. STRAHINIC AND L. TOPISIROVIC. 2001 . The proteolytic activities of two natural isolates of thermophilic lactobacilli, Lactobacillus acidophilus BGRA43 and Lact. delbrueckii BGPF1, and Lact. acidophilus CH2 (Chr. Hansen's strain) and Lact. acidophilus V74 (Visby's strain), were compared. Results revealed that optimal pH for all four proteinases is 6·5, whereas temperature optimum varied among proteinases. Determination of caseinolytic activity done under optimal conditions for each strain revealed that the CH2 and V74 proteinases completely hydrolysed both α_S1-casein and β-casein, showing very low activity towards κ-casein. The BGPF1 proteinase completely hydrolysed only β-casein. The BGRA43 proteinase completely hydrolysed all three casein fractions. The proteolytic activities of whole cells were inhibited by serine proteinase inhibitors, suggesting that all four strains produce serine proteinases. DNA–DNA hybridization and PCR analysis showed that BGPF1 contains the prtB -like proteinase gene. Characterized thermophilic strains BGPF1 and BGRA43 were successfully used as starter cultures for production of yoghurt and acidophilus milk, respectively.     

VANCOMYCIN SUSCEPTIBILITY AS AN AID TO THE IDENTIFICATION OF LACTOBACILLI

Forty strains of lactobacilli isolated from probiotic supplements or functional foods, and two clinical isolates, have been identified by API 50 CHL and tested for susceptibility to vancomycin. All the Lactobacillus acidophilus (16) and Lact. delbreuckii (two) strains were sensitive to vancomycin, while all the other strains (mainly Lact. rhamnosus, 15) were resistant. Susceptibility to other antibiotics was not species-specific. Differential susceptibility to vancomycin may be helpful in speciation of lactobacilli.     

Lactobacillus casei, Lactobacillus rhamnosus, and Lactobacillus zeae isolates identified by sequence signature and immunoblot phenotype

Species taxonomy within the Lactobacillus casei group of bacteria has been unsettled. With the goal of helping clarify the taxonomy of these bacteria, we investigated the first 3 variable regions of the 16S rRNA gene, the 16S-23S rRNA interspacer region, and one third of the chaperonin 60 gene for Lactobacillus isolates originally designated as L. casei, L. paracasei, L. rhamnosus, and L. zeae. For each genetic region, a phylogenetic tree was created and signature sequence analysis was done. As well, phenotypic analysis of the various strains was performed by immunoblotting. Both sequence signature analysis and immunoblotting gave immediate identification of L. casei, L. rhamnosus, and L. zeae isolates. These results corroborate and extend previous findings concerning these lactobacilli; therefore, we strongly endorse recent proposals for revised nomenclature. Specifically, isolate ATCC 393 is appropriately rejected as the L. casei type strain because of grouping with isolates identified as L. zeae. As well, because all other L. casei isolates, including the proposed neotype isolate ATCC 334, grouped together with isolates designated L. paracasei, we support the use of the single species L. casei and rejection of the name L. paracasei.     

Changes in the Lactobacillus community during Ricotta forte cheese natural fermentation

The loss of microbial biodiversity due to the increase in large-scale industrial processes led to the study of the natural microflora present in a typical little known dairy product. The community of lactobacilli was studied in order to understand the natural fermentation of Ricotta forte cheese. The combined use of RAPD analysis, 16S rDNA sequencing and physiological tests allowed 33 different strains belonging to 10 species of Lactobacillus to be characterized. RAPD analysis revealed the heterogeneity of both the Lact. kefiri and Lact. paracasei species. The sequence analysis of the large 16S/23S rRNA spacer region enabled Lact. plantarum to be distinguished from Lact. paraplantarum, two closely related species belonging to the Lact. plantarum group. The recovery of strains endowed with interesting physiological characteristics, such as strong stress resistance, could improve technological and/or organoleptic characteristics of Ricotta forte cheese and other fermented foods.     

Rapid identification of the three major species of dairy obligate heterofermenters Lactobacillus brevis, Lactobacillus fermentum and Lactobacillus parabuchneri by species-specific duplex PCR

In this study, the biodiversity of 154 strains of lactic acid bacteria, including 112 dairy product isolates presumptively identified as obligately heterofermentative lactobacilli (OHL) by classical microbiological tests, as well as 23 OHL-type strains, was investigated by PCR-based methods and gene sequencing. Using these techniques, 51% of the cheese isolates were actually identified as OHL. The non-OHL isolates were identified to the Leuconostoc, Lactobacillus, Weisella, Pediococcus or Streptococcus genera. Among the OHL cheese isolates, five species were directly identified including three of the most frequently isolated species -Lactobacillus fermentum, Lactobacillus parabuchneri and Lactobacillus brevis- and two rarely isolated species - Lactobacillus diolivorans and Lactobacillus reuteri. A sixth group made up of two dairy isolates was also identified and according to 16S rRNA gene and intergenic spacer region (ISR) sequencing data corresponded to Lactobacillus sp. and may constitute a new species. Species-specific primers were designed for the rapid and reliable detection of the three most frequently isolated species by species-specific duplex PCR.     

Epidemiological surveillance of mycoplasmas belonging to the ‘Mycoplasma mycoides’ cluster: is DGGE fingerprinting of 16S rRNA genes suitable?

Aims:  The analysis by Denaturing Gradient Gel Electrophoresis (DGGE) of the PCR-amplified V3 region of 16S rRNA gene was previously shown to detect and differentiate a large number of human and animal mycoplasmas. In this study, we further assessed the suitability of the technique for epidemiological surveillance of mycoplasmas belonging to the ' Mycoplasma mycoides ' cluster, a phylogenetic group that includes major ruminant pathogens.
Methods and Results:  The V3 region of 16S rRNA genes from approx. 50 field strains was amplified and analysed by DGGE. Detection and identification results were compared with the ones obtained by antigenic testing and sequence analysis.
Conclusions:  The DGGE technique is robust and valuable as a first-line test, but the patterns obtained for strains belonging to the ' M. mycoides ' cluster were too variable within a taxon and in contrast too conserved between taxa to allow an unequivocal identification of isolates without further analysis.
Significance and Impact of the Study:  Issues raised by the quest for a single universal test able to detect and identify any mycoplasma in one clinical sample are thoroughly documented.     

Dominance of Lactobacillus acidophilus in the Facultative Jejunal Lactobacillus Microbiota of Fistulated Beagles

Lactobacilli were isolated from jejunal chyme from five fistulated beagles. Cultivable lactobacilli varied from 10(4) to 10(8) CFU/ml. Seventy-four isolates were identified by partial 16S rRNA gene sequencing and differentiated by repetitive element PCR (Rep-PCR), Lactobacillus acidophilus was dominant, and nearly 80% of 54 isolates shared the same DNA fingerprint pattern.     

Characterization and species recognition of Lactobacillus plantarum strains by restriction fragment length polymorphism (RFLP) of the 16S rRNA gene

Twenty-one strains, labelled Lactobacillus plantarum or Lact. plantarum -like, and isolated from different natural sources, were characterized by restriction fragment length polymorphism (RFLP) of the 16S rRNA gene using Hin dIII and Eco RI cleaved chromosomal DNA, together with Lact. plantarum ATCC 14917^T, Lact. pentosus ATCC 8041^T, Lact. plantarum ATCC 10776 and Lact. plantarum ATCC 8014. The fermentation patterns on API 50CH were recorded at 30°C and 37°C for all strains. The phenotypes were heterogeneous, and the ability to ferment 17 of the 49 carbohydrates varied. The fermentation of some carbohydrates, for example D-raffinose and D-arabitol, was temperature-dependent. Strains having identical API profiles were separated by the plasmid profile. All strains but one (affiliated to Lact. casei ) had identical 16S ribosomal DNA sequences ( Lact. plantarum/Lact. pentosus ). The RFLP study resulted in identical ribopatterns for 17 of the strains, including the type strain of Lact. plantarum (pattern A1). Four strains had related fragment patterns to that of Lact. plantarum sensu stricto; three of these strains had more than 60% DNA: DNA homology to the type strain of Lact. plantarum , and one had less than 50% DNA: DNA homology to Lact. plantarum ATCC 14917^T. Two strains had fragment patterns similar to the type strain of Lact. pentosus , and they had more than 80% DNA: DNA homology to Lact. pentosus ATCC 8041^T. One of the Lact. pentosus strains shared one band with the A1 pattern. The ribopatterns of Lact. plantarum were homogeneous (identical for 85% of the strains), irrespective of phenotype and source of isolation. RFLP of the 16S rRNA genes using Eco RI and Hin dIII might be used for species recognition of Lact. plantarum , but seems less suitable for strain typing.     

Rapid identification of Lactobacillus brevis using the polymerase chain reaction

AIMS: Species-specific PCR was applied to identify Lactobacillus brevis and the sensitivity and the specificity of the protocol were determined. METHODS AND RESULTS: Strains of Lact. brevis obtained from foods, particularly dairy products, and various strain collections, were identified by PCR using primers which amplified a 1340 bp fragment within the 16S rRNA gene. The PCR product was obtained after amplification of all the Lact. brevis strains tested; the size of the amplicon was as expected. No PCR products were observed after amplification from DNA of several lactic acid bacteria (LAB) species. CONCLUSIONS: A PCR method was optimized to identify Lact. brevis. The protocol was highly efficient and sensitive. SIGNIFICANCE AND IMPACT OF THE STUDY: Conventional phenotypic methods often lead to ambiguous identification of LAB species belonging to Lact. brevis. The proposed protocol is sensitive, specific, and can be applied to total DNA extracted by use of chelating matrix with loss of neither sensitivity nor specificity.