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Identification of Lactobacillus crispatus by polymerase chain reaction targeting S-layer protein gene.

AIMS: This study aimed to develop a polymerase chain reaction (PCR) method to identify Lactobacillus crispatus. METHODS AND RESULTS: A primer set (CbsA2F-CbsA2R) for amplifying conserved regions of S-layer genes was designed to identify Lact. crispatus and the specificity of this set was compared with that of another primer set (Cri 16SI-Cri 16SII) which has been reported as a species-specific primer set targeting the 16S rRNA gene. Among species in the Lact. acidophilus A1-A4 groups, when KOD polymerase was used for amplification, the primer set CbsA2F-CbsA2R gave PCR products with Lact. crispatus strains only. However, when Taq polymerase was used, this primer set gave products with one Lact. amylovorus strain as well as with Lact. crispatus strains. The primer set Cri 16SI-Cri 16SII gave PCR products with Lact. crispatus strains and two Lact. acidophilus strains, regardless of whether the polymerase used was KOD or Taq. CONCLUSIONS: A PCR targeting the S-layer gene and amplified with KOD polymerase can identify Lact. crispatus accurately and rapidly. SIGNIFICANCE AND IMPACT OF THE STUDY: To the authors' knowledge, this is the first paper to provide a PCR method for the specific identification of Lact. crispatus.

Bacterial Outer Membrane Proteins↗

[Phosphoenolpyruvate:hexose phosphotransferase systems in Lactobacillus species].

The substrate range of phosphoenolpyruvate:hexose phosphotransferase systems (hexose-PTSs) in Lactobacillus casei subsp. casei LAC3 and L. acidophilus LAC5 was examined. Strain LAC3 demonstrated PTS activities for glucose (Glc), mannose (Man), glucosamine (GcN), 2-deoxyglucose (2DG) and fructose (Fru), while strain LAC5 showed the activities only for Man and Fru. These activities were all constitutive. Growth of both strains was strongly inhibited by 2DG. 2DG-resistant mutants DG329 and DG504 were isolated, respectively, from strains LAC3 and LAC5. Mutant DG329 grown on Glc was defective in all the above-described activities observed with strain LAC3, whereas no defect in PTS activities was found in mutant DG504. Mutant DG329, however, showed some inducible activities for Man and Fru when grown on Man, Fru or Scr. These results strongly suggest that strain LAC3 has inducible PTS(s) specific for Man and/or Fru besides the well-known, broadly specific, constitutive Man-PTS, and also that strain LAC5 lacks the Man-PTS, but has other constitutive PTS(s) specific for Man and/or Fru. L. fermentum LAC12 had the Man-PTS as reported previously (Nagasaki et al., 1992), but had no inducible activities like those found in strain LAC3.

Carbohydrate Metabolism↗

Ecological study of Streptococcus mutans, Streptococcus sobrinus and Lactobacillus spp. at sub-sites from approximal dental plaque from children.

Previous immunofluorescence (IF) studies have indicated that Streptococcus mutans may preferentially colonise specific sub-sites within approximal plaque. The present study aimed to extend these observations to other mutans streptococci and lactobacilli in such gingival margin plaque. Two hundred and seventy approximal plaque samples were taken from 90 teeth (3 from each tooth) in 64 children; three gingival margin sub-sites in relation to the contact area: away from (A), to the side of(S) and below (B) the contact area. Samples were processed by indirect IF using high-titred polyclonal anti-S. mutans 'c', anti-S. sobrinus 'd', anti-L. casei and anti-L. acidophilus antisera. An overall positive association was found between S. mutans 'c' and S. sobrinus 'd' (p < 0.001). Significant differences (p < 0.1) were found between the proportional counts at each sub-site for S. mutans 'c': A = 39%, S = 51% and B = 70%, and for S. sobrinus 'd' 21, 33 and 49%. Mutans streptococci (MS) appeared to preferentially colonise the sub-site below the contact area (B = 81%), compared with sub-sites A and S (48 and 62%, respectively). S. mutans 'c' and S. sobrinus 'd' were detected together at subsites A = 12%, S = 22%, and B = 38%, with proportional counts at B sites being higher than those at A (B > A, p < 0.01, and B > S, p < 0.05). Lactobacillus spp. were isolated rarely, and were usually found together with MS. There was a positive relationship between the presence of lactobacilli or MS and caries (white spot lesions only), although these species could frequently be isolated from noncarious sites. The presence of both S. mutans 'c' and S. sobrinus 'd' were strongly correlated with early caries lesions. In addition, this study confirmed the variation in the microflora at different sub-sites within approximal dental plaque.

Antibodies, Bacterial↗

Fermented products with probiotic qualities.

For several centuries, fermented products derived from plant or animal materials have been an acceptable and essential part of the diet in most parts of the world. Health benefits have also often been associated with them. Probiotics can be defined as fermented food containing specific live microorganisms or a live microbial food or feed supplement, which beneficially effects the human or the host animal by improving its intestinal microbial balance. Nearly all probiotics currently on the market contain Lactobacilli, Streptococci, Enterococci or Bifidobacteria. In contrast to Japan, where freeze-dried microorganisms are consumed by a substantial part of the human population, in Europe, probiotic action towards humans are only claimed for certain fermented dairy products (e.g. yoghurts). Those species that have been extensively studied so far, with several experimental trials on man, are the two yoghurt bacteria Streptocaccus thermophilus and Lactobacillus bulgaricus, L. casei and Bifidobacteria. L. acidophilus has also received important scientific interest, however, only a few human studies have been carried out. From the technological point of view a good probiotic should be stable and viable for long periods under storage, should be able to survive the low pH levels of the stomach, be able to colonise the epithelium of the gastro-intestinal tract of the host, should not be pathogenic and, last but not least, must be capable of exerting a growth promoting effect or a resistance to infectious diseases. The beneficial effects of probiotics are mainly contributed to a direct antagonistic effect against specific groups of microorganisms (Enteropathogenes), by an effect on the metabolism in the gut or by a stimulation of systemic or mucosal immunity. We will present major proven health benefits of milks fermented with those bacterial species and discuss, where possible, the impact of the specific selection and utilisation of particular strains.

Journal Article↗

Detection of Leuconostoc strains at a meat processing plant using polymerase chain reaction.

To simplify the labor-intensive conventional routine testing of samples to detect Leuconostoc at a meat processing plant, we developed polymerase chain reaction (PCR) primers specific for Leuconostoc from 16S rRNA gene sequences. These primers did not detect other common lactic acid bacteria such as Lactobacillus plantarum, Lact. sake, Lact. fermentum, Lact. acidophilus and Weissella viridescens. PCR with this primer detected all Leuconostoc species tested (Leu. mesenteroides subsp. mesenteroides, Leu. pseudomesenteroides, Leu. carnosum, Leu. lactic, Leu. citreum, Leu. amelibiosum, Leu. gelidum), except for Leu. fallax, and no other lactic acid bacteria on agarose gel electrophoresis. The method could identify areas contaminated with Leuconostoc in a large-scale industrial meat processing plant. Of 69 samples analyzed, 34 were positive for Leuconostoc according to the conventional culture method (isolation of LAB producing dextran) and PCR, whereas 29 were negative according to both. Six samples were culture-negative but positive by PCR. No false negative results were generated by PCR. The method is rapid and simple, is useful for routinely monitoring areas contaminated with Leuconostoc in meat processing plants, and could help to prevent the spoilage of meat products.

DNA Primers↗

Introduction and reisolation of selected gram-negative bacteria from fermented edible wastes.

A Lactobacillus fermentation process, using edible food wastes, was tested for its ability to eliminate selected bacterial pathogens. This fermentation process converts food wastes into a feed ingredient for animal consumption. Six gram-negative bacterial pathogens of potential zoonotic importance were tested. These experimental organisms were: Salmonella enteritidis serovar typhimurium, S enteritidis serovar anatum, S cholerae-suis, Yersinia enterocolitica, Y pseudotuberculosis, and Pasteurella multocida. Each organism was introduced into ground waste that had been previously inoculated with L acidophilus, and was mixed. This mixture was divided among 8 containers, and was incubated in duplicate at 5 C, 10 C, 20 C, and 30 C for 96 hours. The temperature of the reactant containers, reduction-oxidation potential, and pH were monitored. Waste samples were obtained initially and subsequently at 24-hour periods for 96 hours. Qualitative and quantitative recovery attempts from each sample were made for the introduced gram-negative bacteria. Pasteurella multocida and the S enteritidis serovars typhimurium and anatum survived the fermentation at 5 C and 10 C, but were killed after 48 hours at 20 C and 30 C. Salmonella cholerae-suis survived at 5 C, but was destroyed by 72 hours at the remaining temperatures. Yersinia enterocolitica was viable through 70 hours, but was killed by 96 hours. Yersinia pseudotuberculosis was not reisolated at any temperature.

Animal Feed↗

[The range of antagonistic effects of Lactobacillus bacterial strains on etiologic agents of bacterial vaginosis].

Bacterial vaginosis is caused by uncontrolled sequential overgrowth of some anaerobic bacteria: Gardnerella vaginalis, Prevotella bivia, Bacteroides spp., Peptostreptococcus spp., Mobiluncus sp. usually occurring in stable numbers in the bacterial flora of healthy women. On the other hand, different species of bacteria belonging to the genus Lactobacillus, most frequently L. plantarum, L. rhamnosus and L. acidophilus, form a group of aerobic bacteria dominating in the same environment. The diversity and density of their populations depend on the age and health conditions. Thanks to their antagonistic and adherence properties bacteria of the genus Lactobacillus can maintain a positive balance role in this ecosystem. The aim of this study was to assess the antagonistic properties of Lactobacillus strains isolated from the vagina of healthy women against most common agents of bacterial vaginosis. It was found that nearly all of the tested Lactobacillus strains exerted distinct antagonistic activity against anaerobic bacteria: Gardnerella vaginalis, Prevotella bivia and Peptostreptococcus anaerobius and quite a number also against Gram-negative rods, while only some of them were able to inhibit Gram-positive aerobic cocci as Enterococcus faecalis or Staphylococcus aureus.

Bacteria, Anaerobic↗

Diversity of the genus Lactobacillus revealed by comparative genomics of five species.

The genus Lactobacillus contains over 80 recognized species, and is characterized by a high level of diversity, reflected in its complex phylogeny. The authors' recent determination of the genome sequence of Lactobacillus salivarius means that five complete genomes of Lactobacillus species are available for comparative genomics: L. salivarius, L. plantarum, L. acidophilus, L. johnsonii and L. sakei. This paper now shows that there is no extensive synteny of the genome sequences of these five lactobacilli. Phylogeny based on whole-genome alignments suggested that L. salivarius was closer to L. plantarum than to L. sakei, which was closest to Enterococcus faecalis, in contrast to 16S rRNA gene relatedness. A total of 593 orthologues common to all five species were identified. Species relatedness based on this protein set was largely concordant with genome synteny-based relatedness. A Lactobacillus supertree, combining individual phylogenetic trees from each of 354 core proteins, had four main branches, comprising L. salivarius-L. plantarum; L. sakei; E. faecalis; and L. acidophilus-L. johnsonii. The extreme divergence of the Lactobacillus genomes analysed supports the recognition of new subgeneric divisions.

Genes, Bacterial↗

Comparison of contact angles and adhesion to hexadecane of urogenital, dairy, and poultry lactobacilli: effect of serial culture passages.

The aim of this study was to examine the hydrophobicities of 23 urogenital, dairy, poultry, and American Type Culture Collection isolates of lactobacilli and to determine the effect on hydrophobicity of serially passaging the strains in liquid medium. To this end, strains were grown after isolation and identification and then serially passaged up to 20 times. Hydrophobicity was assessed through contact angle measurements on lawns of cells by using water, formamide, methylene iodide, 1-bromonaphthalene, and hexadecane as wetting agents and through measurement of their partitioning in a hexadecane-water system. The hydrophobicities of these strains varied widely, with Lactobacillus casei strains being predominantly hydrophilic and L. acidophilus strains being mostly hydrophobic. For some isolates, serial passaging was accompanied by a clear loss of hydrophobic surface properties, whereas for other strains, cultures became heterogeneous in that some cells had already lost their hydrophobic surface properties while others were still hydrophobic. Adhesion of this collection of lactobacilli to hexadecane droplets in microbial adhesion to hexadecane (MATH) tests was driven by their aversion to water rather than by their affinity for hexadecane, as concluded from the fact that hexadecane contact angles were zero for all strains. Furthermore, adhesion of the lactobacilli to hexadecane in MATH tests occurred only when the water contact angle on the cells was above 60 degrees.

Adult↗

Reverse effect of gram-positive bacteria vs. gram-negative bacteria on adjuvant-induced arthritis in germfree rats.

Germfree (GF) F344 rats developed severe adjuvant-induced arthritis with a 100% incidence after a single intradermal injection of heat-killed Mycobacterium bovis (BCG). Specific pathogene-free (SPF) rats developed less severe arthritis with a lower incidence. The rats colonized with Escherichia coli or Bacteroides developed mild disease comparable to that in SPF rats. The rats colonized with Bifidobacterium, Propionibacterium acnes, Lactobacillus casei, L. fermentum, L. murini, and L. acidophilus developed more severe disease than that in GF rats. Furthermore, the rats colonized with a mixture of E. coli and the above lactobacilli developed very mild disease similar to that in SPF rats. These results suggest that gram-negative bacteria, such as E. coli and Bacteroides, may suppress the disease, possibly through their lipopolysaccharides, and may be responsible for the lower susceptibility of SPF rats; gram-positive bacteria, such as Bifidobacterium, P. acnes, and lactobacilli, may enhance the disease, possibly through their peptidoglycans; and E. coli may play a dominant role in modulating the development of adjuvant-induced arthritis.

Animals↗

Bacterial counts of intestinal Lactobacillus species in infants with colic.

Intestinal colonization by lactobacilli is suggested to be a prerequisite to normal mucosal immune functions. An inadequate level of lactobacilli may be involved in appearance of allergic disease of which, infantile colic, is often considered an early clinical manifestation. The aim of the study is to evaluate intestinal lactobacilli in breast-fed infants with infantile colic and healthy infants. Fifty-six breast-fed infants, aged 15-60 days were enrolled in the study and divided into two groups: colicky (30 cases) and healthy (26 cases) according to Wessel's criteria. Stool samples were collected, diluted and cultured on selective media. The colonies were counted, reported as colony forming unit (cfu) per gram of faeces and identified by biochemical methods. Different colonization patterns of lactobacilli were found among colicky and healthy infants. Lactobacillus brevis (4.34 x 10(8) cfu/g) and L. lactis lactis (2.51 x 10(7) cfu/g) were found only in colicky infants while L. acidophilus (2.41 x 10(7) cfu/g) was found only in healthy infants. Lactobacillus brevis and L. lactis lactis might be involved in the pathogenesis of infantile colic increasing meteorism and abdominal distension. Further studies are required to understand how the observed differences may be involved in the pathogenesis of this common disorder.

Breast Feeding↗

[Influence of selected Lactobacillus sp. on Clostridium difficile strains with different toxigenicity profile].

This study was performed for determination of antagonistic activity of Lactobacillus spp. (L. plantarum 2017405, L. rhamnosus GG, L. acidophilus DSM 21007 and L. fernmentumn 353) on Clostridiunl difficile strains belonging to different toxigenicity profiles. Forty strains C. difficile isolated from patients suffering from antibiotic associated diarrhea (AAD) were used. Among C. difficile strains 13 produced toxin A and B (A+B+CDT-), 14 produced only toxin B (AB'CDT), 9 produced toxins A and B and possessing of binary toxin genes (A+B+CDT-) and 4 were non-toxigenic (A-B-CDT-). We did not observe relationship between degree of antagonistic activity Lactobacillus spp. and profile of toxigenicity of C. difficile strains.

Antibiosis↗

The genome sequence of the probiotic intestinal bacterium Lactobacillus johnsonii NCC 533.

Lactobacillus johnsonii NCC 533 is a member of the acidophilus group of intestinal lactobacilli that has been extensively studied for their "probiotic" activities that include, pathogen inhibition, epithelial cell attachment, and immunomodulation. To gain insight into its physiology and identify genes potentially involved in interactions with the host, we sequenced and analyzed the 1.99-Mb genome of L. johnsonii NCC 533. Strikingly, the organism completely lacked genes encoding biosynthetic pathways for amino acids, purine nucleotides, and most cofactors. In apparent compensation, a remarkable number of uncommon and often duplicated amino acid permeases, peptidases, and phosphotransferase-type transporters were discovered, suggesting a strong dependency of NCC 533 on the host or other intestinal microbes to provide simple monomeric nutrients. Genome analysis also predicted an abundance (>12) of large and unusual cell-surface proteins, including fimbrial subunits, which may be involved in adhesion to glycoproteins or other components of mucin, a characteristic expected to affect persistence in the gastrointestinal tract (GIT). Three bile salt hydrolases and two bile acid transporters, proteins apparently critical for GIT survival, were also detected. In silico genome comparisons with the >95% complete genome sequence of the closely related Lactobacillus gasseri revealed extensive synteny punctuated by clear-cut insertions or deletions of single genes or operons. Many of these regions of difference appear to encode metabolic or structural components that could affect the organisms competitiveness or interactions with the GIT ecosystem.

Biological Transport↗

Molecular quantification of lactic acid bacteria in fermented milk products using real-time quantitative PCR.

Real-time quantitative PCR assays were developed for the absolute quantification of lactic acid bacteria (LAB) (Streptococcus thermophilus, Lactobacillus delbrueckii, L. casei, L. paracasei, L. rhamnosus, L. acidophilus and L. johnsonii) in fermented milk products. The results of molecular quantification and classic bacterial enumeration did not differ significantly with respect to S. thermophilus and the species of the L. casei group which were detected in the six commercial fermented products tested, thus showing that DNA extraction was efficient and that genomic DNA solutions were free of PCR inhibitors. For L. delbrueckii, the results of bacterial enumeration were generally lower by a factor 10 to 100 than those of PCR quantification, suggesting a loss of viability during storage of the dairy products at 1-8 degrees C for most of the strains in this species. Real-time quantitative assays enabled identification of the species of lactic acid bacterial strains initially present in commercial fermented milk products and their accurate quantification with a detection threshold of 10(3) cells per ml of product.

Base Sequence↗

Fermentation of aqueous plant seed extracts by lactic Acid bacteria.

The effects of lactic acid bacterial fermentation on chemical and physical changes in aqueous extracts of cowpea (Vigna unguiculata), peanut (Arachis hypogea), soybean (Glycine max), and sorghum (Sorghum vulgare) were studied. The bacteria investigated were Lactobacillus helveticus, L. delbrueckii, L. casei, L. bulgaricus, L. acidophilus, and Streptococcus thermophilus. Organisms were inoculated individually into all of the seed extracts; L. bulgaricus and S. thermophilus were also evaluated together as inocula for fermenting the legume extracts. During fermentation, bacterial population and changes in titratable acidity, pH, viscosity, and color were measured over a 72-h period at 37 degrees C. Maximum bacterial populations, titratable acidity, pH, and viscosity varied depending upon the type of extract and bacterial strain. The maximum population of each organism was influenced by fermentable carbohydrates, which, in turn, influenced acid production and change in pH. Change in viscosity was correlated with the amount of protein and titratable acidity of products. Color was affected by pasteurization treatment and fermentation as well as the source of extract. In the extracts inoculated simultaneously with L. bulgaricus and S. thermophilus, a synergistic effect resulted in increased bacterial populations, titratable acidity, and viscosity, and decreased pH in all the legume extracts when compared to the extracts fermented with either of these organisms individually. Fermented extracts offer potential as substitutes for cultured dairy products.

Journal Article↗

Lactic acid-mediated suppression of Helicobacter pylori by the oral administration of Lactobacillus salivarius as a probiotic in a gnotobiotic murine model.

OBJECTIVES: We examined whether or not the lactobacilli administered to treat Helicobacter pylori (H. pylori) infection can suppress the colonization of H. pylori, and we also sought to elucidate the mechanism of such suppression. METHODS: We used an in vitro culture system and an H. pylori-infected gnotobiotic murine model. RESULTS: Among the lactobacillus species examined in vitro, Lactobacillus salivarius (L. salivarius) but not L. casei or L. acidophilus proved to be capable of producing a high amount of lactic acid and thus completely inhibiting the growth of H. pylori in a mixed culture. The validity of L. salivarius as a probiotic to suppress H. pylori and thus reduce the inflammatory response was again confirmed in vivo by using an H. pylori-infected gnotobiotic murine model. CONCLUSION: Based on our findings, L. salivarius was found to be a potentially effective probiotic against H. pylori.

Animals↗

Lactic acid bacteria isolated from dairy products inhibit genotoxic effect of 4-nitroquinoline-1-oxide in SOS-chromotest.

Antigenotoxic activity against 4-nitroquinoline-1-oxide (4-NQO) of lactic acid bacteria isolated from commercial dairy products was studied using SOS-Chromotest. The supernatants from bacteria-genotoxin co-incubations in general exhibited a strong suppression on SOS-induction produced by 4-NQO on the tester organism Escherichia coli PQ37 (sfiA::lacZ). High genotoxicity inhibition (>75%) was found for 31/67 of the examined bacteria and the maximum values of some strains within the species were as follows: Lactobacillus casei, 99.1%; L. plantarum, 93.3%; L. rhamnosus, 93.4%; L. acidophilus, 90.9%; L. delbrueckii subsp. bulgaricus, 85.7% and Bifidobacterium bifidum, 89.6%; Strains with low antigenotoxicity (5-60%) were evidenced in both L. acidophilus and L. delbrueckii subsp. bulgaricus, whereas some inactive strains were found only in L. casei and L. delbrueckii subsp. bulgaricus. Cell exposure to 100 degrees C for 15 min prevented antigenotoxicity and no effect was evidenced for cell-free spent media. The active strains survived at 0.1 mM 4-NQO exposure and generally presented some relevant functional properties, such as tolerance to bile (0.5%) or acid environment (pH 2.0) and adherence to Caco-2 enterocytes. Antigenotoxicity was always associated with modification of the 4-NQO absorbance profile.

4-Nitroquinoline-1-oxide↗