Natural populations of lactic acid bacteria associated with silage fermentation as determined by phenotype, 16S ribosomal RNA and recA gene analysis

One hundred and fifty-six strains isolated from corn (Zea mays L.), forage paddy rice (Oryza sativa L.), sorghum (Sorghum bicolor L.) and alfalfa (Medicago sativa L.) silages prepared on dairy farms were screened, of which 110 isolates were considered to be lactic acid bacteria (LAB) according to their Gram-positive and catalase-negative characteristics and, mainly, the lactic acid metabolic products. These isolates were divided into eight groups (A-H) based on the following properties: morphological and biochemical characteristics, γ-aminobutyric acid production capacity, and 16S rRNA gene sequences. They were identified as Weissella cibaria (36.4%), Weissella confusa (9.1%), Leuconostoc citreum (5.3%), Leuconostoc lactis (4.9%), Leuconostoc pseudomesenteroides (8.0%), Lactococcus lactis subsp. lactis (4.5%), Lactobacillus paraplantarum (4.5%) and Lactobacillus plantarum (27.3%). W. cibaria and W. confusa were mainly present in corn silages, and L. plantarum was dominant on sorghum and forage paddy rice silages, while L. pseudomesenteroides, L. plantarum and L. paraplantarum were the dominant species in alfalfa silage. The corn, sorghum and forage paddy rice silages were well preserved with lower pH values and ammonia-N concentrations, but had higher lactic acid content, while the alfalfa silage had relatively poor quality with higher pH values and ammonia-N concentrations, and lower lactic acid content. The present study confirmed the diversity of LAB species inhabiting silages. It showed that the differing natural populations of LAB on these silages might influence fermentation quality. These results will enable future research on the relationship between LAB species and silage fermentation quality, and will enhance the screening of appropriate inoculants aimed at improving such quality.     

Molecular diversity of Lactobacillus spp. and other lactic acid bacteria in the human intestine as determined by specific amplification of 16S ribosomal DNA.

A Lactobacillus group-specific PCR primer, S-G-Lab-0677-a-A-17, was developed to selectively amplify 16S ribosomal DNA (rDNA) from lactobacilli and related lactic acid bacteria, including members of the genera Leuconostoc, Pediococcus, and WEISSELLA: Amplicons generated by PCR from a variety of gastrointestinal (GI) tract samples, including those originating from feces and cecum, resulted predominantly in Lactobacillus-like sequences, of which ca. 28% were most similar to the 16S rDNA of Lactobacillus ruminis. Moreover, four sequences of Leuconostoc species were retrieved that, so far, have only been detected in environments other than the GI tract, such as fermented food products. The validity of the primer was further demonstrated by using Lactobacillus-specific PCR and denaturing gradient gel electrophoresis (DGGE) of the 16S rDNA amplicons of fecal and cecal origin from different age groups. The stability of the GI-tract bacterial community in different age groups over various time periods was studied. The Lactobacillus community in three adults over a 2-year period showed variation in composition and stability depending on the individual, while successional change of the Lactobacillus community was observed during the first 5 months of an infant's life. Furthermore, the specific PCR and DGGE approach was tested to study the retention in fecal samples of a Lactobacillus strain administered during a clinical trial. In conclusion, the combination of specific PCR and DGGE analysis of 16S rDNA amplicons allows the diversity of important groups of bacteria that are present in low numbers in specific ecosystems to be characterized, such as the lactobacilli in the human GI tract.     

Phylogenetic position of Cowdria ruminantium (Rickettsiales) determined by analysis of amplified 16S ribosomal DNA sequences.

The 16S ribosomal DNA sequence of Cowdria ruminantium, the causative agent of heartwater disease in ruminants, was determined. An analysis of this sequence showed that C. ruminantium forms a tight phylogenetic cluster with the canine pathogen Ehrlichia canis and the human pathogen Ehrlichia chaffeensis. Although a close relationship between the genus Cowdria and several members of the tribe Ehrlichieae has been suspected previously, the tight phylogenetic cluster with E. canis and E. chaffeensis is surprising in view of known differences in host preference and target cells.     

Detection of Desulfotomaculum in an Italian rice paddy soil by 16S ribosomal nucleic acid analyses

Two specific primers were developed for the amplification of 16S rRNA genes of Desulfotomaculum lineage 1 to detect members of the genus Desulfotomaculum in rice field soil. The combination of both primers in PCR allowed the specific amplification and cloning of ten 16S rDNA sequences of this group from rice paddy soil DNA extracts. The phylogenetic analysis showed that these sequences formed a deeply branching cluster within Desulfotomaculum lineage 1, together with two sequences from the database and two sequences from a hydrocarbon-contaminated aquifer. Dissimilarity values to validly described species, including recently isolated strains of Desulfotomaculum from rice paddy microcosms, were higher than 12%. Within the new cluster the cloned sequences formed three separate groups which were each represented by at least two sequences with identities of >/=99% while one sequence represented an additional group. The sequences should represent sulfate-reducing organisms because they clearly fell into the physiologically coherent group of Gram-positive sulfate reducers. The relative abundance of bacteria of the Desulfotomaculum lineage 1 in rice paddy soil and root samples was estimated with rRNA dot blot hybridizations of extracted RNA. The relative RNA content of Desulfotomaculum lineage 1 was 0.55% in the bulk soil and 1% in the rice root samples, respectively, of the total 16S rRNA content (probe Eub338). Hybridization of rRNA with a probe targeting the new cluster represented by the cloned sequences confirmed the high abundance of 16S rRNA sequences from this cluster in the rice paddy field samples. Another hybridization probe detecting Desulfotomaculum acetoxidans and two closely related Desulfotomaculum isolates from rice paddy soil indicated that these bacteria were less abundant.     

Survival of silage lactic acid bacteria in the goat gastrointestinal tract as determined by denaturing gradient gel electrophoresis

Aims: To determine the survival rate of silage lactic acid bacteria (LAB) in the ruminant gastrointestinal tract. Methods and Results: Wilted Italian ryegrass (Lolium multiflorum Lam.) silage (containing 1·9 × 10⁶ CFU LAB g^?1) was fed ad libitum to three goats equipped with rumen cannulae. Silage was given alone or with concentrates at a 1 : 1 ratio on a dry matter basis. Rumen fluid was then obtained 2, 4 and 8 h after the morning feeding. Denaturing gradient gel electrophoresis was performed to compare LAB communities in silage, rumen fluid and faeces. The LAB detected in the wilted silage included Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus murinus and Lactobacillus sakei. Bands indicative of Lact. murinus were detected in either the rumen fluid or faeces, whereas the bands indicative of Lact. plantarum, Lact. brevis and Lact. sakei were not. Although the rumen fluid LAB counts and volatile fatty acid concentrations were higher in goats fed silage plus concentrates compared with those fed silage alone, the LAB communities themselves remained unaffected. Sampling times and goat‐to‐goat variations did not affect the LAB communities found in the rumen fluid. Conclusion: LAB communities found in the gut are not remarkably affected by the consumption of silage LAB, even when the silage is accompanied by concentrates that facilitate gut fermentation. Significance and Impact of the Study: Although silage can improve probiotic function, it may be difficult for silage LAB to survive the digestive process in the ruminant gastrointestinal tract.     

Phylogenetic affiliations ofRhodoferax fermentans and related species of phototrophic bacteria as determined by automated 16S rDNA sequencing

16S rDNA sequences of strains ofRhodoferax fermentans were analyzed and compared with those of species of the generaRubrivivax andRhodocyclus. Approximately 1.5-kb fragments of 16S rDNA from crude cell lysates were amplified by the polymerase chain reaction (PCR) and sequenced directly by usingTth DNA polymerase with the linear PCR sequencing protocol, followed by on-line detection with an automated laser fluorescent DNA sequencer. Pairwise sequence comparisons and distance matrix tree analysis showed thatRhodoferax fermentans, Rubrivivax gelatinosus, andRhodocyclus species belong to three separate lineages within the beta subclass of theProteobacteria, thereby confirming the phylogenetic validity of the genusRhodoferax, as well as of the generaRubrivivax andRhodocyclus.     

Phylogenetic analysis of the genera Pseudonocardia and Actinobispora based on 16S ribosomal DNA sequences

The 16S rDNA sequences of 11 strains, nine type strains of validated Pseudonocardia species and Actinobispora yunnanensis, and two strains of unnamed Pseudonocardia species, were determined and compared with those of representatives of the family Pseudonocardiaceae. The phylogenetic analysis indicated that all of the validated species of the genera Pseudonocardia and Actinobispora consistently formed a monophyletic unit and separated well from the other genera of the family Pseudonocardiaceae. One unnamed Pseudonocardia strain was related to members of the genus Pseudonocardia, whereas the other unnamed Pseudonocardia strain formed a distinct clade within the radiation of the genus Amycolatopsis.     

Novel bacterial diversity recovered from the rhizosphere of oilseed rape (Brassica napus) determined by the analysis of 16S ribosomal DNA

Soil was sampled to a distance of 2.5 mm beneath a root mat of oilseed rape (Brassica napus) in a model rhizosphere system. DNA was extracted and the 16S rDNA amplified, cloned and sequenced. Phylogenetic analysis of these sequences with those held on-line, revealed that 37% of the clones fell within the Holophaga / Acidobacterium phylum, 17% were within the proteobacteria, 14% of the clones were close relatives of Bacillus megaterium and 5% were related to Verrucomicrobium spinosum. An additional eleven clones (21%) could not be assigned to any known phylum and may represent novel bacterial lineages. This study highlights the diverse nature of rhizosphere soils and reinforces the role that molecular approaches play in unravelling such diversity.     

Microbial diversity in an in situ reactor system treating monochlorobenzene contaminated groundwater as revealed by 16S ribosomal DNA analysis

A molecular approach based on the construction of 16S ribosomal DNA clone libraries was used to investigate the microbial diversity of an underground in situ reactor system filled with the original aquifer sediments. After chemical steady state was reached in the monochlorobenzene concentration between the original inflowing groundwater and the reactor outflow, samples from different reactor locations and from inflowing and outflowing groundwater were taken for DNA extraction. Small-subunit rRNA genes were PCR-amplified with primers specific for Bacteria, subsequently cloned and screened for variation by restriction fragment length polymorphism (RFLP). A total of 87 bacterial 16S rDNA genes were sequenced and subjected to phylogenetic analysis. The original groundwater was found to be dominated by a bacterial consortium affiliated with various members of the class of Proteobacteria, by phylotypes not affiliated with currently recognized bacterial phyla, and also by sporulating and non-sporulating sulfate-reducing bacteria. The most occurring clone types obtained from the sediment samples of the reactor were related to the beta-Proteobacteria, dominated by sequences almost identical to the widespread bacterium Alcaligenes faecalis, to low G+C gram-positive bacteria and to Acidithiobacillus ferrooxidans (formerly Thiobacillus ferrooxidans) within the gamma subclass of Proteobacteria in the upper reactor sector. Although bacterial phylotypes originating from the groundwater outflow of the reactors also grouped within different subdivisions of Proteobacteria and low G+C gram-positive bacteria, most of the 16S rDNA sequences were not associated with the sequence types observed in the reactor samples. Our results suggest that the different environments were inhabited by distinct microbial communities in respect to their taxonomic diversity, particular pronounced between sediment attached microbial communities from the reactor samples and free-living bacteria from the groundwater in- and outflow.     

Phylogenetic interrelationships of members of the genera Aeromonas and Plesiomonas as determined by 16S ribosomal DNA sequencing: lack of congruence with results of DNA-DNA hybridizations.

The phylogenetic interrelationships of members of the genera Aeromonas and Plesiomonas were investigated by using small-subunit ribosomal DNA (rDNA) sequencing. Members of the genus Aeromonas formed a distinct line within the gamma subclass of the Proteobacteria. Plesiomonas shigelloides also clustered within the confines of the gamma subclass of the Proteobacteria but exhibited a closer association with members of the family Enterobacteriaceae than with members of the family Aeromonadaceae. Species of the genus Aeromonas exhibited very high levels of overall sequence similarity (ca. 98 to 100%) with each other. Several of the relationships derived from an analysis of the rDNA sequence data were in marked disagreement with the results of chromosomal DNA-DNA pairing experiments. Diagnostic rDNA signatures that have possible value for differentiating most Aeromonas species were discerned.     

Phylogenetic analysis of Piscirickettsia salmonis by 16S, internal transcribed spacer (ITS) and 23S ribosomal DNA sequencing

Piscirickettsia salmonis, the etiologic agent of piscirickettsiosis, is a systemic disease of salmonid fish. Variations in virulence and mortality have been observed during epizootics at different geographical regions and in laboratory experiments with isolates from these different locations. This raises the possibility that biogeographical patterns of genetic variation might be a significant factor with this disease. To assess the genetic variability the 16S ribosomal DNA, the internal transcribed spacer (ITS) and the 23S ribosomal DNA of isolates from 3 different hosts and 3 geographic origins were amplified using the polymerase chain reaction (PCR). Results of this analysis confirm that P. salmonis is a member of the gamma subgroup of the Proteobacteria and show that the isolates form a tight monophyletic cluster with 16S rDNA similarities ranging from 99.7 to 98.5%. The ITS regions were 309 base pairs (bp), did not contain tRNA genes, and varied between isolates (95.2 to 99.7% similarity). Two-thirds of the 23S rRNA gene was sequenced from 5 of the isolates, yielding similarities ranging from 97.9 to 99.8%. Phylogenetic trees were constructed based on the 16S rDNA, ITS and 23S rDNA sequence data and compared. The trees were topologically similar, suggesting that the 3 types of molecules provided similar phylogenetic information. Five of the isolates are closely related (> 99.4% 16S rDNA similarity, 99.1% to 99.7% ITS and 99.3 to 99.8% 23S rDNA similarities). The sequence of one Chilean isolate, EM-90, was unique, with 16S rDNA similarities to the other isolates ranging from 98.5 to 98.9%, the ITS from 95.2 to 96.9% and the 23S rDNA from 97.6 to 98.5%.     

链状亚历山大藻共附生细菌多样性

采用非分离培养分析方法,直接提取链状亚历山大藻（Alexandrium catenella）共附生细菌总DNA,以之为模板进行PCR扩增获得细菌16S rDNA基因片段,并构建16S rDNA克隆文库。通过16S rDNA限制性酶切片段长度多态性（restriction fragment length polymorphism,RFLP）和测序方法对链状亚历山大藻共附生细菌的多样性进行了研究。84个16S rDNA克隆片段经限制性内切酶HaeⅢ酶切分析,得到30种不同的酶切指纹类型。挑选50个克隆子进行测序获得其16S rDNA部分序列,并对16S rDNA序列进行聚类分析构建了系统进化树。结果表明,链状亚历山大藻共附生的细菌多样性较强,优势细菌类群为变形菌α亚群（α-Proteobacteria）和拟杆菌（Bacteroidetes）,其中玫瑰杆菌（Roseobacter sp.）在α-Proteobacteria中占绝对优势。     

A survey of lactic acid bacteria in Italian silage

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

Metabolite profiles of lactic acid bacteria in grass silage

The metabolite production of lactic acid bacteria (LAB) on silage was investigated. The aim was to compare the production of antifungal metabolites in silage with the production in liquid cultures previously studied in our laboratory. The following metabolites were found to be present at elevated concentrations in silos inoculated with LAB strains: 3-hydroxydecanoic acid, 2-hydroxy-4-methylpentanoic acid, benzoic acid, catechol, hydrocinnamic acid, salicylic acid, 3-phenyllactic acid, 4-hydroxybenzoic acid, (trans, trans)-3,4-dihydroxycyclohexane-1-carboxylic acid, p-hydrocoumaric acid, vanillic acid, azelaic acid, hydroferulic acid, p-coumaric acid, hydrocaffeic acid, ferulic acid, and caffeic acid. Among these metabolites, the antifungal compounds 3-phenyllactic acid and 3-hydroxydecanoic acid were previously isolated in our laboratory from liquid cultures of the same LAB strains by bioassay-guided fractionation. It was concluded that other metabolites, e.g., p-hydrocoumaric acid, hydroferulic acid, and p-coumaric acid, were released from the grass by the added LAB strains. The antifungal activities of the identified metabolites in 100 mM lactic acid were investigated. The MICs against Pichia anomala, Penicillium roqueforti, and Aspergillus fumigatus were determined, and 3-hydroxydecanoic acid showed the lowest MIC (0.1 mg ml(-1) for two of the three test organisms).     

应用16SrDNA-RFLP方法分析宁夏地区稻田土壤细菌的多样性

水稻是宁夏地区主要粮食作物, 水稻种植也具有维持生态系统平衡, 防止土地荒漠化等重要的生态功能。而稻田土壤细菌是维持土壤生态功能的基础。但长期以来缺乏对干旱地区稻田土壤细菌多样性的认识。本研究采用非培养技术提取稻田土壤样品总DNA, 构建其16S rDNA克隆文库, 用PCR-RFLP分析进一步测序后聚类分析细菌群落的多样性。从稻田土样中分离获得了大于23 kb的DNA片段。PCR-RFLP共得到74种酶切带型, 序列分析发现77.3%的克隆序列与环境中未培养细菌的16S rDNA序列有较高的相似性, 仅有22.7%的克隆序列与数据库中可培养细菌有较高的相似性, 表明宁夏稻区土壤中的多数细菌尚未培养。系统发育研究发现74个序列分属于12个类群, 其中变形细菌所占比例最大(37.8%), 依次为酸杆菌(16.2%)、放线菌(12.2%)、拟杆菌(10.8%)、绿屈挠菌(10.8%)、浮游霉菌(8.1%), 另外有少量厚壁菌门、芽单胞菌门和疣微菌门细菌克隆。在变形细菌的序列中包括、、γ和δ 4个类型, 比例分别为13.5%、5.4%、12.2%和6.8%。表明宁夏稻区土壤中优势细菌类群为变形杆菌和酸杆菌, 且土壤细菌类群具有丰富的多样性。     

Phylogenetic analysis of rumen bacteria by comparative sequence analysis of cloned 16S rRNA genes

Comparative DNA sequence analysis of 16S rRNA genes (rDNA) was undertaken to further our understanding of the make-up of bacterial communities in the rumen fluid of dairy cattle. Total DNA was extracted from the rumen fluid of 10 cattle fed haylage/corn silage/concentrate rations at two different times. Rumen samples were collected on two separate occasions from five cows each. In experiment 1, 31 cloned rDNA sequences were analysed. In experiment 2, DNA extractions were amplified using either 12 or 30 cycles of PCR in order to examine biases introduced during the reactions. A set of 53 sequences were analysed in experiment 2 from DNA amplified using 12 cycles and 49 sequences from PCR using 30 cycles. Sequences from the 5' end of 16S rRNA gene were compared with existing sequences in the Ribosomal Database Project. Clones from experiment 1 produced a data set in which 55% of the sequences were similar to low G+C Gram-positive bacteria related to the genus Clostridia, the majority of which were closely related to bacteria in Cluster XIV. Approximately 30% of the cloned sequences were related to bacteria in the Prevotella-Bacteroides group. Clones from experiment 2 produced a data set in which the majority of sequences were related to the Prevotella-Bacteroides group, regardless of the number of cycles of PCR. The remaining sequences clustered with members of the genus Clostridia. The majority of rDNA sequences analysed in this study represent novel rumen bacteria which have not yet been isolated.     

Phylogenetic analysis of antimicrobial lactic acid bacteria from farmed seabass Dicentrarchus labrax

The use of lactic acid bacteria (LAB) in the prevention or reduction of fish diseases is receiving increasing attention. In the present study, 47 LAB strains were isolated from farmed seabass ( Dicentrarchus labrax ) and were phenotypically and phylogenetically analysed by 16S rDNA and randomly amplified polymorphic DNA - polymerase chain reaction (RAPD-PCR). Their antimicrobial effect was tested in vitro against a wide variety of pathogenic and spoilage bacteria. Most of the strains isolated were enterococci belonging to the following species: Enterococcus faecium (59%), Enterococcus faecalis (21%), Enterococcus sanguinicola (4 strains), Enterococcus mundtii (1 strain), Enterococcus pseudoavium (1 strain), and Lactococcus lactis (1 strain). An Aerococcus viridans strain was also isolated. The survey of their antimicrobial susceptibility showed that all isolates were sensitive to vancomycin and exhibited resistance to between 4 and 10 other antibiotics relevant for therapy in human and animal medicine. Different patterns of resistance were noted for skin and intestines isolates. More than 69% (32 strains) of the isolates inhibited the growth of the majority of pathogenic and spoilage bacteria tested, including Listeria monocytogenes, Staphylococcus aureus, Aeromonas hydrophila, Aeromonas salmonicida, Vibrio anguillarum, and Carnobacterium sp. To our knowledge, this is the first report of bioactive enterococcal species isolated from seabass that could potentially inhibit the undesirable bacteria found in food systems.