Similarity and differences in the Lactobacillus acidophilus group identified by polyphasic analysis and comparative genomics

A set of lactobacilli were investigated by polyphasic analysis. Multilocus sequence analysis, DNA typing, microarray analysis, and in silico whole-genome alignments provided a remarkably consistent pattern of similarity within the Lactobacillus acidophilus complex. On microarray analysis, 17 and 5% of the genes from Lactobacillus johnsonii strain NCC533 represented variable and strain-specific genes, respectively, when tested against four independent isolates of L. johnsonii. When projected on the NCC533 genome map, about 10 large clusters of variable genes were identified, and they were enriched around the terminus of replication. A quarter of the variable genes and two-thirds of the strain-specific genes were associated with mobile DNA. Signatures for horizontal gene transfer and modular evolution were found in prophages and in DNA from the exopolysaccharide biosynthesis cluster. On microarray hybridizations, Lactobacillus gasseri strains showed a shift to significantly lower fluorescence intensities than the L. johnsonii test strains, and only genes encoding very conserved cellular functions from L. acidophilus hybridized to the L. johnsonii array. In-silico comparative genomics showed extensive protein sequence similarity and genome synteny of L. johnsonii with L. gasseri, L. acidophilus, and Lactobacillus delbrueckii; moderate synteny with Lactobacillus casei; and scattered X-type sharing of protein sequence identity with the other sequenced lactobacilli. The observation of a stepwise decrease in similarity between the members of the L. acidophilus group suggests a strong element of vertical evolution in a natural phylogenetic group. Modern whole-genome-based techniques are thus a useful adjunct to the clarification of taxonomical relationships in problematic bacterial groups.     

Hydrogen peroxide production by Lactobacillus johnsonii NCC 533 and its role in anti-Salmonella activity

The human intestinal isolate Lactobacillus johnsonii NCC 533 (La1) is a probiotic strain with well-documented antimicrobial properties. Previous research has identified the production of lactic acid and bacteriocins as important factors, but that other unidentified factors are also involved. We used the recently published genome sequence of L. johnsonii NCC 533 to search for novel antipathogen factors and identified three potential gene products that may catalyze the synthesis of the known antimicrobial factor hydrogen peroxide, H(2)O(2). In this work, we confirmed the ability of NCC 533 as well as eight different L. johnsonii strains and Lactobacillus gasseri to produce H(2)O(2) when resting cells were incubated in the presence of oxygen, and that culture supernatant containing NCC 533-produced H(2)O(2) was effective in killing the model pathogen Salmonella enterica serovar Typhimurium SL1344 in vitro.     

Integration and distribution of Lactobacillus johnsonii prophages

In Lactobacillus johnsonii strain NCC533, two prophages were integrated into tRNA genes and one was disrupted by integration. In a survey, the prophages were restricted to strains sharing an essentially identical restriction pattern. Microarray analysis showed that the prophage DNA represents about 50% of the NCC533 strain-specific DNA.     

Group-specific comparison of four lactobacilli isolated from human sources using differential blast analysis

Lactic acid bacteria (LAB) have been used in fermentation processes for centuries. More recent applications including the use of LAB as probiotics have significantly increased industrial interest. Here we present a comparative genomic analysis of four completely sequenced Lactobacillus strains, isolated from the human gastrointestinal tract, versus 25 lactic acid bacterial genomes present in the public database at the time of analysis. Lactobacillus acidophilus NCFM, Lactobacillus johnsonii NCC533, Lactobacillus gasseri ATCC33323, and Lactobacillus plantarum WCFS1are all considered probiotic and widely used in industrial applications. Using Differential Blast Analysis (DBA), each genome was compared to the respective remaining three other Lactobacillus and 25 other LAB genomes. DBA highlighted strain-specific genes that were not represented in any other LAB used in this analysis and also identified group-specific genes shared within lactobacilli. Initial comparative analyses highlighted a significant number of genes involved in cell adhesion, stress responses, DNA repair and modification, and metabolic capabilities. Furthermore, the range of the recently identified potential autonomous units (PAUs) was broadened significantly, indicating the possibility of distinct families within this genetic element. Based on in silico results obtained for the model organism L. acidophilus NCFM, DBA proved to be a valuable tool to identify new key genetic regions for functional genomics and also suggested re-classification of previously annotated genes.     

Host specific diversity in Lactobacillus johnsonii as evidenced by a major chromosomal inversion and phage resistance mechanisms

Genetic diversity and genomic rearrangements are a driving force in bacterial evolution and niche adaptation. We sequenced and annotated the genome of Lactobacillus johnsonii DPC6026, a strain isolated from the porcine intestinal tract. Although the genome of DPC6026 is similar in size (1.97 mbp) and GC content (34.8%) to the sequenced human isolate L. johnsonii NCC 533, a large symmetrical inversion of approximately 750 kb differentiated the two strains. Comparative analysis among 12 other strains of L. johnsonii including 8 porcine, 3 human and 1 poultry isolate indicated that the genome architecture found in DPC6026 is more common within the species than that of NCC 533. Furthermore a number of unique features were annotated in DPC6026, some of which are likely to have been acquired by horizontal gene transfer (HGT) and contribute to protection against phage infection. A putative type III restriction-modification system was identified, as were novel Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) elements. Interestingly, these particular elements are not widely distributed among L. johnsonii strains. Taken together these data suggest intra-species genomic rearrangements and significant genetic diversity within the L. johnsonii species and indicate towards a host-specific divergence of L. johnsonii strains with respect to genome inversion and phage exposure.     

Oxygen Relieves the CO2 and Acetate Dependency of Lactobacillus johnsonii NCC 533

Oxygen relieves the CO₂ and acetate dependency of Lactobacillus johnsonii NCC 533. The probiotic Lactobacillus johnsonii NCC 533 is relatively sensitive to oxidative stress; the presence of oxygen causes a lower biomass yield due to early growth stagnation. We show however that oxygen can also be beneficial to this organism as it relieves the requirement for acetate and CO₂ during growth. Both on agar- and liquid-media, anaerobic growth of L. johnsonii NCC 533 requires CO₂ supplementation of the gas phase. Switching off the CO₂ supply induces growth arrest and cell death. The presence of molecular oxygen overcomes the CO₂ dependency. Analogously, L. johnsonii NCC 533 strictly requires media with acetate to sustain anaerobic growth, although supplementation at a level that is 100-fold lower (120 microM) than the concentration in regular growth medium for lactobacilli already suffices for normal growth. Analogous to the CO₂ requirement, oxygen supply relieves this acetate-dependency for growth. The L. johnsonii NCC 533 genome indicates that this organism lacks genes coding for pyruvate formate lyase (PFL) and pyruvate dehydrogenase (PDH), both CO₂ and acetyl-CoA producing systems. Therefore, C1- and C2- compound production is predicted to largely depend on pyruvate oxidase activity (POX). This proposed role of POX in C2/C1-generation is corroborated by the observation that in a POX deficient mutant of L. johnsonii NCC 533, oxygen is not able to overcome acetate dependency nor does it relieve the CO₂ dependency.     

Analysis of the genome sequence of Lactobacillus gasseri ATCC 33323 reveals the molecular basis of an autochthonous intestinal organism

This study presents the complete genome sequence of Lactobacillus gasseri ATCC 33323, a neotype strain of human origin and a native species found commonly in the gastrointestinal tracts of neonates and adults. The plasmid-free genome was 1,894,360 bp in size and predicted to encode 1,810 genes. The GC content was 35.3%, similar to the GC content of its closest relatives, L. johnsonii NCC 533 (34%) and L. acidophilus NCFM (34%). Two identical copies of the prophage LgaI (40,086 bp), of the Sfi11-like Siphoviridae phage family, were integrated tandomly in the chromosome. A number of unique features were identified in the genome of L. gasseri that were likely acquired by horizontal gene transfer and may contribute to the survival of this bacterium in its ecological niche. L. gasseri encodes two restriction and modification systems, which may limit bacteriophage infection. L. gasseri also encodes an operon for production of heteropolysaccharides of high complexity. A unique alternative sigma factor was present similar to that of B. caccae ATCC 43185, a bacterial species isolated from human feces. In addition, L. gasseri encoded the highest number of putative mucus-binding proteins (14) among lactobacilli sequenced to date. Selected phenotypic characteristics that were compared between ATCC 33323 and other human L. gasseri strains included carbohydrate fermentation patterns, growth and survival in bile, oxalate degradation, and adhesion to intestinal epithelial cells, in vitro. The results from this study indicated high intraspecies variability from a genome encoding traits important for survival and retention in the gastrointestinal tract.     

Single bioreactor gastrointestinal tract simulator for study of survival of probiotic bacteria

The aim of the present study was to design an in vitro model system to evaluate the probiotic potential of food. A single bioreactor system-gastrointestinal tract simulator (GITS) was chosen for process simulation on account of its considerable simplicity compared to multi-vessel systems used in previous studies. The bioreactor was evaluated by studying the viability of four known probiotic bacteria (Lactobacillus acidophilus La-5, Lactobacillus johnsonii NCC 533, Lactobacillus casei strain Shirota, and Lactobacillus rhamnosus GG) as a function of their physiological state. L. acidophilus and L. johnsonii survived in GITS better when introduced at an early stationary or exponential phase compared to being previously stored for 2 weeks at 4 degrees C. These two species were more resistant to bile salts and survived better than L. casei and L. rhamnosus GG. The latter two species gave large losses (up to 6 log) in plate counts independent of growth state due to the bile. However, experiments with some commercial probiotic products containing Lb. GG bacteria showed much better survival compared with model food (modified deMan-Rogosa-Sharpe growth medium), thus demonstrating the influence of the food matrix on the viability of bacteria. The study demonstrated that GITS can be successfully used for evaluation of viability of probiotic bacteria and functionality of probiotic food.     

Iron requirement of Lactobacillus spp. in completely chemically defined growth media

A completely chemically-defined growth medium, containing guanine, thymine, cytidine, 2'-deoxyadenosine and 2'-deoxyuridine as DNA precursors, was developed for Lactobacillus johnsonii, on the basis of statistically designed techniques suitable for other lactobacilli. Particular focus was given to the nucleotide composition of different defined media, and to the specific nucleotide requirements of Lact. johnsonii. Most of the lactobacilli tested grew in a medium containing five free bases, four ribonucleosides or five deoxyribonucleosides. Adenine and guanine were replaceable by inosine. The requirement for thymine and cytosine was satisfied with uracil. The presence of inosine and uracil was identified as being essential for the growth of different Lactobacillus species, displaying their inability to synthesize purines and pyrimidines de novo. Defined recipes with different nucleotide composition were used to investigate iron requirements of lactobacilli. Only marginal differences in growth were observed in iron-depleted media supplemented with five free bases, four ribonucleosides or five deoxyribonucleosides; iron depletion had a greater effect on growth when inosine and uracil were supplied as the only nucleotide sources. The results suggest that iron plays a role in the pyrimidine and purine metabolism of lactobacilli. Lactobacillus spp., particularly Lact. johnsonii, require iron under particular environmental conditions with limited or specific nucleotide sources.     

Modification of the technical properties of Lactobacillus johnsonii NCC 533 by supplementing the growth medium with unsaturated fatty acids

The aim of this study was to investigate the influence of supplementing growth medium with unsaturated fatty acids on the technical properties of the probiotic strain Lactobacillus johnsonii NCC 533, such as heat and acid tolerance, and inhibition of Salmonella enterica serovar Typhimurium infection. Our results showed that the membrane composition and morphology of L. johnsonii NCC 533 were significantly changed by supplementing a minimal Lactobacillus medium with oleic, linoleic, and linolenic acids. The ratio of saturated to unsaturated plus cyclic fatty acids in the bacterial membrane decreased by almost 2-fold when minimal medium was supplemented with unsaturated fatty acids (10 μg/ml). The subsequent acid and heat tolerance of L. johnsonii decreased by 6- and 20-fold when the strain was grown in the presence of linoleic and linolenic acids, respectively, compared with growth in oleic acid (all at 10 μg/ml). Following acid exposure, significantly higher (P < 0.05) oleic acid content was detected in the membrane when growth medium was supplemented with linoleic or linolenic acid, indicating that saturation of the membrane fatty acids occurred during acid stress. Cell integrity was determined in real time during stressed conditions using a fluorescent viability kit in combination with flow cytometric analysis. Following heat shock (at 62.5°C for 5 min), L. johnsonii was unable to form colonies; however, 60% of the bacteria showed no cell integrity loss, which could indicate that the elevated heat inactivated vital processes within the cell, rendering it incapable of replication. Furthermore, L. johnsonii grown in fatty acid-enriched minimal medium had different adhesion properties and caused a 2-fold decrease in S. enterica serovar Typhimurium UK1-lux invasion of HT-29 epithelial cells compared with bacteria grown in minimal medium alone. This could be related to changes in the hydrophobicity and fluidity of the membrane. Our study shows that technical properties underlying probiotic survivability can be affected by nutrient composition of the growth medium.     

Effects of culture conditions on the growth and auto-aggregation ability of vaginal Lactobacillus johnsonii CRL 1294

AIMS: To evaluate the effects of different physico-chemical factors on the growth and auto-aggregating ability of vaginal Lactobacillus johnsonii CRL 1294. METHODS AND RESULTS: L. johnsonii CRL 1294 was cultivated in different culture media, initial pH and temperature of incubation. The growth parameters were estimated by the Gompertz model, being optimal (higher final biomass and growth rate, and shorter lag phase) at an initial pH of 6.5 and at a temperature of 37 degrees C, both in LAPTg and MRS. The auto-aggregation ability, which was assessed by a model of exponential association, was evidenced in all the growth phases, being higher at pH 5 or 6.5. CONCLUSIONS: The growth of L. johnsonii CRL 1294 was affected in different way by all the physico-chemical factors tested. However, the auto-aggregation ability increased mainly at low initial pH of growth media. SIGNIFICANCE AND IMPACT OF THE STUDY: The auto-aggregation ability under different culture conditions of a vaginal Lactobacillus strain was systematically and statistically evaluated for the first time. The higher cellular aggregation evidenced at low pH could be a fundamental characteristic in the acidic vaginal environment to promote the protective role of lactobacilli.     

The probiotic Lactobacillus johnsonii NCC 533 produces high-molecular-mass inulin from sucrose by using an inulosucrase enzyme

Fructansucrase enzymes polymerize the fructose moiety of sucrose into levan or inulin fructans, with beta(2-6) and beta(2-1) linkages, respectively. The probiotic bacterium Lactobacillus johnsonii strain NCC 533 possesses a single fructansucrase gene (open reading frame AAS08734) annotated as a putative levansucrase precursor. However, (13)C nuclear magnetic resonance (NMR) analysis of the fructan product synthesized in situ revealed that this is of the inulin type. The ftf gene of L. johnsonii was cloned and expressed to elucidate its exact identity. The purified L. johnsonii protein was characterized as an inulosucrase enzyme, producing inulin from sucrose, as identified by (13)C NMR analysis. Thin-layer chromatographic analysis of the reaction products showed that InuJ synthesized, besides the inulin polymer, a broad range of fructose oligosaccharides. Maximum InuJ enzyme activity was observed in a pH range of 4.5 to 7.0, decreasing sharply at pH 7.5. InuJ exhibited the highest enzyme activity at 55 degrees C, with a drastic decrease at 60 degrees C. Calcium ions were found to have an important effect on enzyme activity and stability. Kinetic analysis showed that the transfructosylation reaction of the InuJ enzyme does not obey Michaelis-Menten kinetics. The non-Michaelian behavior of InuJ may be attributed to the oligosaccharides that were initially formed in the reaction and which may act as better acceptors than the growing polymer chain. This is only the second example of the isolation and characterization of an inulosucrase enzyme and its inulin (oligosaccharide) product from a Lactobacillus strain. Furthermore, this is the first Lactobacillus strain shown to produce inulin polymer in situ.     

Identification of lactobacilli isolated from the cloaca and vagina of laying hens and characterization for potential use as probiotics to control Salmonella Enteritidis

AIMS: To select Lactobacillus strains from laying hens for potential use as probiotic to control Salmonella Enteritidis infection. METHODS AND RESULTS: One hundred and eighty-six lactobacilli were isolated from the cloaca and vagina of laying hens, and identified at the species level by a polyphasic taxonomic approach. All isolates belonged to the Lactobacillus acidophilus, Lactobacillus reuteri or Lactobacillus salivarius phylogenetic groups, with the L. reuteri group being the most predominant group. Based on genetic diversity, about 50 representative strains were selected and tested for in vitro properties that could be predictive for probiotic activity in laying hens. Salmonella inhibition was shown to be species dependent, and correlated to some extent with the production of lactic acid. A selection of strains was evaluated in a S. Enteritidis challenge experiment. Two strains, L. reuteri R-17485 and Lactobacillus johnsonii R-17504 significantly decreased the colonization of chicks by S. Enteritidis in caeca, liver and spleen. CONCLUSIONS: Lactobacilli isolated from laying hens were observed to inhibit Salmonella growth in vitro, most probably through production of lactic acid, and to decrease in vivo the S. Enteritidis colonization of chicks. SIGNIFICANCE AND IMPACT OF THE STUDY: The data demonstrate that Lactobacillus isolates from laying hens may have probiotic potential in reducing S. Enteritidis infection.     

Identification and characterization of novel surface proteins in Lactobacillus johnsonii and Lactobacillus gasseri

We have identified and sequenced the genes encoding the aggregation-promoting factor (APF) protein from six different strains of Lactobacillus johnsonii and Lactobacillus gasseri. Both species harbor two apf genes, apf1 and apf2, which are in the same orientation and encode proteins of 257 to 326 amino acids. Multiple alignments of the deduced amino acid sequences of these apf genes demonstrate a very strong sequence conservation of all of the genes with the exception of their central regions. Northern blot analysis showed that both genes are transcribed, reaching their maximum expression during the exponential phase. Primer extension analysis revealed that apf1 and apf2 harbor a putative promoter sequence that is conserved in all of the genes. Western blot analysis of the LiCl cell extracts showed that APF proteins are located on the cell surface. Intact cells of L. johnsonii revealed the typical cell wall architecture of S-layer-carrying gram-positive eubacteria, which could be selectively removed with LiCl treatment. In addition, the amino acid composition, physical properties, and genetic organization were found to be quite similar to those of S-layer proteins. These results suggest that APF is a novel surface protein of the Lactobacillus acidophilus B-homology group which might belong to an S-layer-like family.     

Genotypic and phenotypic studies of murine intestinal lactobacilli: species differences in mice with and without colitis

Lactobacilli represent components of the commensal mammalian gastrointestinal microbiota and are useful as probiotics, functional foods, and dairy products. This study includes systematic polyphasic analyses of murine intestinal Lactobacillus isolates and correlation of taxonomic findings with data from cytokine production assays. Lactobacilli were recovered from mice with microbiota-dependent colitis (interleukin-10 [IL-10]-deficient C57BL/6 mice) and from mice without colitis (Swiss Webster and inducible nitric oxide synthetase-deficient C57BL/6 mice). Polyphasic analyses were performed to elucidate taxonomic relationships among 88 reference and murine gastrointestinal lactobacilli. Genotypic tests included single-locus analyses (16S ribosomal DNA sequencing and 16S-23S rRNA intergenic spacer region PCR) and genomic DNA profiling (repetitive DNA element-based PCR), and phenotypic analyses encompassed more than 50 tests for carbohydrate utilization, enzyme production, and antimicrobial resistance. From 20 mice without colitis, six Lactobacillus species were recovered; the majority of the mice were colonized with L. reuteri or L. murinus (72% of isolates). In contrast, only, L. johnsonii was isolated from 14 IL-10-deficient mice. Using an in vitro assay, we screened murine isolates for their ability to inhibit tumor necrosis factor alpha (TNF-alpha) secretion by lipopolysaccharide-activated macrophages. Interestingly, a subpopulation of lactobacilli recovered from mice without colitis displayed TNF-alpha inhibitory properties, whereas none of the L. johnsonii isolates from IL-10-deficient mice exhibited this effect. We propose that differences among intestinal Lactobacillus populations in mammals, combined with host genetic susceptibilities, may account partly for variations in host mucosal responses.     

Species-specific oligonucleotide probes for the detection and identification of Lactobacillus isolated from mouse faeces

AIMS: To develop species-specific monitoring techniques for rapid detection and identification of Lactobacillus isolated from mouse faeces. METHODS AND RESULTS: The specificity of oligonucleotide probes was evaluated by dot blot hybridization to 16S rDNA and 23S rDNA amplified by PCR from 12 Lactobacillus type strains and 100 strains of Lactobacillus isolated from mouse faeces. Oligonucleotide probes specific for each Lactobacillus species hybridized only with targeted rDNA. The Lactobacillus strains isolated from mouse faeces were identified mainly as Lactobacillus intestinalis, L. johnsonii, L. murinus and L. reuteri using species-specific probes. 16S rDNA of eight unidentified isolates were sequenced and two new probes were designed. Four of eight strains of unhybridized Lactobacillus were identified as L. johnsonii/gasseri group, and the remaining four strains as L. vaginalis. CONCLUSIONS: The species-specific probe set of L. intestinalis, L. johnsonii, L. murinus, L. reuteri and L. vaginalis in this study was efficient for rapid identification of Lactobacillus isolated from mouse faeces. SIGNIFICANCE AND IMPACT OF THE STUDY: The oligonucleotide probe set for Lactobacillus species harboured in the mouse intestine, can be used for rapid identification of lactobacilli and monitoring of the faecal Lactobacillus community.     

A Chinese rhesus macaque (Macaca mulatta) model for vaginal Lactobacillus colonization and live microbicide development

Background  We sought to establish a nonhuman primate model of vaginal Lactobacillus colonization suitable for evaluating live microbial microbicide candidates.
Methods  Vaginal and rectal microflora in Chinese rhesus macaques ( Macaca mulatta ) were analyzed, with cultivable bacteria identified by 16S rRNA gene sequencing. Live lactobacilli were intravaginally administered to evaluate bacterial colonization.
Results  Chinese rhesus macaques harbored abundant vaginal Lactobacillus , with Lactobacillus johnsonii as the predominant species. Like humans, most examined macaques harbored only one vaginal Lactobacillus species. Vaginal and rectal Lactobacillus isolates from the same animal exhibited different genetic and biochemical profiles. Vaginal Lactobacillus was cleared by a vaginal suppository of azithromycin, and endogenous L. johnsonii was subsequently restored by intravaginal inoculation. Importantly, prolonged colonization of a human vaginal Lactobacillus jensenii was established in these animals.
Conclusions  The Chinese rhesus macaque harbors vaginal Lactobacillus and is a potentially useful model to support the pre-clinical evaluation of Lactobacillus -based topical microbicides.