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.     

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.     

Evolution of the bacterial species Lactobacillus delbrueckii: a partial genomic study with reflections on prokaryotic species concept

The species Lactobacillus delbrueckii consists at present of three subspecies, delbrueckii, lactis and bulgaricus, showing a high level of DNA-DNA hybridization similarity but presenting markedly different traits related to distinct ecological adaptation. The internal genetic heterogeneity of the bacterial species L. delbrueckii was analyzed. Phenotypic and several genetic traits were investigated for 61 strains belonging to this species. These included 16S rDNA sequence mutations, expression of beta-galactosidase and of the cell wall-anchored protease, the characterization of the lactose operon locus and of the sequence of lacR gene, galactose metabolism, and the distribution of insertion sequences. The high genetic heterogeneity of taxa was confirmed by every trait investigated: the lac operon was completely deleted in the subsp. delbrueckii, different mutation events in the repressor gene of the operon led to a constitutive expression of lacZ in the subsp. bulgaricus. Structural differences in the same genetic locus were probably due to the presence of different IS elements in the flanking regions. The different expression of the cell wall-anchored protease, constitutive in the subsp. bulgaricus, inducible in the subsp. lactis, and absent in the subsp. delbrueckii was also a consequence of mutations at the gene level. The galT gene for galactose metabolism was found only in the subsp. lactis, while no specific amplification product was detected in the other two subspecies. All these data, together with the absence of a specific IS element, ISL6, from the major number of strains belonging to the subsp. bulgaricus, confirmed a deep internal heterogeneity among the three subspecies. Moreover, this evidence and the directional mutations found in the 16S rDNA sequences suggested that, of the three subspecies, L. delbrueckii subsp. lactis is the taxon closer to the ancestor. Limitations of the current prokaryotic species definition were also discussed, based on presented evidences. Our results indicate the need for an accurate investigation of internal heterogeneity of bacterial species. This study has consequences on the prokaryotic species concept, since genomic flexibility of prokaryotes collides with a stable classification, necessary from a scientific and applied point of view.     

Identification of lactobacillus delbrueckii and subspecies by hybridization probes and PCR

Three methods addressing two different target sites were compared for identification and differentiation of the subspecies Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus delbrueckii subsp. lactis/delbrueckii. A PCR method - three primer pairs that enable direct identification of the species and the two subspecies, respectively - was derived from a DNA fragment showing significant similarities to parts of the addAB genes of Bacillus sutbtilis. In addition, two oligonucleotide probes for the two subspecies were designed from that DNA region. Further, two oligonucleotide probes targeting the 16S rDNA were developed for subspecies differentiation by a one base-pair difference following identification of the species. Moreover, these probes were demonstrated to be applicable for in situ hybridization experiments. The results obtained by the different methods were in good agreement.     

Use of PCR-based methods for rapid differentiation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis.

Two PCR-based methods, specific PCR and randomly amplified polymorphic DNA PCR (RAPD-PCR), were used for rapid and reliable differentiation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis. PCR with a single combination of primers which targeted the proline iminopeptidase (pepIP) gene of L. delbrueckii subsp. bulgaricus allowed amplification of genomic fragments specific for the two subspecies when either DNA from a single colony or cells extracted from dairy products were used. A numerical analysis of the RAPD-PCR patterns obtained with primer M13 gave results that were consistent with the results of specific PCR for all strains except L. delbrueckii subsp. delbrueckii LMG 6412(T), which clustered with L. delbrueckii subsp. lactis strains. In addition, RAPD-PCR performed with primer 1254 provided highly polymorphic profiles and thus was superior for distinguishing individual L. delbrueckii strains.     

Isolation, taxonomic identification and hydrogen peroxide production by Lactobacillus delbrueckii subsp. lactis T31, isolated from Mongolian yoghurt: inhibitory activity on food-borne pathogens

AIMS: The aim of this work was to isolate lactic acid bacteria (LAB) strains from Mongolian tarag (a traditionally homemade yoghurt) displaying antimicrobial activities against food-borne pathogens, identify inhibitory substances and study the kinetics of their production. METHODS AND RESULTS: Inhibitory substance-producing bacterial strains were isolated from tarag. From 300 bacterial clones, 31 were able to inhibit the growth of the indicator strain Lactobacillus bulgaricus 340. One of the most active strains was identified as Lactobacillus delbrueckii subsp. lactis strain T31 by using cluster analysis of amplified fragment length polymorphism (AFLP) DNA fingerprints. The antimicrobial substance was inactivated by catalase, demonstrating the production of hydrogen peroxide (H(2)O(2)). Production of H(2)O(2) was studied under aerated and nonaerated culture conditions. The amount of H(2)O(2) in the culture supernatant increased during bacterial growth and reached a maximum (5.12 mmol l(-1)) at the early stationary phase under aerated conditions (agitated cultures). H(2)O(2) was not detected in the culture performed without agitation. In mixed cultures performed in milk with either Lact. delbrueckii subsp. lactis T31 in the presence of Escherichia coli, or Lact. delbrueckii subsp. lactis T31 in the presence of Listeria innocua under aerated and nonaerated conditions, a significant decrease in pathogen count was observed in aerated cultures. SIGNIFICANCE AND IMPACT OF THE STUDY: The significant decrease in Listeria viability observed in aerated mixed cultures of Lact. delbrueckii subsp. lactis T31 is mainly because of H(2)O(2) production. Lactobacillus delbrueckii subsp. lactis T31 could be used as a protective culture in food industries or as a probiotic to prevent intestinal and urogenital infections.     

Cell-wall protein profiles of dairy thermophilic lactobacilli

SDS–PAGE fingerprinting of cell-wall proteins extracted from 119 strains belonging to different species of lactic acid bacteria have been compared. The method of extraction and electrophoretic separation utilized in this work was found to be a reliable and rapid way for characterizing thermophilic lactobacilli species and strains. A protein of approximately 50 kDa was found to be characteristic for all the Lact. helveticus strains, and two cell-wall proteins of about 20 and 30 kDa were typical of the species Lact. delbrueckii , but the discrimination between the subspecies lactis and bulgaricus was not possible by the electrophoretic technique used. The other thermophilic species studied in this work presented cell-wall protein patterns that permitted their differentiation from both Lact. helveticus and Lact. delbrueckii .     

Lacticin, a bacteriocin produced by Lactobacillus delbrueckii subsp. lactis

Twenty-one strains of Lactobacillus delbrueckii and L. helveticus were tested for bacteriocin production against each other. Lactobacillus delbrueckii subsp. lactis JCM 1106 and 1107 produced an inhibitory agent active against L. delbrueckii subsp. bulgaricus JCM 1002 and NIAI yB-62, L. delbrueckii subsp. lactis JCM 1248 and L. delbrueckii subsp. delbrueckii JCM 1012. Lactobacillus delbrueckii subsp. lactis JCM 1248 inhibited only the growth of L. delbrueckii subsp. bulgaricus NIAI yB-62. These agents were sensitive to proteolytic enzymes and heating (at 60°C for 10min). These agents were considered to be bacteriocins and designated lacticin A and B.     

Ribotyping of Fusobacterium necrophorum strains isolated from bovine and ovine hepatic abscesses

A microbiological study was made of 100 strains of Fusobacterium necrophorum isolated from hepatic abscesses in bovine and ovine herds. Differences between the biological activity and ribotypes within the two F. necrophorum subspecies were studied. Conventional methods identified 89 isolates as F. necrophorum subsp. necrophorum and 11 as F. necrophorum subsp. funduliforme. For ribotyping, 50 strains (35 F.n. subsp. necrophorum, 11 F.n. subsp. funduliforme and 4 reference strains) were digested with restriction endonucleases (HindIII, EcoRI and BamHI) and examined after hybridization with digoxigenin-labelled cDNA probe transcribed from a 16 and 23S rRNAs from Escherichia coli. The most discriminating restriction endonuclease enzymes for ribotyping were EcoRI and BamHI. The presence or absence of two distinct band of 5 kb (EcoRI) and 10.5 kb (BamHI) differentiated the two subspecies. This technique also revealed genetic differences between isolates which could be used in the epidemiological study of clinical processes caused by F. necrophorum.     

Differentiation of Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus delbrueckii subsp. lactis by SDS-PAGE of cell-wall proteins

AIMS: In the present study, a method based on SDS-PAGE fingerprinting of surface layer proteins was developed to identify Lactobacillus delbrueckii subsp. bulgaricus and subsp. lactis dairy isolates. METHODS AND RESULTS: The two subspecies, identified by species-specific PCR, were characterized by different SDS-PAGE cell-wall protein profiles; subspecies bulgaricus showed one band of about 31 kDa which, in some cases, was observed at a doublet, and subspecies lactis showed one band of about 21 kDa or 18 kDa. CONCLUSION: The sensitivity of this procedure for discriminating between the two subspecies was very high. The different types of SDS-PAGE profile for cell-wall proteins of the strains studied in this work did not seem to be correlated to the different dairies of origin. SIGNIFICANCE AND IMPACT OF THE STUDY: The method appears to be an efficient taxonomic tool. It has the advantage of easy gel interpretation over fingerprinting of whole-cell protein extracts, and may be used as an alternative to established PCR-based techniques which, though rapid and safe, require expensive instruments and reagents.     

Characterization of dominant microbiota of a Ghanaian fermented milk product, nyarmie, by culture- and nonculture-based methods

AIMS: To characterize the predominant micro-organisms in a Ghanaian traditional fermented dairy product, nyarmie, made from cows' milk, using both culture- and nonculture-based methods. METHODS AND RESULTS: Samples of nyarmie were analysed from three production sites in Accra, by determining the counts on selective culture media. The microbial diversity occurring in nyarmie was also evaluated by 16S/18S ribosomal DNA PCR amplification and denaturing gradient gel electrophoresis. Results showed that nyarmie contained lactococci and lactobacilli in the range of 10(8) and 10(10) CFU ml(-1), respectively, and yeasts at around 10(7) CFU ml(-1). The pH ranged between 3.49 and 4.25. The predominant lactic acid bacteria (LAB) in nyarmie were Leuconostocmesenteroides ssp. mesenteroides, Streptococcus thermophilus, Lactobacillus delbrueckii ssp. bulgaricus, Lact.helveticus, Lact. delbrueckii ssp. lactis and Lactococcus lactis, while Saccharomyces cerevisiae was the predominant yeast species. Lactobacillus delbrueckii ssp. delbrueckii was not detected by cultivation but its predominance was revealed by PCR-DGGE analysis. CONCLUSIONS: The flora in products from different producers varied in the LAB composition present and may result in variations in product quality. SIGNIFICANCE AND IMPACT OF THE STUDY: Development and use of starter cultures for nyarmie may be beneficial in improving the consistency of product quality.     

Integrated polymerase chain reaction-based procedures for the detection and identification of species and subspecies of the Gram-positive bacterial genus Lactococcus

AIMS: Five species of the Gram-positive bacterial genus Lactococcus (Lactococcus lactis, L. garvieae, L. plantarum, L. piscium and L. raffinolactis) are currently recognized. The aim of this work was to develop a simple approach for the identification of these species, as well as to differentiate the industrially important dairy subspecies L. lactis subsp. lactis and L. lactis subsp. cremoris. METHODS AND RESULTS: Methods were devised based on specific polymerase chain reaction (PCR) amplifications that exploit differences in the sequences of the 16S ribosomal RNA genes of each species, followed by restriction enzyme cleavage of the PCR products. The techniques developed were used to characterize industrial cheese starter strains of L. lactis and the results were compared with biochemical phenotype and DNA sequence data. CONCLUSIONS: The PCR primers designed can be used simultaneously, providing a simple scheme for screening unknown isolates. Strains of L. lactis show heterogeneity in the 16S ribosomal RNA gene sequence. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides an integrated set of methods for differentiation and identification of lactococcal species associated with agricultural, veterinary, medical and processed food industries.     

Differentiation of Lactobacillus helveticus,Lactobacillus delbrueckii subsp bulgaricus,subsp lactis and subsp delbrueckii using physiological and genetic tools and reclassification of some strains from the ATCC collection

Several physiological tests of glucose metabolism and genetic tools including species specific probes and 16S rDNA sequences were used to identify strains of L. helveticus and the group of L. delbrueckii with its three subspecies lactis, bulgaricus, and delbrueckii. These species are important for the milk industry as fermenting lactic acid bacteria. The identification procedure was applied to the different strains of these species available from the ATCC collection and allowed to reclassify part of them.     

Comparison of methods for discrimination between strains of Listeria monocytogenes from epidemiological surveys.

Total cellular DNA from 28 strains of Listeria monocytogenes isolated from food implicated in food-borne illness and from patients with listeriosis was digested with the restriction endonucleases HindIII, HaeIII, and EcoRI. Following agarose gel electrophoresis, the fragments were subjected to Southern blot hybridization with a digoxigenin-labeled cDNA probe transcribed from Escherichia coli 16S and 23S rRNA. The patterns of bands from genomic (DNA fingerprints) and rDNA fingerprints (ribotypes) were used for classifying L. monocytogenes strains, and the resulting subtypes were compared with serotyping and multilocus enzyme electrophoresis classification schemes. A total of 15 distinct and identical groups were obtained when genomic DNA was digested with either HindIII or HaeIII. The most discriminating enzyme for ribotyping of strains was EcoRI, which divided the 28 strains of L. monocytogenes into 6 ribotype groups. DNA fingerprinting and ribotyping differentiated L. monocytogenes from other Listeria spp., including L. ivanovii, L. welshimeri, and L. innocua as well as the lactic acid bacteria Lactococcus lactis subsp. lactis and subsp. cremoris. L. monocytogenes strains isolated from four independent food-borne illness incidents were analyzed by all typing methods. Patient and product isolates were not distinguishable by serotyping, ribotyping, or multilocus enzyme electrophoresis. DNA fingerprinting was the only method capable of differentiating these strains, or conversely, of proving relatedness of patient-product pairs of isolates. This method was a relatively simple, sensitive, reproducible, and highly discriminating method for epidemiological tracking of L. monocytogenes implicated in food-borne illness.     

Diversity in specificity of the extracellular proteinases in Lactobacillus helveticus and Lactobacillus delbrueckii subsp. bulgaricus

AIMS: To investigate the diversity in specificity of cell-bound extracellular proteinases in Lactobacillus helveticus and Lactobacillus delbrueckii subsp. bulgaricus. METHODS AND RESULTS: HPLC analysis of whole-cell preparations of 14 Lact. delbrueckii subsp. bulgaricus and eight Lact. helveticus strains incubated with alpha (s1)-casein (f 1-23) detected at least six distinct proteolytic patterns. Differences between groups were found in both the primary and secondary specificity toward alpha(s1)-casein (f 1-23) and its breakdown products. No correlation was found between the o-phthaldialdehyde (OPA) general proteolysis analysis and alpha(s1)-casein (f 1-23) cleavage profiles. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF STUDY: Using the alpha(s1)-CN (f 1-23) method, six patterns of proteolysis were found in the dairy lactobacilli tested. Understanding the influence of Lactobacillus proteinase specificity on casein degradation should facilitate efforts to develop starter cultures that predictably improve the functional properties of Mozzarella cheese.     

Exopolysaccharides produced by lactic acid bacteria of kefir grains

A Lactobacillus delbrueckii subsp. bulgaricus HP1 strain with high exopolysaccharide activity was selected from among 40 strains of lactic acid bacteria, isolated from kefir grains. By associating the Lactobacillus delbrueckii subsp. bulgaricus HP1 strain with Streptococcus thermophilus T15, Lactococcus lactis subsp. lactis C15, Lactobacillus helveticus MP12, and Sacharomyces cerevisiae A13, a kefir starter was formed. The associated cultivation of the lactobacteria and yeast had a positive effect on the exopolysaccharide activity of Lactobacillus delbrueckii subsp. bulgaricus HP1. The maximum exopolysaccharide concentration of the starter culture exceeded the one by the Lactobacillus delbrueckii subsp. bulgaricus HP1 monoculture by approximately 1.7 times, and the time needed to reach the maximum concentration (824.3 mg exopolysacharides/l) was shortened by 6 h. The monomer composition of the exopolysaccharides from the kefir starter culture was represented by glucose and galactose in a 1.0:0.94 ratio, which proves that the polymer synthesized is kefiran.     

Differentiation of Enterococcus spp. by cell membrane fatty acid methyl ester profiling, biotyping and ribotyping

AIMS: Gas chromatographic analysis of cell membrane fatty acid methyl esters (FAME), biochemical profiling (biotyping) and EcoRI restriction endonuclease profiling of DNA containing ribosomal RNA sequences (ribotyping) were compared for differentiation of Enterococcus spp. METHODS AND RESULTS: FAME profiling, biotype profiling and ribotyping of 41 strains from retail Swiss-type cheeses and five strains from culture collections resulted in 17, 25 and 26 groups, respectively, with only two pairs of strains having the same FAME group, biotype profile and ribogroup. CONCLUSION: Substantial overlap occurred in groupings assigned by the three methods. SIGNIFICANCE AND IMPACT OF THE STUDY: Differentiation of Enterococcus spp. strains increases if multiple methods are used.     

Cloning and expression analysis of the 28 kDa protein from Lactobacillus delbrueckii subsp. lactis ATCC 4797 hypothesized to influence lactacin B production

AIMS: A cell wall-associated lactacin B inducer protein (IP) was purified from Lactobacillus delbrueckii subsp. lactis ATCC 4797 (Lact. lactis) by chromatofocusing and gel filtration HPLC (Barefoot et al. 1994). METHODS AND RESULTS: N-terminal sequence of the purified IP was used to design an oligonucleotide (24-mer) for gene identification by Southern and colony hybridizations. Southern hybridization on Lact. lactis chromosomal DNA digested with EcoRI and PstI produced a single 4-5 kbp DNA fragment. Colony hybridizations with 6250 clones produced four positive recombinants for the proposed IP. Sequence of the DNA isolated from RU43e9 revealed a 4623 bp DNA fragment containing three open reading frames (ORF) potentially encoding enzymes that function in glycolysis. One ORF, coding for an active triosephosphate isomerase (Tpi), showed 98% homology to the N-terminal domain of the HPLC purified IP. PCR primers were designed to amplify the ORF encoding the proposed IP for subcloning, protein expression, purification and bacteriocin enhancing assays on pure cultures of Lactobacillus acidophilus N2. CONCLUSIONS: The regions flanking the Tpi gene (data not shown) were also sequenced and it is concluded that the proposed IP reported by Barefoot et al. (1994) is located on an operon containing several glycolytic enzymes that function in glycolysis. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings of this study do not support previously published research (Barefoot et al. 1994) hypothesizing that a purified IP from Lact. lactis, homologous to a Bacillus stearothermophilus Tpi, is capable of enhancing bacteriocin synthesis in Lact. acidophilus N2.