Isolation of cultivable thermophilic lactic acid bacteria from cheeses made with mesophilic starter and molecular comparison with dairy-related Lactobacillus helveticus strains

Aims:  To isolate cultivable thermophilic lactic acid bacteria from cheeses made with mesophilic starter and compare them with dairy-related Lactobacillus helveticus strains using molecular typing methods.
Methods and Results:  The number of thermophilic bacteria in seven commercial cheeses manufactured with mesophilic starters was estimated to be <10 CFU g⁻¹. Implementation of an enumeration step in the isolation method made it possible to isolate one thermophilic strain from each of five of seven cheeses. Comparing repetitive sequence PCR (rep-PCR) profiles of the isolates with dairy-related Lact. helveticus strains indicated that one isolate was a Lact. helveticus . Partial sequencing of 16S rRNA confirmed this, and the remaining four strains were identified as Lactobacillus delbrueckii , Lactobacillus fermentum and Enterococcus faecium . The rep-PCR profile of the isolated Lact. helveticus was identical to the rep-PCR profile of the Lact. helveticus adjunct culture used in the specific cheese, but their pulsed field gel electrophoresis profiles differed slightly.
Conclusion:  It was possible to isolate cultivable thermophilic bacteria from ripened cheeses manufactured with mesophilic starter and thermophilic adjunct cultures by using an enumeration step.
Significance and Impact of the Study:  Isolation of cultivable thermophilic bacteria from ripened cheeses made with mesophilic starters offers an original source for new dairy-relevant cultures.     

Lactobacillus rhamnosus GG inhibits invasion of cultured human respiratory cells by prtF1-positive macrolide-resistant group A streptococci

Aims:  This study was designed to determine whether the probiotic strain Lactobacillus GG, which is extensively used in the treatment and prevention of intestinal disorders, is able to inhibit invasion of cultured human respiratory cells by macrolide-resistant group A streptococci (GAS) carrying the prtF1 gene, which encodes the fibronectin (Fn)-binding invasin F1.
Methods and Results:  Eight prtF1 -positive erythromycin-resistant GAS strains were used to infect A549 monolayers in competition and displacement assays with Lactobacillus GG. Live (L-LGG) and heat-killed (HK-LGG) lactobacilli and their spent culture supernatant (SCS) significantly reduced ( P  <   0·001) GAS invasion efficiency in both assays. No antibacterial activity of Lactobacillus GG against GAS was detected. Both L-LGG and HK-LGG and all prtF1 -positive GAS induced a strong agglutination reaction using Fn-coated particles.
Conclusions:  Lactobacillus GG exerts an antagonistic action against GAS by inhibiting cell invasion. Competitive binding of Lactobacillus GG and GAS to Fn might be involved in the inhibition process.
Significance and Impact of the Study:  The finding that Lactobacillus GG can prevent in vitro invasion of respiratory cells by GAS suggests new applications for this probiotic strain and warrants further studies of its capacity to prevent GAS throat infections.     

Diversity of environmental Mycobacterium isolates from hemodialysis water as shown by a multigene sequencing approach

Here we used a multigene sequencing approach for the identification and molecular typing of environmental mycobacteria of the fast-growing subgroup. Strains were isolated from hemodialysis water and clinical samples. Eleven type strains of related species of the genus were also included in this study. To gain further insight into the diversity of the environmental mycobacteria, we analyzed several housekeeping genes (16S rRNA, ITS1, gyrB, hsp65, recA, rpoB, and sodA). No individual phylogenetic tree allowed good discrimination of all of the species studied. However, a concatenated and a consensus analysis, combining the genes, allowed better discrimination of each strain to the species level, and the increase in sequence size also led to greater tree robustness. This approach is useful not only for the discrimination and identification of environmental mycobacteria but also for their molecular typing and studies of population genetics. Our results demonstrate high genetic diversity among the isolates obtained, which are probably new species of the genus.     

Lactobacilli antagonize biological effects of enterohaemorrhagic Escherichia coli in vitro

Aims:  To assess the effect of two lactobacilli on the biological activity of enterohaemorrhagic Escherichia coli (EHEC) in vitro .
Methods and Results:  Strains CIDCA 133 ( Lactobacillus delbrueckii subsp. lactis ) and CIDCA 83114 ( Lactobacillus plantarum ) were studied. Hep-2 cells were used as an in vitro model to assess the biological effect of a clinical isolate of EHEC. Preincubation of cell monolayers with lactobacilli before EHEC prevented detachment of eukaryotic cells and minimizes both F-actin rearrangements and morphological alterations. Interestingly, the protective effect could not be ascribed to pathogen exclusion. In addition, viability of the lactobacilli was not necessary for protection and other species of the genus Lactobacillus failed to protect eukaryotic cells.
Conclusions:  Our results suggest that lactobacilli are antagonizing virulence mechanisms of EHEC either by modification of the microenvironment or by interfering with the signalling cascades triggered by the pathogen.
Significance and Impact of the Study:  Our findings give a rationale basis for the use of specific probiotic strains for the prophylaxis and prevention of intestinal infections due to EHEC.     

Strain typing with ISLpl1 in lactobacilli

Twenty-seven Lactobacillus plantarum ssp. plantarum, 11 Lactobacillus paraplantarum and five Lactobacillus casei-related strains, isolated from various autochthonous Serbian and Montenegro-fermented foods, were identified using phenotypical characterization and current PCR methods based on PCR of the recA gene or the 23S-5S rRNA gene intragenic spacer (IS) region. The strains were genotypically characterized by a new method based on the insertion sequence element ISLpl11 that grouped these lactobacilli into 10 IS-fingerprinting groups. Between six and 23 copies of the ISLpl1 were found in each strain and the ISLpl1-fingerprint groups correlated well with the origin of the strains. The method proved suitable for strain typing of lactic acid bacteria at the infraspecies level.     

BOOK REVIEWS

Book reviewed in this article:
Willows: the genus Salix. Christopher Newsholme. 224 pp., 65 colour plates, 159 black-and-white figures.
Blooms of Bressingham Garden Plants. Alan and Adrian Bloom. 320 pp., 750 + colour photographs.     

Lactobacillus intestinalis (ex Hemme 1974) sp. nov., nom. rev., isolated from the intestines of mice and rats

The genetic and phenotypic properties of 10 strains identified as Lactobacillus intestinalis sp. nov. were examined. These strains constitute a distinct species which can be differentiated from all of the previously described homofermentative species in the genus Lactobacillus by their carbohydrate fermentation pattern. The guanine-plus-cytosine contents of their DNAs are 33 to 35 mol%. DNAs from 10 other Lactobacillus species did not exhibit significant levels of relatedness to representative strains of the new species. The name Lactobacillus intestinalis (ex Hemme) sp. nov., nom. rev. is proposed for these isolates, and strain Th4 (= ATCC 49335 = JCM 7548) is the type strain.     

Validation of the dorsal air pouch model to predict and examine immunostimulatory responses in the gut

Aims:  To validate the use of the air pouch system to predict and examine early immune responses induced by the presumptive probiotics Lactobacillus paracasei subsp. paracasei B112, DC205, DC215 and DC412 strains in the gut mucosa.
Methods and Results:  Only the DC412 strain interacted strongly with the cells forming the air pouch lining tissue and induced early innate immune responses such as polymorphonuclear (PMN) cell recruitment, phagocytosis and tumour necrosis factor alpha (TNF-α) production that equal the respective responses induced by the probiotic Lactobacillus acidophilus NCFB 1748. The strains exhibiting strong immunoregulatory activity in the air pouch also interacted strongly with the gut-associated lymphoid tissue (GALT). The strain DC412 exerts its effect on the intestine through stimulation of Toll-like receptor (TLR)2/TLR4-mediated signalling events leading to secretion of a certain profile of cytokines in which gamma interferon (IFN-γ), TNF-α, interleukin (IL)-6 and IL-10 are included. The probiotic Lact. acidophilus NCFB 1748 induces the same cytokine profile in addition to IL-12B, and this response is potentially mediated by the synergy of TLR2 and TLR9.
Conclusion:  The strain DC412 possesses the in vitro and in vivo characteristics of a probiotic micro-organism.
Significance and Impact of the Study:  The dorsal mouse or rat air pouch may be used as an alternative and rapid method for the initial discrimination and selection of potential probiotic Lactobacillus strains.     

Diversity of Environmental Mycobacterium Isolates from Hemodialysis Water as Shown by a Multigene Sequencing Approach

Here we used a multigene sequencing approach for the identification and molecular typing of environmental mycobacteria of the fast-growing subgroup. Strains were isolated from hemodialysis water and clinical samples. Eleven type strains of related species of the genus were also included in this study. To gain further insight into the diversity of the environmental mycobacteria, we analyzed several housekeeping genes (16S rRNA, ITS1, gyrB, hsp65, recA, rpoB, and sodA). No individual phylogenetic tree allowed good discrimination of all of the species studied. However, a concatenated and a consensus analysis, combining the genes, allowed better discrimination of each strain to the species level, and the increase in sequence size also led to greater tree robustness. This approach is useful not only for the discrimination and identification of environmental mycobacteria but also for their molecular typing and studies of population genetics. Our results demonstrate high genetic diversity among the isolates obtained, which are probably new species of the genus.     

Identification of lactobacilli isolated in traditional ripe wheat sourdoughs by using molecular methods

Eleven sourdoughs from Molise region (Southern-Italy) were subjected to microbiological analyses in order to select predominant lactobacilli species to be utilised as starter culture for bread production. A multiple approach was used, consisting of the growth in different culture media, DGGE analysis, 16S rRNA gene sequencing and RAPD-PCR typing. Forty-three lactobacilli were identified and four different species, facultatively or obligately heterofermentative lactobacilli, were found. Lactobacillus plantarum and Lactobacillus brevis represented the prevailing lactobacilli, while Lactobacillus casei and Lactobacillus paracasei ssp. paracasei were detected only in few samples. The use of different media demonstrated that there is no efficient medium for the study of sourdoughs and the cultivation in different substrates remains the best tool to obtain a picture of lactic acid bacteria population. DGGE and 16S rRNA gene sequencing allowed to obtain a reliable identification of strains, while RAPD-PCR resulted a suitable method for typing lactobacilli at strain level.     

Phylogeny of the defined murine microbiota: altered Schaedler flora.

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

Taxonomic study of the Lactobacillus acidophilus group, with recognition of Lactobacillus gallinarum sp. nov. and Lactobacillus johnsonii sp. nov. and synonymy of Lactobacillus acidophilus group A3 (Johnson et al. 1980) with the type strain of Lactobacillus amylovorus (Nakamura 1981).

Biochemical properties and DNA-DNA reassociation studies of Lactobacillus acidophilus strains isolated from humans and animals indicate that these include six genomospecies. Two new species can be differentiated from the established species of the genus Lactobacillus: L. gallinarum sp. nov. (type strain, ATCC 33199) and L. johnsonii sp. nov. (type strain, ATCC 33200). Furthermore, it was clarified that L. acidophilus group A3 (Johnson et al. 1980) is synonymous with L. amylovorus.     

Lactobacillus tucceti sp. nov., a new lactic acid bacterium isolated from sausage

Following the application of several molecular techniques strain R 19c, isolated from sausage by Reuter in 1970 and deposited at the DSMZ as Lactobacillus sp., has been identified as pertaining to a new species. It showed singular ISR-DdeI and ISR-HaeIII profiles that allowed its differentiation from 68 lactic acid bacteria reference strains analyzed. Phylogenetic analysis based on 16S rRNA gene sequences places this strain in the genus Lactobacillus within the Lactobacillus alimentarius group. Species L. versmoldensis is the closest phylogenetic neighbor with 96.3% sequence similarity. DNA-DNA hybridization experiments confirmed the independent status at species level of this strain. Species-specific primers for PCR detection of this new species have been developed. Phenotypically it can be distinguished from the closest relative L. versmoldensis by several traits such as the peptidoglycan type (L-Lys-Gly-D-Asp), acid production from L-rhamnose, D-mannitol and L-fucose and its inability to ferment d-galactose, d-melibiose and d-sucrose. The name Lactobacillus tucceti sp. nov. is proposed with strain R 19c(T) (=DSM 20183(T)= CECT 5920(T)) as the type strain.     

Lactobacillus alvi sp. nov., isolated from the intestinal tract of chicken

Strain R54(T) was isolated from the gizzard of hens. The isolate was Gram-positive, facultative anaerobic, gas-forming, catalase-negative, nonmotile, nonspore-forming and short-rod-shaped. The optimal temperature for growth was 40 °C and the DNA G+C content was 42.7 mol%. The 16S rRNA gene sequences similarity showed that strain R54(T) was most closely related to Lactobacillus ingluviei LMG 20380(T) (97.5%), followed by Lactobacillus coleohominis CIP 106820(T) (96.1%), Lactobacillus secaliphilus DSM 17896(T) (95.6%) and Lactobacillus gastricus LMG 22113(T) (95.4%). The DNA-DNA relatedness between strain R54(T) and L. ingluviei LMG 20380(T) , was 43.3%. The predominant cellular fatty acids of strain R54(T) were C(18:1 ω9c) (64.9%) and C(16:0) (20.0%), and the major polar lipid group was phospholipids. On the basis of polyphasic taxonomy approach, strain R54(T) represents a novel species of the genus Lactobacillus, for which the name Lactobacillus alvi sp. nov. is proposed (type strain R54(T) = KCCM 90099(T) = JCM 17644(T)).     

Deoxyribonuclease activities in Lactobacillus delbrueckii

DNase activity was examined in the extracellular and subcellular fractions of six non-transformable strains belonging to Lactobacillus delbrueckii subsp. lactis (L. lactis) and Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus) and compared with the activity present in Lactobacillus johnsonii NCK 65, a transformable strain of Lactobacillus. In the extracellular fraction of the L. delbrueckii strains, a common protein band of 36 kDa was detected, while a band of 29 kDa was found in the same fraction of L. johnsonii. No nuclease activity was detected in the cytoplasmic fraction of this strain, indicating that the localization of the DNase activity could be a key factor in the uptake of foreign DNA.     

Fructooligosaccharides metabolism and effect on bacteriocin production in Lactobacillus strains isolated from ensiled corn and molasses

Fructo- (FOS) and galacto-oligosaccharides have been used to promote the growth of probiotics, mainly those from Lactobacillus genus. However, only few reports have evaluated the effect of prebiotics on bacteriocins activity and production. In this work, we characterized the effect of FOS supplementation on the growth, lactic and acetic acids production, and antimicrobial activity of crude extracts obtained from Lactobacillus strains isolated from ensiled corn and molasses. Seven out of 28 isolated Lactobacillus, belonging to Lactobacillus casei, Lactobacillus plantarum, and Lactobacillus brevis, showed antimicrobial activity against Listeria innocua. Among them, the strain L. plantarum LE5 showed antimicrobial activity against Listeria monocytogenes and Enteroccocus faecalis; while the L. plantarum LE27 strain showed antimicrobial effect against L. monocytogenes, E. faecalis, Escherichia coli and Salmonella enteritidis. This antimicrobial activity in most of the cases was obtained only after FOS supplementation. In summary, these results show the feasibility to increase the antimicrobial activity of Lactobacillus bacteriocins by supplementing the growth medium with FOS.     

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.     

Genetic diversity of the genus Lactobacillus bacteria from the human gastrointestinal microbiome

The species and strain genetic diversity of bacterial cultures belonging to the genus Lactobacillus, which were isolated from the gastrointestinal microbiome of the human population living in the former Soviet Union in the years 1960-1980, was studied. The bacteria demonstrated probiotic characteristics. Phylogenetic analysis of sequences of the gene coding for 16S rRNA detected earlier by us, showed that the gene found in bacteria isolated from the intestinal content of healthy adults and represented by species L. plantarum, L. helveticus, L. casei/paracasei, L. rhamnosus, and L. fermentum has high homology (97-100%) with this gene in type representatives of the species. The genotypic and strain diversity of cultures was studied using RAPD-PCR and nonspecific primers. This method with the use of the ERIC-1 primer gave reliable and reproducible results as compared that using with M13 and MSP primers and allowed the identification of examined bacteria belonging to the genus Lactobacillus at the level of species and certification at the strain level.     

Multilocus sequence typing of Lactobacillus casei reveals a clonal population structure with low levels of homologous recombination

Robust genotyping methods for Lactobacillus casei are needed for strain tracking and collection management, as well as for population biology research. A collection of 52 strains initially labeled L. casei or Lactobacillus paracasei was first subjected to rplB gene sequencing together with reference strains of Lactobacillus zeae, Lactobacillus rhamnosus, and other species. Phylogenetic analysis showed that all 52 strains belonged to a single compact L. casei-L. paracasei sequence cluster, together with strain CIP107868 (= ATCC 334) but clearly distinct from L. rhamnosus and from a cluster with L. zeae and CIP103137(T) (= ATCC 393(T)). The strains were genotyped using amplified fragment length polymorphism, multilocus sequence typing based on internal portions of the seven housekeeping genes fusA, ileS, lepA, leuS, pyrG, recA, and recG, and tandem repeat variation (multilocus variable-number tandem repeats analysis [MLVA] using nine loci). Very high concordance was found between the three methods. Although amounts of nucleotide variation were low for the seven genes (pi ranging from 0.0038 to 0.0109), 3 to 12 alleles were distinguished, resulting in 31 sequence types. One sequence type (ST1) was frequent (17 strains), but most others were represented by a single strain. Attempts to subtype ST1 strains by MLVA, ribotyping, clustered regularly interspaced short palindromic repeat characterization, and single nucleotide repeat variation were unsuccessful. We found clear evidence for homologous recombination during the diversification of L. casei clones, including a putative intragenic import of DNA into one strain. Nucleotides were estimated to change four times more frequently by recombination than by mutation. However, statistical congruence between individual gene trees was retained, indicating that recombination is not frequent enough to disrupt the phylogenetic signal. The developed multilocus sequence typing scheme should be useful for future studies of L. casei strain diversity and evolution.