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[Interaction between lactobacilli and the intestinal mucosa].

The study of the distribution of lactobacilli in the lumen and on the mucosa of the intestine of CBA mice, as well as the interaction of lactobacilli with the mucosa under normal conditions and in experimental postradiation dysbacteriosis, showed that under normal conditions lactobacilli could be found practically in all portions of the intestine of the mice both in the lumen and on the mucosa. Lactobacilli were closely associated with the mucosa, and this association was not affected by the treatment of the mucosa with trypsin and detergents. In postradiation dysbacteriosis the association of lactobacilli with the mucosa became weaker; as a result, the number of bacteria associated with the mucosa decreased 10 times. Exogenic streptomycin-resistant lactic acid bacteria, subjected to multiple passages through the organism of the animals, could take on the intestinal mucosa.

Animals↗

Stimulation of cell defense mechanism of bovine endometrium by temporal colonization with selected strains of lactobacilli.

The aim of the experiments was to assess the response to topical administration of selected live strains of lactobacilli of the cells responsible for the resistance of bovine endometrium. Experimental cows (n = 8) at 8 to 12 days of the estrous received one intrauterine dose of 20 ml of a suspension of lactobacilli in 1% glucose solution. Group I (n = 4) was treated with the strain Lactobacillus spp. G 013 (5.5 x 10(8) CFU/ml) and Group II (n = 4) with the strain Lactobacillus casei CCM 1753 (1.2 x 10(8) CFU/ml). Control cows (n = 4) received 20 ml of 1% glucose solution. Samples of endometrial tissue were obtained by biopsy or from slaughtered cows on post-treatment days 5 or 6 and/or 10 or 12. Colonization of the uterine cavity with lactobacilli for up to 12 days was confirmed by bacteriological examination and scanning electron microscopy. Highly significant increases (P < 0.01) were found in numbers of all cell types under study. The pronounced cellular infiltration of endometrium was mostly due to the accumulation of mononuclear cells, particularly lymphocytes forming often indistinctly demarcated lymphoid nodules. Also marked was the infiltration of mast cells and macrophages. The cellular infiltration of endometrium persisted still on post-treatment day 12. No signs of alteration of epithelial cells were observed. No principal differences in the effects on endometrium were found between the two lactobacilli strains. The proved stimulatory effect of lactobacilli on endometrial cell defense mechanisms demonstrated in our experiments and inhibitory effects of the former on the growth of pathogenic microorganisms are promising for practical application in the prevention and alternative therapy of bovine endometritis.

Animals↗

Do vaginal lactobacilli prevent preterm labour?

OBJECTIVE: To determine the prevalence of Lactobacillus spp. in vaginal flora during pregnancy and to assess the protective effects of lactobacilli against preterm labour. DESIGN: Cross-sectional analysis of Lactobacillus spp. in the vaginal flora of the pregnant coloured population of the Western Cape. PARTICIPANTS: A total of 480 consecutive pregnant women, aged 13-48 years, seen at their first visit to the Tygerberg Hospital antenatal clinic. MAIN OUTCOME MEASURES: Preterm labour, i.e. before 34 and 37 weeks' gestation, premature rupture of membranes, intra-uterine growth retardation and perinatal deaths. RESULTS: A total of 163 patients had negative cultures and 317 positive cultures for lactobacilli, aerobes or both. Delivery before 37 weeks occurred in 18% and 20% of the two groups, respectively. Lactobacillus only was cultured from 116 patients and Lactobacillus and/or other aerobes from 201 patients. Preterm labour occurred in 20% of the first group and in 19% of the second group. The perinatal outcome in patients from whom lactobacilli only were cultivated did not differ from patients from whom other aerobes and lactobacilli or other aerobes only were cultured. CONCLUSION: In patients at high risk for preterm labour, the presence of lactobacilli in the vagina does not seem to play a protective role.

Adolescent↗

Inhibition of Salmonella typhimurium attachment to chicken cecal mucus by intestinal isolates of Enterobacteriaceae and lactobacilli.

The ability of selected strains of Enterobacteriaceae or lactobacilli isolated from the intestines of adult chickens to inhibit in vitro attachment of Salmonella typhimurium 3333/O to cecal mucus in the presence or absence of D-mannose was determined. Attachment in the absence of mannose was reduced by prior exposure of mucus to cultures of two isolates of Enterobacteriaceae, an Escherichia coli and a Hafnia alvei strain, but not to a third isolate, an Enterobacter agglomerans strain. Attachment of S. typhimurium was not inhibited when mannose was present in the blocking or attachment step. Formation of fimbriae by the two inhibitory Enterobacteriaceae strains and the S. typhimurium strain, as indicated by titers of mannose-sensitive hemagglutination of guinea pig erythrocytes was optimal in Z biphasic medium (consisting of tryptone, yeast extract, dextrose, and NaCl) incubated anaerobically at 42 C. Fimbriae of each of three strains prepared from these cultures also inhibited attachment. These are characteristics consistent with attachment and inhibition of attachment mediated by a mannose-sensitive adhesin associated with type 1 fimbriae on bacterial cells of Enterobacteriaceae strains. Attachment in the presence of mannose was significantly reduced by prior exposure of mucus to cultures of a Lactobacillus salivarius strain and a Lactobacillus delbrueckii delbrueckii strain but not to a strain of Lactobacillus for which the species had not been determined. Washed cells or spent culture supernatant fluid from brain-heart infusion broth, Z broth, or Z biphasic cultures of the inhibitory strains of lactobacilli incubated at 37 or 42 C inhibited this form of attachment. Of 27 intestinal isolates of Enterobacteriaceae and 21 of lactobacilli, the lactobacilli strains were generally more hydrophobic than the Enterobacteriaceae as determined by adherence to hexadecane. The lactobacilli isolates did not agglutinate guinea pig erythrocytes. The data suggest more than one mechanism for mediating attachment of inhibitory bacterial strains and for subsequent attachment of S. typhimurium.

Adhesins, Bacterial↗

In vitro studies of porcine lactobacilli for possible probiotic use.

Fourteen strains of lactobacilli isolated from the gut of suckling pigs were identified and studied to determine their susceptibility to antimicrobial feed additives, acid tolerance, adherence to epithelial cells from the porcine intestine and antimicrobial activity. Four strains were identified as Lactobacillus casei subsp. casei, two strains as Lactobacillus rhamnosus as well as Lactobacillus reuteri and three strains as Lactobacillus salivarius. The remaining strains could not be identified. Antimicrobial susceptibility tests showed susceptibility of the lactobacilli to many of the antimicrobial feed additives commonly used in pigs. Strains resistant to cyadox and nourseothricin occurred in high numbers. The tested lactobacilli were acid-resistant at pH 3. Thirteen strains of lactobacilli exhibited strong adherence to epithelial cells from the porcine intestine while one strain was classed as weakly-adherent. All lactobacilli showed inhibitory activity against indicator bacteria in the presence of glucose. Lactate and acetate were the principal final products of glucose fermentation in all strains. Only three strains produced H2O2 in detectable amounts.

Animals↗

Effect of an enzymatic rinse on salivary levels of Streptococcus mutans and lactobacilli in periodontally treated patients.

Root surface caries is prevalent in patients with both treated and untreated periodontal disease. The major etiologic factor has been identified as microbial plaque. In periodontally treated patients, significantly higher root caries prevalence and incidence have been found in patients with high levels of Streptococcus mutans and Lactobacilli in saliva. Reducing the levels of S. mutans and Lactobacilli in saliva may lower the risk of root caries development. The purpose of this investigation was to determine the effect of an oral enzymatic rinse on the salivary counts of S. mutans and Lactobacilli in periodontally treated patients. Fifteen adult subjects participated in a double-blind, cross-over designed clinical trial. Each subject had previously undergone comprehensive periodontal therapy and had been maintained on a regular program of supportive periodontal therapy. Paraffin-stimulated whole saliva was collected from each participant. Each subject was then randomly given either the enzymatic rinse product or a control rinse and instructed to rinse with one tablespoonful twice a day for 2 weeks, after which saliva samples were taken. After a washout period, salivary samples were again taken, and the subjects received the alternate rinse product. Two weeks later, final salivary samples were taken. The salivary samples were serially diluted and incubated aerobically on selective culture media. S. mutans and Lactobacilli were counted on the basis of colonial morphology. Pretreatment and posttreatment salivary counts of S. mutans and Lactobacilli were analyzed using the Wilcoxon matched-pairs signed-rank test at the 5% level of significance. Analysis of data revealed that neither the test nor the control rinse significantly lowered salivary counts of either species in the sample population.

Adult↗

Molecular identification of vaginal lactobacilli isolated from Bulgarian women.

Lactobacilli play an important role in maintaining the vaginal health of women. The development of suitable bacterial replacement therapies for the treatment of vaginosis requires knowledge of the vaginal lactobacilli species representation. The aim of this study was to identify at the species level vaginal Lactobacillus isolates obtained from Bulgarian women in childbearing age by using different molecular methods. Twenty-two strains of lactobacilli isolated from vaginal samples were identified and grouped according to their genetic relatedness. A combined approach, which included amplified ribosomal DNA restriction analysis (ARDRA), ribotyping and polymerase chain reaction (PCR) with species-specific oligonucleotide primers was applied. All vaginal isolates were grouped into 5 clusters in comparison with a set of 21 reference strains based on the initial ARDRA results, which was then confirmed by ribotyping. Finally, the strains were subjected to PCR analysis with eight different species-specific primer pairs, which allowed most of them to be classified as belonging to one of the following species: Lactobacillus crispatus, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus helveticus and Lactobacillus plantarum. In conclusion, this study suggests that the most straightforward identification strategy for vaginal lactobacilli would be grouping by ARDRA or ribotyping, followed by PCR specific primers identification at species level.

Adolescent↗

Use of MRSD medium and the hydrophobic grid membrane filter technique to differentiate between pediococci and lactobacilli in fermented meat and starter cultures.

Modifications of MRS medium were made by incorporation of 0.1 M L-arginine-HCl, 0.0025% phenol red, 100 IU polymyxin B sulfate, by deletion of meat extract, use of only 1.2% (w/v) glucose and increase of Mn2+ to 1000 ppm. In addition, adoption of the hydrophobic grid membrane filter (HGMF) system with 0.025% Fast Green FCF dye and adjustment of the agar medium to pH 5.5 gave MRSD (differential) medium. Incubation at 25 degrees C anaerobically under N2 or CO2 followed by a post-growth staining procedure involving use of 0.4% (w/v) bromocresol purple yielded conditions under which pediococci colonies were blue whereas homo- and heterofermentative lactobacilli were green in color. Under these conditions, 7 pediococci, 16 lactobacilli, and 18 commercial meat starter cultures were successfully analyzed by plate count to yield a differential assessment of the lactobacilli and pediococci present without interference from the 9 other genera tested. Streptococcus lactis and Leuconostoc spp. produced blue and green colonies, respectively, at 25 degrees C which might interference but these organisms are not present in significant numbers in fermented meats. Pediococcus parvulus and Streptococcus faecalis produced green and blue colonies, respectively, but their very poor growth at 25 degrees C prevented their interference. Use of the developed MRSD medium was described for enumeration of both pediococci and lactobacilli in starter cultures and in fermenting dry sausages to enable documentation of starter culture performance.

Arginine↗

Bacteriophages of lactobacilli.

Lactobacilli are members of the bacterial flora of lactic starter cultures used to generate lactic acid fermentation in a number of animal or plant products used as human or animals foods. They can be affected by phage outbreaks, which can result in faulty and depreciated products. Two groups of phages specific of Lactobacillus casei have been thoroughly studied. 1. The first group is represented by phage PL-1. This phage behaves as lytic in its usual host L. casei ATCC 27092, but can lysogenize another strain, L. casei ATCC 334. Bacterial receptors of this phage are located in a cell-wall polysaccharide and rhamnose is the main component of the receptors. Ca2+ and adenosine triphosphate (ATP) are indispensable to ensure the injection of the phage DNA into the bacterial cell. The phage DNA is double-stranded, mostly linear, but with cohesive ends which enables it to be circularized. The vegetative growth of PL-1 proceeds according to the classical mode. Cell lysis is produced by an N-acetyl-muramidase at the end of vegetative growth. 2. The second group is represented by the temperate phage phi FSW of L. casei ATCC27139. It has been shown how virulent phages originate from this temperate phage in Japanese dairy plants. The lysogenic state of phi FSW can be altered either by point mutations or by the insertion of a mobile genetic element called ISL 1, which comes from the bacterial chromosome. This is the first transposable element that has been described in lactobacilli. Lysogeny appears to be widespread among lactobacilli since one study showed that 27% of 148 strains studied, representing 15 species, produced phage particles after induction by mitomycin C. Similarly, 23 out of 30 strains of Lactobacillus salivarius are lysogenic and produce, after induction by mitomycin C, temperate phages, killer particles, or defective phages. Temperate phages have also been found in 10 out of 105 strains of Lactobacillus bulgaricus or Lactobacillus lactis after induction by mitomycin C. Phages so far studied of the latter 2 and closely related lactobacilli, either temperate or isolated as lytic, may be divided into 4 unrelated groups called a, b, c and d. Most of these phages are found in group a and an unquestionable relationship has already been shown between lytic phages and temperate phages that belong to this group. Lytic phage LL-H of L. lactis LL 23, isolated in Finland, is one of the most representative of those of group a and has been extensively studied on the molecular level.

Bacteriophages↗

Prebiotic effects of oligosaccharides on selected vaginal lactobacilli and pathogenic microorganisms.

The purpose of this study was to select endogenous human vaginal lactobacilli strains on the basis of the main probiotic properties observed in the vaginal environment in order to use them for the evaluation of the potential prebiotic properties of oligosaccharides. From vaginal samples of 50 women with a normal flora, 17 lactobacilli strains were first isolated because of their high level of hydrogen peroxide production. Then six strains were selected mainly for their ability (i) to adhere to vaginal cells, (ii) to produce compounds in sufficient amount, such as lactic acid, having an inhibitory action on pathogens, and less importantly, (iii) to demonstrate arginine deiminase activity. These six strains were found to belong to three distinct species: Lactobacillus crispatus, L. jensenii and L. vaginalis. One strain of each species was chosen as a potential vaginal probiotic strain with regard to our criteria. These three strains were then used to evaluate the prebiotic properties of different oligosaccharide series: two fructooligosaccharide series (FOS Actilight and FOS Raftilose) and two glucooligosaccharide series varying by their osidic linkages (alpha-1,6/alpha-1,4 GOS and alpha-1,2/alpha-1,6/alpha-1,4 GOS). The test was based on the ability of the oligosaccharides to promote the growth of the three beneficial strains selected but not of pathogenic microorganisms often encountered in urogenital infections such as Candida albicans, Escherichia coli and Gardnerella vaginalis. Oligosaccharide hydrolysis was followed by HPLC analysis. This revealed that two oligosaccharide series (FOS Actilight DP3 and all alpha-1,6/alpha-1,4 GOS DP > or = 4) were used only by the lactobacilli strains, the pathogenic microorganisms being unable to metabolise them. The selected lactobacilli and oligosaccharides are good candidates for incorporation in a formula to prevent vaginal infections.

Journal Article↗

Numbers and strains of lactobacilli in some probiotic products.

The numbers and types of Lactobacillus strains in probiotic feed or food products for sale in Europe were compared with the information stated on the product labels. The labels of all 10 products gave information on the genus and species of Lactobacillus, and seven gave information on the numbers of a specified strain(s). Lactobacilli were recovered using a recently validated method, developed as part of the European Community Project SMT4 CT98-2235 for the official control of probiotic microorganisms used as feed additives. Polymerase chain reaction (PCR)-based methods were used to assess the accuracy of labelling with regard to genus and species, and pulsed-field gel electrophoresis (PFGE) was used to identify strains. Five products were mislabelled with respect to the numbers and three with respect to species of lactobacilli. In four cases, the specified strains were not detected. Four fermented milks sold under three trademarks contained the same strain, which was named differently on each label. As safety and functionality of probiotics are strain dependent, these results demonstrated the need to control lactobacilli present in commercially probiotic human food products, not only at the species but also at the strain level, to ensure their quality and protect the consumer. The usefulness of the official methods developed for animal feed additives to identify lactobacilli in probiotic foods for humans was demonstrated.

Colony Count, Microbial↗

Hydrogen peroxide-producing Lactobacilli in the vaginal flora of pregnant women with preterm labor with intact membranes.

OBJECTIVES: To investigate the role of vaginal infection in preterm delivery, we studied characteristics of vaginal discharge related to hydrogen peroxide-producing Lactobacilli. METHODS: Vaginal specimens were obtained from 66 women with normal pregnancy and 30 women with preterm labor with intact membranes. pH, leukocyte counts on wet smear, and scores by Nugent criteria on Gram stain were measured. Lactobacilli were tested for their production of hydrogen peroxide. RESULTS: Leukocyte levels in wet smears and Nugent scores of Gram-stained smear of women with preterm labor with intact membranes were significantly higher than those of normal pregnant women (P<0.01, P<0.05). Hydrogen peroxide-producing Lactobacilli levels in the vaginal flora of women with preterm labor with intact membranes were significantly lower (P<0.01). CONCLUSION: Distribution of hydrogen peroxide-producing Lactobacilli in vaginal flora as defense factors for infection may have an important role in the pathophysiology of preterm labor.

Adult↗

Quantification of total bacteria, enterobacteria and lactobacilli populations in pig digesta by real-time PCR.

Jejunum digesta samples were taken from weaning pigs in order to evaluate real-time PCR (qPCR) as a method for quantifying pig gut bacteria. Total bacteria, lactobacilli and enterobacteria were quantified by qPCR and the results were compared with those obtained with traditional methods: 4',6-diamidino-2-phenylindole (DAPI staining) for total bacteria, selective culture for lactobacilli and enterobacteria. Real-time PCR showed higher values in terms of 16S rRNA gene copies than DAPI counts or CFU. Despite the differences, the lactobacilli:enterobacteria ratio was similar between methods (2.5 +/- 0.58 for qPCR and 3.1 +/- 0.71 for selective culture, P = 0.39). Possible reasons for the higher PCR counts are discussed considering both an overestimation with PCR by quantification of dead bacteria or free DNA and also an underestimation with conventional methods. Inherent differences in the pre-treatment of the samples could partially explain the discrepancies observed. Regardless of the numerical differences between methods, values obtained by qPCR and traditional methods showed a significant correlation for lactobacilli and total bacteria. In the light of these results, real-time PCR seems a valid method to quantify microbial shifts in the gastrointestinal tract.

Animals↗

Citrate utilization by homo- and heterofermentative lactobacilli.

Citrate utilization by several homo- and heterofermentative lactobacilli was determined in Kempler and McKay and in calcium citrate media. The last medium with glucose permitted best to distinguish citrate-fermenting lactobacilli. Lactobacillus rhamnosus ATCC 11443, Lactobacillus zeae ATCC 15820 and Lactobacillus plantarum ATCC 8014 used citrate as sole energy source, whereas in the other strains, glucose and citrate were cometabolized. Some lactobacilli strains produced aroma compounds from citrate. Citrate transport experiments suggested that all strains studied presented a citrate transport system inducible by citrate. The levels of induction were variable between several strains. Dot blot experiment showed that lactobacilli do not present an equivalent plasmid coding for citrate permease.

Acetoin↗

HLA class II alleles and salivary numbers of mutans streptococci and lactobacilli among young adults in Japan.

To investigate the genetic predisposition toward the accumulation of oral microorganisms, the association between the alleles of HLA-DQA1, -DQB1 and -DRB1 and the salivary numbers of mutans streptococci and lactobacilli were examined in 102 young adults in Japan. HLA-DQA1*0102, DQB1*0604, DRB1*0802, and DRB1*1302 were weakly associated with the numbers of lactobacilli in saliva samples (P<0.05). HLA-DQB1*0601 was associated with the salivary numbers of mutans streptococci (P<0.01). Two HLA class II haplotypes, HLA-[DRB1*0802-DQA1*0301-DQB1*0302] and HLA-[DRB1*1302-DQA1*0102-DQB1*0604], were also weakly associated with the salivary numbers of lactobacilli (P<0.05). These observations suggest that HLA class II alleles may be related to the salivary populations of oral microorganisms, such as mutans streptococci and lactobacilli.

Adolescent↗

Decreasing prevalence of salivary lactobacilli in Swedish schoolchildren 1987-1998.

The prevalence and number of salivary lactobacilli was determined in all schoolchildren starting the 7th grade (12-13 yr; n = 1,578) in comprehensive school located in Kumla, Sweden between 1987 and 1998. Whole saliva samples were collected and transferred to dip-slides (Dentocult-LB) and incubated in room temperature for 7 d. Caries data were collected from the dental records and from bitewing radiographs. Both the number of lactobacilli and the prevalence of manifest caries and restorations exhibited a decreasing tendency and were significantly lower in 1998 than in 1987. The mean caries prevalence (DFS) declined from 5.2 to 1.8. In 1987, 45% of the children harboured high or very high lactobacilli counts ( > or =10(5) CFU/ml) in their saliva compared to approximately 10-15% at the later examinations. The proportion of children with no detectable counts varied between 4-29% during the study period, while children with low counts (10(3)-10(4) CFU/ml saliva) constituted a clear majority at the recent samplings. With such low levels in the population, the use of lactobacilli counts as a didactic tool in dietary counselling must be called into question.

Adolescent↗

In situ delivery of passive immunity by lactobacilli producing single-chain antibodies.

Lactobacilli have previously been used to deliver vaccine components for active immunization in vivo. Vectors encoding a single-chain Fv (scFv) antibody fragment, which recognizes the streptococcal antigen I/II (SAI/II) adhesion molecule of Streptococcus mutans, were constructed and expressed in Lactobacillus zeae (American Type Culture Collection (ATCC) 393). The scFv antibody fragments secreted into the supernatant or expressed on the surface of the bacteria showed binding activity against SAI/II in enzyme-linked immunosorbent assay (ELISA), and surface scFv-expressing lactobacilli agglutinated SAI/II-expressing S. mutans in vitro without affecting the corresponding SAI/II knockout strain. Lactobacilli expressing the scFv fragment fused to an E-tag were visualized by scanning electron microscopy (SEM) using beads coated with a monoclonal anti-E-tag antibody, and they bound directly to beads coated with SAI/II. After administration of scFv-expressing bacteria to a rat model of dental caries development, S. mutans bacteria counts and caries scores were markedly reduced. As lactobacilli are generally regarded as safe (GRAS) microorganisms, this approach may be of considerable commercial interest for in vivo immunotherapy.

Animals↗

Pre- and post-treatment levels of salivary mutans streptococci and lactobacilli in pre-school children.

OBJECTIVES: To examine the effect of operative and restorative treatment of dental caries on the levels of caries associated microorganisms in saliva and to relate alterations to the type and extent of treatment. DESIGN: Longitudinal. SETTING: Paediatric Dentistry Department at a central hospital in Sweden. SUBJECTS AND METHODS: One hundred and eight pre-school children with severe dental caries scheduled for treatment under general anaesthesia. Chair-side tests were used to estimate the levels of salivary mutans streptococci, lactobacilli and buffer capacity before the surgery and at recall appointments 1 and 6 months after treatment. Caries were assessed according to WHO guidelines and the number of extracted teeth and filled surfaces during surgery were recorded. RESULTS: The results demonstrate that the post-treatment levels of mutans streptococci and lactobacilli were significantly reduced (P < 0.001) compared to pretreatment levels. Lactobacilli levels were more dramatically reduced than mutans streptococci. The reduction of mutans streptococci was positively correlated to the number of extracted teeth (P < 0.01), but not to the number of restored or ground surfaces. Lactobacilli reduction was not significantly related to the type of treatment. CONCLUSION: The findings suggest that extensive operative and restorative dental care effectively reduces the levels of caries associated with microorganisms during a period of at least 6 months.

Anesthesia, Dental↗