Identification and characterization of Lactic Acid Bacteria isolated from Tibetan Qula cheese

Fourteen strains of Lactic Acid Bacteria (LAB) isolated from Qula, a Tibetan traditional yak cheese, were divided into four groups (A-D) according to morphological and biochemical characteristics. On the basis of the 16S rRNA gene sequence analysis, group A and group B strains were placed in the cluster making up the genus Leuconostoc, which together with Leuconostoc mesenteroides and Leuconostoc pseudomesenteroides, formed a distinct cluster. The group C strain was clearly identified as Enterococcus faecium by forming a very well defined cluster with this species. The group D strains were placed in the lactobacilli cluster with Lactobacillus plantarum and Lactobacillus pentosus being the closely related species. On the basis of DNA-DNA hybridization, strains in the groups A, B, C and D were identified as Leuconostoc mesenteroides subsp. dextranicum, Leuconostoc pseudomesenteroides, Enterococcus faecium and Lactobacillus plantarum, respectively. Leuconostoc mesenteroides subsp. dextranicum was the dominate member of the population.     

Lactobacillus perolens sp. nov., a soft drink spoilage bacterium.

Lactic acid bacteria that are able to spoil soft drinks with low pH comprise a limited number of acidotolerant or acidophilic species of the genera Lactobacillus, Leuconostoc and Weissella. Various Gram-positive rods causing turbidity and off-flavour were isolated from orange lemonades. Physiological and biochemical studies including SDS-PAGE whole-cell protein analysis showed a homogeneous group of organisms. The 16S rRNA gene sequence analysis of two representatives revealed that they formed a phylogenetically distinct line within the genus Lactobacillus. All strains were facultatively heterofermentative, producing L-lactic acid. Based on the data presented a new species L. perolens is proposed. The name refers to the off-flavour caused by high amounts of diacetyl. The type strain of L. perolens is DSM 12744 (LMG 18936). A rRNA targeted oligonucleotide probe was designed that allows a fast and reliable identification of L. perolens.     

Molecular diversity of lactic acid bacteria from cassava sour starch (Colombia)

Lactic acid bacteria and more particularly lactobacilli and Leuconostoc, are widely found in a wide variety of traditional fermented foods of tropical countries, made with cereals, tubers, meat or fish. These products represent a source of bacterial diversity that cannot be accurately analysed using classical phenotypic and biochemical tests. In the present work, the identification and the molecular diversity of lactic acid bacteria isolated from cassava sour starch fermentation were assessed by using a combination of complementary molecular methods: Randomly Amplified Polymorphic DNA fingerprinting (RAPD), plasmid profiling, hybridization using rRNA phylogenetic probes and partial 16S rDNA sequencing. The results revealed a large diversity of bacterial species (Lb. manihotivorans, Lb. plantarum, Lb. casei, Lb. hilgardii, Lb. buchneri, Lb. fermentum, Ln. mesenteroides and Pediococcus sp.). However, the most frequently isolated species were Lb. plantarum and Lb. manihotivorans. The RAPD analysis revealed a large molecular diversity between Lb. manihotivorans or Lb. plantarum strains. These results, observed on a rather limited number of samples, reveal that significant bacterial diversity is generated in traditional cassava sour starch fermentations. We propose that the presence of the amylolytic Lb. manihotivorans strains could have a role in sour starch processing.     

Identification of lactobacilli residing in chicken ceca with antagonism against Campylobacter

Bacteriocins produced by Lactobacillus salivarius have been recently recognized as a natural means to control Campylobacter and Salmonella in live poultry. This finding is of relevance since Campylobacter jejuni and Campylobacter coli are the predominant species isolated from poultry that are associated with human campylobacteriosis. In the present work, lactic acid bacteria (LAB) isolated from the cecum of twenty Tunisian chickens were identified and those isolates with antagonism against Campylobacter were further characterized. Following their preliminary confirmation as LAB, 150 strains were identified by combining morphological criteria, biochemical tests, and molecular methods, the latter inluding intergenic 16S- 23S PCR, specific lactobacilli PCR, and a biphasic approach. Most of the LAB isolated belonged to the genus Lactobacillus, among them Lb. sakei (33.3%), Lb. salivarius (19.4%), Lb. reuteri (8.6%), and Lb. curvatus (8.6%). The other LAB strains included those of the genus Weissella (16.7%), Enterococcus faecalis (5.3%), Leuconostoc mesenteroides (2.7%), Lactococcus graviae (2.7%), and Streptococcus sp. (2.7%). The Lactobacilli strains were tested for their antagonism against C. jejuni and C. coli. The activity of three of them, Lb. salivarius SMXD51, Lb. salivarius MMS122, and Lb. salivarius MMS151, against the aforementioned target strains could be ascribed to the production of bacteriocins.     

Identification and characterization of Leuconostoc fallax strains isolated from an industrial sauerkraut fermentation

Lactic acid bacterial strains were isolated from brines sampled after 7 days of an industrial sauerkraut fermentation, and six strains were selected on the basis of susceptibility to bacteriophages. Bacterial growth in cabbage juice was monitored, and the fermentation end products were identified, quantified, and compared to those of Leuconostoc mesenteroides. Identification by biochemical fingerprinting, endonuclease digestion of the 16S-23S intergenic transcribed spacer region, and sequencing of variable regions V1 and V2 of the 16S rRNA gene indicated that the six selected sauerkraut isolates were Leuconostoc fallax strains. Random amplification of polymorphic DNA fingerprints indicated that the strains were distinct from one another. The growth and fermentation patterns of the L. fallax isolates were highly similar to those of L. mesenteroides. The final pH of cabbage juice fermentation was 3.6, and the main fermentation end products were lactic acid, acetic acid, and mannitol for both species. However, none of the L. fallax strains exhibited the malolactic reaction, which is characteristic of most L. mesenteroides strains. These results indicated that in addition to L. mesenteroides, a variety of L. fallax strains may be present in the heterofermentative stage of sauerkraut fermentation. The microbial ecology of sauerkraut fermentation appears to be more complex than previously indicated, and the prevalence and roles of L. fallax require further investigation.     

Isolation and characterization of IS1165, an insertion sequence of Leuconostoc mesenteroides subsp. cremoris and other lactic acid bacteria.

We have cloned and characterized an insertion sequence from Leuconostoc mesenteroides subsp. cremoris strain DB1165. This element, designated IS1165, is 1553 bp, has imperfect inverted repeat ends, contains an open reading frame of 1236 bp, and is not related to any previously described insertion sequence. The copy number of IS1165 varies from 4 to 13 in L. mesenteroides subsp. cremoris strains allowing genetic fingerprinting of strains based on location and number of bands on hybridization. IS1165 or closely related elements have been detected by hybridization in L. lactis, L. oenos, Pediococcus sp., Lactobacillus helveticus, and Lb. casei but not in Lactococcus.     

Diversity of Lactobacillus and Bifidobacterium in feces of herbivores, omnivores and carnivores

The Lactobacillus and Bifidobacterium population in the feces of 26 animals (16 species) were studied by culture-dependent and culture-independent techniques. Lactobacilli were detected from a few herbivores, all carnivores and some omnivores. Lactobacillus johnsonii, Lactobacillus reuteri, Lactobacillus salivarius, Lactobacillus vaginalis and Lactobacillus ingluviei were the most dominant lactobacilli in carnivores. These species were, however, not predominant in herbivores and omnivores. Lactobacillus brevis, Lactobacillus casei, Lactobacillus parabuchneri, Lactobacillus plantarum, Lactobacillus sakei, Leuconostoc mesenteroides and Leuconostoc pseudomesenteroides, usually present in raw plant material, were present in omnivores but not in carnivores. Bifidobacteria were detected in only four herbivores and two omnivores. Bifidobacterium pseudolongum was the only Bifidobacterium species detected in herbivores. Bifidobacteria detected in the two omnivores are phylogenetically not closely related to known species and are possible novel species in the genus.     

Intrageneric relationships of members of the genus Fusobacterium as determined by reverse transcriptase sequencing of small-subunit rRNA.

The phylogenetic interrelationships of 14 members of the genus Fusobacterium were investigated by performing a comparative analysis of the 16S rRNA sequences of these organisms. The sequence data revealed considerable intrageneric heterogeneity. The four species Fusobacterium nucleatum (including F. nucleatum subsp. nucleatum, F. nucleatum subsp. polymorphum, "F. nucleatum subsp. fusiforme," and "F. nucleatum subsp. animalis"), Fusobacterium alocis, Fusobacterium periodonticum, and Fusobacterium simiae, which colonize oral cavities, exhibited high levels of sequence homology with each other and formed a distinct group within the genus. Fusobacterium mortiferum, Fusobacterium varium, and Fusobacterium ulcerans also formed a phylogenetically coherent group, as did the two species Fusobacterium gonidiaformans and Fusobacterium necrophorum. Fusobacterium russii and Fusobacterium necrogenes displayed no specific relationship with any of the other fusobacteria. The sequence data are discussed in the context of previous physiological and chemical findings.     

Ehrlichia ewingii sp. nov., the etiologic agent of canine granulocytic ehrlichiosis.

The 16S rRNA gene was amplified, cloned, and sequenced from the blood of two dogs that were experimentally infected with the etiologic agent of canine granulocytic ehrlichiosis. The 16S rRNA sequence was found to be unique when it was compared with the sequences of other members of the genus Ehrlichia. The most closely related species were Ehrlichia canis (98.0% related) and the human ehrlichiosis agent (Ehrlichia chaffeensis) (98.1% related); all other species in the genus were found to be phylogenetically much more distant. Our results, coupled with previous serologic data, provide conclusive evidence that the canine granulocytic ehrlichiosis agent is a new species of the genus Ehrlichia that is related to, but is distinct from, E. canis and all other members of the genus. We propose the name Ehrlichia ewingii sp. nov.; the Stillwater strain is the type strain.     

Characterization of Leuconostoc gasicomitatum sp. nov., associated with spoiled raw tomato-marinated broiler meat strips packaged under modified-atmosphere conditions

Lactic acid bacteria (LAB) associated with gaseous spoilage of modified-atmosphere-packaged, raw, tomato-marinated broiler meat strips were identified on the basis of a restriction fragment length polymorphism (RFLP) (ribotyping) database containing DNAs coding for 16S and 23S rRNAs (rDNAs). A mixed LAB population dominated by a Leuconostoc species resembling Leuconostoc gelidum caused the spoilage of the product. Lactobacillus sakei, Lactobacillus curvatus, and a gram-positive rod phenotypically similar to heterofermentative Lactobacillus species were the other main organisms detected. An increase in pH together with the extreme bulging of packages suggested a rare LAB spoilage type called "protein swell." This spoilage is characterized by excessive production of gas due to amino acid decarboxylation, and the rise in pH is attributed to the subsequent deamination of amino acids. Protein swell has not previously been associated with any kind of meat product. A polyphasic approach, including classical phenotyping, whole-cell protein electrophoresis, 16 and 23S rDNA RFLP, 16S rDNA sequence analysis, and DNA-DNA reassociation analysis, was used for the identification of the dominant Leuconostoc species. In addition to the RFLP analysis, phenotyping, whole-cell protein analysis, and 16S rDNA sequence homology indicated that L. gelidum was most similar to the spoilage-associated species. The two spoilage strains studied possessed 98.8 and 99.0% 16S rDNA sequence homology with the L. gelidum type strain. DNA-DNA reassociation, however, clearly distinguished the two species. The same strains showed only 22 and 34% hybridization with the L. gelidum type strain. These results warrant a separate species status, and we propose the name Leuconostoc gasicomitatum sp. nov. for this spoilage-associated Leuconostoc species.     

Phylogenetic analyses of the nitrogen-fixing genus Derxia

Phylogenetic analyses of the 16S rRNA gene sequence indicate that the genus Derxia forms a distinct lineage in the beta-Proteobacteria. On the NJ tree Derxia has a low bootstrap value (30.9%) with Alcaligeneceae, and on the ML tree it shows an independent cluster separated from other families. Moreover, there is below 93.4% 16S rDNA sequence similarity between genus Derxia and the genera of the beta-Proteobacteria. These facts reveal that Derxia is not grouped with any known family of beta-Proteobacteria and should be placed as a separate genus of beta-Proteobacteria. The data on high G+C content (71 mol%), the cellular fatty acid composition, and the physiological characteristics of facultative hydrogen autotrophy and nitrogen fixation are unique for Derxia. The nifH gene sequence was found in this genus and phylogenetically compared among nitrogen-fixing bacteria to indicate that Derxia is clustered with the diazotrophs of beta-Proteobacteria.     

A genus-specific PCR method for differentiation between Leuconostoc and Weissella and its application in identification of heterofermentative lactic acid bacteria from coffee fermentation

A genus-specific PCR analysis method was developed for a rapid and reliable differentiation between the two heterofermentative lactic acid bacteria genera Leuconostoc and Weissella. Primer sets specific for target regions of the 16S rRNA genes were designed and the specificity of the PCR was evaluated using the type strains of 13 species of Leuconostoc and 11 species of Weissella. In addition, the newly developed genus-specific PCR analysis was applied to characterize 72 lactic acid bacteria (LAB) strains isolated from coffee fermentation and which were presumptively classified as Leuconostoc or Weissella species. Additionally, a total of 34 LAB isolates from various other fermented foods were included. The investigations of these strains were conducted to test the effectiveness of correct characterization of field isolates using the genus-specific PCR approach. The correct assignment to one of these two genera by the application of the genus-specific primers was confirmed by further identifying the strains using repetitive extragenic palindromic-PCR and 16S rRNA gene sequencing.     

Isolation and identification of cultivable lactic acid bacteria in traditional yak milk products of Gansu Province in China

Various traditional fermented yak milk and raw milk foods could be considered as an abundant resource for obtaining novel lactic acid bacteria (LAB) with unique properties. Eighty-eight samples of yak milk products were collected from Gansu Province in China. Three hundred and nineteen strains of LAB isolated from these samples were identified by phenotypic methods, 16S rRNA gene sequence analysis and PCR-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) technology. Among the isolates, one hundred and sixty-four isolates (51.41% of the total) were classified under Lactobacilli, and one hundred and fifty-five (48.59%) belonged to cocci. All the isolates were classified to six genera (Lactobacillus, Lactococcus, Leuconostoc, Streptococcus, Enterococcus and Weissella) and twenty-one species. Lactobacillus helveticus (87 strains), Leuconostoc mesenteroides subsp. mesenteroides (49 strains), Streptococcus thermophilus (39 strains), Lactobacillus casei (31 strains) and Lactococcus lactis subsp. lactis (19 strains) were considered as the predominant populations in the yak milk products. The results showed that there were abundant genus and species LAB existing in yak milk products in Gansu Province in China. The obtained LAB pure cultures may be a valuable source for further starter selection.     

Development and use of a selective medium for isolation of Leuconostoc spp. from vegetables and dairy products.

A selective medium (LUSM medium) for the isolation of Leuconostoc spp. was developed. This medium contained 1.0% glucose, 1.0% Bacto Peptone (Difco), 0.5% yeast extract (BBL), 0.5% meat extract (Difco), 0.25% gelatin (Difco), 0.5% calcium lactate, 0.05% sorbic acid, 75 ppm of sodium azide (Sigma), 0.25% sodium acetate, 0.1% (vol/vol) Tween 80, 15% tomato juice, 30 micrograms of vancomycin (Sigma) per ml, 0.20 microgram of tetracycline (Serva) per ml, 0.5 mg of cysteine hydrochloride per ml, and 1.5% agar (Difco). LUSM medium was used successfully for isolation and enumeration of Leuconostoc spp. in dairy products and vegetables. Of 116 colony isolates obtained from fresh raw milk, curdled milk, or various vegetables, 115 were identified as members of the genus Leuconostoc. A total of 89 of these isolates were identified to species; 13.5% of the isolates were Leuconostoc cremoris, 7.9% were Leuconostoc mesenteroides subsp. mesenteroides, 11.2% were Leuconostoc mesenteroides subsp. dextranicum, 16.9% were Leuconostoc mesenteroides subsp. paramesenteroides, 10.1% were leuconostoc lactis, and 40.4% were Leuconostoc oenos. When we compared the counts obtained for two Leuconostoc strains, Leuconostoc dextranicum 181 and L. cremoris JLL8, on MRS agar and LUSM medium, we found no significant difference between the values obtained on the two media.     

Nucleoside deoxyribosyltransferase and inosine phosphorylase activity in lactic acid bacteria

Phosphate-independent nucleoside deoxyribosyltransferase activity was detected in Aerococcus viridans, Leuconostoc mesenteroides, two species of Pediococcus, eight obligately homofermentative and three obligately heterofermentative lactobacilli. In cellfree extracts of one strain of Lactococcus, one strain of Streptococcus and three strains of facultatively heterofermentative lactobacilli no phosphate-independent nucleoside deoxyribosyltransferase could be detected and inosine nucleoside phosphorylase was the only enzyme catalysing pentosyl transfer to purines. A classification based on the presence or absence of these enzymes separates Streptococcus and Lactococcus from the remaining genera which is in agreement with studies using anti-aldolase sera and 16S rRNA oligonucleotide catalogues.Abbreviations NCIMB National Collection of Industrial and Marine bacteria - NCDO National Collection of Dairy organisms - MRS deMan Rogosa Sharpe - Pipes Piperazine-N,N-bis[2-ethanesulphonic acid]     

Specific enumeration of lactic acid bacteria in fermenting grape must and wine by colony hybridization with non-isotopic DNA probes

A. LONVAUD-FUNEL, A. JOYEUX AND O. LEDOUX. 1991. Total DNA extracted from lactic acid bacteria commonly found in musts and wines was randomly labelled with digoxigenin. It was assayed for the detection of several species by dot-blot hybridization. The method proved to be specific as there was no cross-hybridization between most of the species belonging to the genera Leuconostoc, Pediococcus and Lactobacillus , homofermentative and heterofermentative ( Lact. plantarum, Lact. casei, Leuc. mesenteroides, Leuc. oenos, Ped. damnosus, Ped. pentosaceus ). However, it failed for some Lact. brevis strains which strongly hybridized with Lact. hilgardii.
Colony hybridization was performed directly on plates soon after enumeration. Eight probes of the most common species were used; it was possible to follow the evolution of each species during the vinification of two red wines. According to the phase of alcoholic fermentation, then malolactic fermentation, the predominance or regression of bacilli and cocci could be established.