Association study of FUT2 (rs601338) with celiac disease and inflammatory bowel disease in the Finnish population.

Homozygosity for a nonsense mutation in the fucosyltransferase 2 (FUT2) gene (rs601338G>A) leads to the absence of ABH blood groups (FUT2 non-secretor status) in body fluids. As the secretor status has been shown to be a major determinant for the gut microbial spectrum, assumed to be important in the gut immune homeostasis, we studied the association of rs601338-FUT2 with celiac disease (CelD) and inflammatory bowel disease (IBD) in the Finnish population. Rs601338 was genotyped in CelD (n = 909), dermatitis herpetiformis (DH) (n = 116), ulcerative colitis (UC) (n = 496) and Crohn's disease (CD) (n = 280) patients and healthy controls (n = 2738). CelD showed significant genotypic [P = 0.0074, odds ratio (OR): 1.28] and recessive (P = 0.015, OR: 1.28) association with the rs601338-AA genotype. This was also found in the combined CelD+DH dataset (genotype association: P = 0.0060, OR: 1.28; recessive association: P < 0.011, OR: 1.28). The A allele of rs601338 showed nominal association with dominant protection from UC (P = 0.044, OR: 0.82) and UC+CD (P = 0.035, OR: 0.84). The frequency of non-secretors (rs601338-GG) in controls, CelD, DH, UC and CD datasets was 14.7%, 18%, 18.1%, 14.3% and 16.1%, respectively. No association was evident in the DH or CD datasets alone. In conclusion, FUT2 non-secretor status is associated with CelD susceptibility and FUT2 secretor status may also play a role in IBD in the Finnish population.

Improving the storage stability of Bifidobacterium breve in low pH fruit juice.

Bifidobacterial food applications are limited since bifidobacteria are sensitive to e.g. acidic conditions prevalent in many food matrices. The aim of the present study was to investigate whether a low pH selection step alone or combined to UV mutagenesis could improve the viability of an acid sensitive Bifidobacterium strain, B. breve 99, in low pH food matrices. Furthermore, the potential of carriers and an oat fibre preparation to further improve the stability was studied. The best performing low pH tolerant variants in the present study were generated by UV-mutagenesis with 70-700µJ/cm(2) followed by incubation in growth medium at pH 4.5. The most promising variants regarding the low pH tolerance showed, in repeated tests with cells grown without pH control, about one Log-value better survival in pH 3.8 fruit juice after one week storage at 4°C compared to wild-type B. breve 99. Cells grown with pH control, PDX formulated and then frozen showed poorer viability in low pH fruit juice than cells grown with no pH control. For frozen concentrates pH 3.8 was too stressful and no or small differences between the variants and the wild-type strain were seen. The differences detected at pH 3.8 with the cells grown without pH control were also seen with the frozen concentrates at pH 4.5. Some improvement in the stability could be achieved by using a combination of trehalose, vitamin C and PDX as a freezing carrier material, whereas a significant improvement in the stability was seen when oat fibre was added into the fruit juice together with the frozen cells. Due to the initial very poor fruit juice tolerance of B. breve 99 the obtained improvement in the stability was not enough for commercial applications. However, the same methods could be applied to initially better performing strains to further improve their stability in the fruit juice.

Effect of the fermentation pH on the storage stability of Lactobacillus rhamnosus preparations and suitability of in vitro analyses of cell physiological functions to predict it.

AIMS: To investigate how cell physiological functions can predict the stability of freeze-dried probiotics. In addition, the effect of the fermentation pH on the stability of probiotics was investigated. METHODS AND RESULTS: Fermenter-grown (pH 5.8 or 5.0) Lactobacillus rhamnosus cells were freeze-dried and their survival was evaluated during storage at 37 degrees C, in apple juice and during acid [hydrochloric acid (HCl) and malic acid] and bile exposure. Cells grown at pH 5.0 were generally coping better with acid-stress than cells grown at pH 5.8. Cells were more sensitive to malic acid compared with HCl. Short-term stability results of Lact. rhamnosus cells in malic acid correlated well with the long-term stability results in apple juice, whereas the results of cell membrane integrity studies were in accordance with bile exposure results. CONCLUSIONS: Malic acid exposure can prove useful in evaluating the long-term stability of probiotic preparations in apple juice. Fermentation at reduced pH may ensure a better performance of Lact. rhamnosus cells during the subsequent acid-stress. SIGNIFICANCE AND IMPACT OF THE STUDY: The beneficial effect of lowered fermentation pH to Lact. rhamnosus stability during storage in apple juice and the usefulness of malic acid test in predicting the stability were shown.

Influence of oral doxycycline therapy on the diversity and antibiotic susceptibility of human intestinal bifidobacterial population.

AIM: To evaluate the influence of doxycycline therapy on the composition and antibiotic susceptibility of intestinal bifidobacteria. METHODS AND RESULTS: Faecal samples were collected from nine subjects receiving doxycycline therapy and ten control subjects, and analysed for bifidobacteria by culturing and PCR-DGGE (denaturing gradient gel electrophoresis). A marked decrease in the diversity (average number of amplicons detected by PCR-DGGE 0.8 in the antibiotic vs 4.3 in the control group) of Bifidobacterium populations was observed during doxycycline therapy. The proportion of a tetracycline-resistant bifidobacterial population was higher in the antibiotic group than in the control group (83%vs 26%). Based on the tet gene PCR, resistance could be associated with the presence of tet(W). In two subjects, strains representing highly similar genetic fingerprints but different tetracycline susceptibilities were detected. A mutation causing lack of functionality in the tet(W) was observed in one of the susceptible strains. CONCLUSIONS: Doxycycline therapy had a drastic effect on the diversity and tetracycline susceptibility of intestinal Bifidobacterium populations. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of broad-spectrum antibiotics increased the pool of tetracycline-resistant commensal bacteria in the intestine. The detection of resistance genes alone is not sufficient for the evaluation of bacterial antibiotic resistance.

Comparison of three test media for antimicrobial susceptibility testing of bifidobacteria using the Etest method.

The performance of three test media for antimicrobial susceptibility testing of bifidobacteria using the Etest was compared. All Bifidobacterium strains (n=42) displayed good growth on trypticase-phytone-yeast extract agar (TPY). Most strains showed good growth on lactic acid bacteria susceptibility test medium supplemented with cysteine (LSM+cys); Bifidobacterium bifidum showed moderate growth. Growth of seven strains was inadequate on Brucella blood agar (BRU) and an additional eight strains showed moderate growth. The minimum inhibitory concentrations (MICs) for tetracycline were highest on BRU and lowest on LSM+cys (agreement 57%), whereas the MICs for streptomycin were lowest on BRU and highest on TPY (agreement 40%). Occasional mismatches (agreement 71-91%) between the test media were also detected for the beta-lactam antibiotics. This study describes test medium-dependent variation of MICs and the applicability of LSM+cys for antimicrobial susceptibility testing of bifidobacteria.

Influence of fermentation time, cryoprotectant and neutralization of cell concentrate on freeze-drying survival, storage stability, and acid and bile exposure of Bifidobacterium animalis ssp. lactis cells produced without milk-based ingredients.

AIMS: To investigate the stability of Bifidobacterium animalis ssp. lactis VTT E-012010 (=Bb-12) during freeze-drying, storage and acid and bile exposure. The effect of harvesting time and composition and pH of the cryoprotectant on the survival was evaluated. The procedure was performed by using a milk-free culture medium and cryoprotectants to produce cells for nonmilk-based applications. METHODS AND RESULTS: Bifidobacterial cells were grown in fermenters in general edible medium for 15 or 22 h. The cell mass was freeze-dried either as non-neutralized or neutralized using sucrose, betaine or reconstituted skim milk (control) as cryoprotectants. For stability studies freeze-dried powders were stored at 37, 5 and -20 degrees C for 2-6 months. In addition, acid and bile tolerance of the powders was tested. Sucrose-formulated B. animalis ssp. lactis preparations had an excellent stability during storage at refrigerated and frozen temperatures for 5-6 months. They also had a good survival during storage at 37 degrees C for 2 months as well as during exposure to pH 3 and 1% bile acids. No difference was observed between 15 and 22 h grown cells or between non-neutralized and neutralized cells. Betaine proved to be a poor cryoprotectant compared with sucrose. CONCLUSIONS: Fermentation time and neutralization of cell concentrate before freeze-drying had no impact on the storage stability and bile and acid tolerance of freeze-dried bifidobacterial cells. The nonmilk-based production protocol using sucrose as a cryoprotectant yielded powdery preparations with excellent stability in adverse conditions (storage at elevated temperatures and during acid and bile exposure). SIGNIFICANCE AND IMPACT OF THE STUDY: The results indicate that it is feasible to develop nonmilk-based production technologies for probiotic cultures. This provides new possibilities for the development of nondairy-based probiotic products.

Application of a microplate scale fluorochrome staining assay for the assessment of viability of probiotic preparations.

Cell viability in probiotic preparations is traditionally assessed by the plate count technique. Additionally, fluorescent staining combined with epifluorescence microscopy or flow cytometry has been developed for the viability assessment, but the currently available assays are either laborious or require highly sophisticated equipment. The aim of this study was to investigate the applicability of a microplate scale fluorochrome assay for predicting the cell state of freeze-dried Lactobacillus rhamnosus and Bifidobacterium animalis subsp. lactis preparations. In addition to viability assessment with LIVE/DEAD BacLight Bacterial Viability Kit, DiBAC(4)3 stain was used for the kinetic measurement of changes in bifidobacterial cell membrane functions during exposure to low pH. The microplate scale fluorochrome assay results on the viability and cell numbers of probiotic preparations correlated well with the results obtained with the culture-based technique and (with few exceptions) with epifluorescence microscopy. The assay was applicable also for the viability assessment of stressed (acid-treated) cells provided that the cell density in treatments was adjusted to the optimal measurement level of the fluorometer. The microplate scale fluorochrome assay offers a rapid and robust tool for the viability assessment of probiotic preparations, and enables also kinetic measurements.

Genetic heterogeneity and functional properties of intestinal bifidobacteria.

AIMS: The aim of the present study was to compare several molecular methods for the identification and genotyping of bifidobacteria, and further to investigate genetic heterogeneity and functional properties of bifidobacterial isolates from intestinal samples of Finnish adult subjects. METHODS AND RESULTS: A total of 153 intestinal bifidobacterial isolates were included in initial screening and 34 isolates were further characterized. Identification results obtained with PCR-ELISA and ribotyping were well in accordance with each other, while randomly amplified polymorphic DNA (RAPD) gave tentative identification only to Bifidobacterium bifidum and to 65% of the B. longum isolates. The most commonly detected species were B. longum biotype longum followed by B. adolescentis and B. bifidum. In addition, B. animalis (lactis), B. angulatum and B. pseudocatenulatum were found. Ribotyping and pulsed-field gel electrophoresis (PFGE) proved to be discriminatory methods for bifidobacteria, but also RAPD revealed intraspecies heterogeneity. Besides two B. animalis (lactis) isolates with very close similarity to a commercially available probiotic strain, none of the intestinal isolates showed optimal survival in all tolerance (acid, bile and oxygen) or growth performance tests. CONCLUSIONS: Several species/strains of bifidobacteria simultaneously colonize the gastrointestinal tract of healthy Finnish adults and intestinal Bifidobacterium isolates were genetically heterogeneous. Functional properties of bifidobacteria were strain-dependent. SIGNIFICANCE AND IMPACT OF THE STUDY: Applicability of ribotyping with the automated RiboPrinter System for identification and genotyping of bifidobacteria was shown in the present study.

Stationary-phase acid and heat treatments for improvement of the viability of probiotic lactobacilli and bifidobacteria.

AIMS: To investigate whether sublethal treatments of stationary-phase probiotic cultures enhance their survival during lethal treatments and to adapt these treatments to the fermenter-scale production of probiotic cultures. METHODS AND RESULTS: Conditions for acid and heat pretreatments were screened for three Lactobacillus and two Bifidobacterium strains. Strains were sublethally treated both at laboratory scale and at fermenter scale in a strain-specific manner and exposed to a subsequent lethal treatment. At laboratory scale viability improvement was detected in each strain. However, improvement was more pronounced in the Lactobacillus than in the Bifidobacterium strains. At fermenter scale three strains were tested: for the two Lactobacillus strains a marked improvement in viability was obtained whereas for the Bifidobacterium strain the improvement was either minor or not detected. CONCLUSIONS: Development of treatments for viability enhancement of probiotic strains is feasible, but strain-specific optimization is necessary to obtain notable improvements. SIGNIFICANCE AND IMPACT OF THE STUDY: Strain-specific treatments were developed for the viability enhancement of stationary-phase probiotic cells both at laboratory and fermenter scale. These results can be utilised in the production of probiotic cultures with improved viability.

Plasma enterolactone or intestinal Bifidobacterium levels do not explain adenoma formation in multiple intestinal neoplasia (Min) mice fed with two different types of rye-bran fractions.

The study was designed to evaluate whether two types of rye-bran fractions result in distinct bifidogenic effect or enterolactone production in multiple intestinal neoplasia (Min) mice and whether these parameters are associated with intestinal tumorigenesis in this animal model. The experimental diets were a non-fibre diet (control), a rye-bran diet, and diets containing either the soluble extract or the insoluble fraction prepared from rye bran. The main result on adenoma formation in these experiments was the observation that the soluble extract increased number (P=0.012) and size (P=0.008) of adenomas in the distal small intestine when compared with the non-fibre group. All rye-supplemented diets supported similarly the in vivo growth of Bifidobacterium (10(8)-10(9) colony forming units/g) in Min mice, whereas the non-fibre diet lowered intestinal Bifidobacterium below the level of detection. The results show that water solubility does not affect the bifidogenicity of rye bran. Mean plasma enterolactone concentration was highest in the rye-bran group (30.0 nmol/l; P=0.002), which along with the soluble-extract group (16.2 nmol/l; P=0.024) differed significantly from the non-fibre diet group (7.5 nmol/l). Thus, the mice fed with the rye bran were the best enterolactone producers. In conclusion, rye bran and rye fractions influence adenoma formation in Min mice to a varying degree but plasma enterolactone levels or the production of bifidogenic bacteria do not mediate the effect.

Adhesion of bifidobacteria to granular starch and its implications in probiotic technologies.

Adhesion of 19 Bifidobacterium strains to native maize, potato, oat, and barley starch granules was examined to investigate links between adhesion and substrate utilization and to determine if adhesion to starch could be exploited in probiotic food technologies. Starch adhesion was not characteristic of all the bifidobacteria tested. Adherent bacteria bound similarly to the different types of starch, and the binding capacity of the starch (number of bacteria per gram) correlated to the surface area of the granules. Highly adherent strains were able to hydrolyze the granular starches, but not all amylolytic strains were adherent, indicating that starch adhesion is not a prerequisite for efficient substrate utilization for all bifidobacteria. Adhesion was mediated by a cell surface protein(s). For the model organisms tested (Bifidobacterium adolescentis VTT E-001561 and Bifidobacterium pseudolongum ATCC 25526), adhesion appeared to be specific for alpha-1,4-linked glucose sugars, since adhesion was inhibited by maltose, maltodextrin, amylose, and soluble starch but not by trehalose, cellobiose, or lactose. In an in vitro gastric model, adhesion was inhibited both by the action of protease and at pH values of < or =3. Adhesion was not affected by bile, but the binding capacity of the starch was reduced by exposure to pancreatin. It may be possible to exploit adhesion of probiotic bifidobacteria to starch granules in microencapsulation technology and for synbiotic food applications.

Permanent jejunal fistula: promising method for obtaining small intestinal chyme without disturbing intestinal function.

Accurate information on changes in small intestinal microflora in dogs is rather limited because of difficulties in obtaining samples of small intestinal chyme. In the study reported here, intussuscepted nipple valves were surgically placed into the jejunum of seven laboratory beagles to obtain intestinal juice samples. The influence of the fistula on intestinal motility was determined by use of barium-impregnated polyethylene spheres (BIPS) and on microflora by use of bacterial culturing. The BIPS were fed two weeks before surgery and again five weeks after surgery. Bacterial samples were collected before (fecal samples), during (small intestinal samples) and 11 weeks after surgery. There were no surgical complications, and the animals tolerated the fistula well. Mean orocolic transit percentage was 93% before and 83% after surgery, and notable changes in gastrointestinal motility were not seen, except in one dog. The surgery did not markedly alter the bacterial flora in feces. Microflora did change in small intestinal samples; however, methodologic factors may explain most of these differences. In conclusion, the nipple valve is a promising method that creates easy and safe long-term access to the jejunum and appears not to have an influence on intestinal function.

The Prevotella intermedia group organisms in young children and their mothers as related to maternal periodontal status.

Currently, the Prevotella intermedia group includes three biochemically and phylogenetically related species: Prevotella intermedia, Prevotella nigrescens, and the newly described Prevotella pallens. The two first-named species are mentioned with varying emphasis in connection with periodontal diseases, while such a connection of P. pallens is not known. Mothers serve as a plausible source of bacteria to their children, and conceivably, a mother with periodontitis as a recurrent reservoir of periodontally infecting organisms. In the present study, 23 mothers and their young children were examined for the presence of the P. intermedia group organisms in relation to maternal periodontal status (I: periodontal health, II: initial periodontitis, and III: advanced periodontitis). Species differentiation was based on established biochemical methods, electrophoretic mobility patterns, SDS-PAGE, and DNA hybridization. P. intermedia was not recovered from children but nearly exclusively from mothers in group III, thus confirming its association with periodontitis. P. nigrescens and P. pallens were frequently found in mothers and children. To determine bacterial transmission between a mother and her child, 72 isolates from 13 mother-child pairs were analyzed by arbitrarily primed PCR (AP-PCR). Similar AP-PCR types of P. nigrescens and/or P. pallens were recovered from 3/4 pairs in group I, 2/5 pairs in group II, and none in group III. Our results indicate that different species within the P. intermedia group have a different colonization pattern in childhood and that the periodontal status reflects qualitatively their presence in maternal saliva. Intra-familial transmission of P. nigrescens and P. pallens can occur in early childhood, however similar AP-PCR types were most obvious within periodontally healthy mother-child pairs.

Probiotic bacteria: safety, functional and technological properties.

During the past two decades probiotic (health promoting) micro-organisms have been increasingly included in various types of food products, especially in fermented milks. Several aspects, including safety, functional and technological characteristics, have to be taken into consideration in the selection process of probiotic micro-organisms. Safety aspects include specifications such as origin (healthy human GI-tract), non-pathogenicity and antibiotic resistance characteristics. Functional aspects include viability and persistence in the GI-tract, immunomodulation, antagonistic and antimutagenic properties. Before probiotic strains, chosen on the basis of their good safety and functional characteristics, can benefit the consumer, they must first be able to be manufactured under industrial conditions. Furthermore, they have to survive and retain their functionality during storage, and also in the foods into which they are incorporated without producing off-flavours. Factors related to the technological and sensory aspects of probiotic food production are of utmost importance since only by satisfying the demands of the consumer can the food industry succeed in promoting the consumption of functional probiotic products in the future.

Beta-lactamase production in Prevotella intermedia, Prevotella nigrescens, and Prevotella pallens genotypes and in vitro susceptibilities to selected antimicrobial agents.

The present study investigated the beta-lactamase production of 73 Prevotella intermedia, 84 Prevotella nigrescens, and 14 Prevotella pallens isolates and their in vitro susceptibilities to six antimicrobial agents. The P. intermedia and P. nigrescens isolates were recovered from oral and extraoral samples obtained from subjects in two geographic locations from 1985 to 1995. The clonality of the beta-lactamase-positive and beta-lactamase-negative isolates and the clustering of the genotypes were studied by arbitrarily primed-PCR fingerprinting. beta-Lactamase production was detected in 29% of P. intermedia isolates, 29% of P. nigrescens isolates, and 57% of P. pallens isolates. No difference in the frequencies of beta-lactamase production by P. intermedia and P. nigrescens between isolates from oral and extraoral sites, between isolates obtained at different time periods, or between P. intermedia isolates from different geographic locations was observed. However, the P. nigrescens isolates from the United States were significantly more frequently (P = 0.015) beta-lactamase positive than those from Finland. No association between the genotypes and beta-lactamase production or between the genotypes and the sources of the isolates was found. The penicillin G MICs at which 90% of the isolates were inhibited were 8 microg/ml for P. intermedia, 8 microg/ml for P. nigrescens, and 16 microg/ml for P. pallens. For the beta-lactamase-negative isolates, the corresponding values were 0.031, 0.031, and 0.125 microg/ml, and for the beta-lactamase-positive isolates, the corresponding values were 16, 8, and 32 microg/ml. All isolates were susceptible to amoxicillin-clavulanate, cefoxitin, metronidazole, azithromycin, and trovafloxacin. The MICs of amoxicillin-clavulanate and cefoxitin were relatively higher for the beta-lactamase-positive population than for the beta-lactamase-negative population.

Biochemical and genetic characterization of a Prevotella intermedia/nigrescens-like organism.

Thirty-three previously non-typable faintly pigmented Gram-negative anaerobic bacterial isolates, biochemically most closely related to Prevotella intermedia and Prevotella nigrescens, were analysed for enzymic reactions, cellular fatty acid (CFA) composition, electrophoretic mobility of malate and glutamate dehydrogenases, hybridization with P. intermedia and P. nigrescens species-specific oligonucleotide probes and, for genetic heterogeneity, by arbitrarily primed PCR (AP-PCR). P. intermedia ATCC 25611T and P. nigrescens ATCC 33563T were run in parallel for comparison. Twenty-nine isolates originated from the normal oral flora of 18 subjects (including five mother-child pairs), and four isolates from various infections. Except for a negative lipase reaction, enzymic profiles of the test isolates were similar to those of P. intermedia and P. nigrescens. Clustering of CFAs, electrophoretic mobility patterns, hybridization with DNA probes for P. intermedia and P. nigrescens, and AP-PCR band patterns of the test isolates differed from those of the type strains of P. intermedia and P. nigrescens, suggesting the existence, in humans, of a new anaerobic species of pigmented, moderately saccharolytic, indole-positive Gram-negative rods.

Phylogenetic characterization and proposal of a new pigmented species to the genus Prevotella: Prevotella pallens sp. nov.

Complete 16S rRNA gene sequences of three representative strains of anaerobic, Gram-negative, pigmented, moderately saccharolytic, indole-positive bacteria isolated from the oral cavity of humans were determined. According to comparative analyses of the rRNA sequence data, this organism represents a previously unknown species within the genus Prevotella. In addition, 22 representative strains and 21 reference strains (including 11 Prevotella intermedia and 10 Prevotella nigrescens strains) were subjected to multilocus enzyme electrophoretic analysis. The strains were consistently separated into three clearly distinct groups, corresponding to their previous entities. On the basis of the present phylogenetic results that confirmed our biochemical and genetic data, we propose a new species, Prevotella pallens. The type strain is NCTC 13042 (= AHN 10371).

Mutacin production by Streptococcus mutans may promote transmission of bacteria from mother to child.

The production of bacteriocin-like inhibitory substances, mutacins, by mutans streptococci varies among isolates. To find if the degree of mutacin activity of an isolate was related to its transmission between mother and her child, 19 mothers and their 18-month- to 3-year-old children were sampled for their oral mutans streptococci. In addition, the stability of mutacin activity was studied with isolates from the mothers and with isolates from five unrelated 5-year-old children in 5- to 7-year follow-up studies. A total of 145 oral mutans streptococcal isolates were serotyped by immunodiffusion, ribotyped, and mutacin typed by the stab culture technique. Mutacin was produced by 88% of the strains against more than 1 of the 14 indicator strains, representing mutans streptococci, Streptococcus sanguis, Streptococcus salivarius, Streptococcus oralis, Streptococcus gordonii, and Streptococcus pyogenes. Streptococcus mutans isolates showed more inhibitory activity than did Streptococcus sobrinus isolates. Identical ribotypes had similar mutacin activity profiles within a subject, initially and in the follow-up studies, in all but two cases. The mothers harbored a total of 37 different mutans streptococcal ribotypes. Six children were negative for mutans streptococci. Transmission was probable in 9 of 20 mother-child pairs on the basis of the presence of identical strains, as determined by ribotyping and bacteriocin (mutacin) typing. S. mutans strains shared between a mother and her child showed a broader spectrum of inhibitory activity than did nontransmitted strains. In conclusion, the mutacin activity of clinical isolates is reasonably stable, and this virulence factor seems to be of clinical importance in early colonization by S. mutans.

Detection of Porphyromonas gingivalis from saliva by PCR by using a simple sample-processing method.

Simple sample-processing methods for PCR detection of Porphyromonas gingivalis, a major pathogen causing adult periodontitis, from saliva were studied. The ability to detect P. gingivalis from 118 salivary samples by PCR after boiling and Chelex 100 processing was compared with bacterial culture. P. gingivalis was detected three times more often by PCR than by culture. Chelex 100 processing of saliva proved to be effective in preventing PCR inhibition and was applied to determine the occurrence of P. gingivalis in saliva samples from 263 Finnish subjects between 5 and 80 years of age. The occurrence of P. gingivalis increased with age, and it was detected by PCR in the saliva of 5.0% of subjects between 5 and 10 years of age, 13.8% of subjects between 11 and 20 years of age, 13.4% of subjects between 21 and 30 years of age, and 63.3% of subjects between 31 and 80 years of age. The results indicate that P. gingivalis is a rare finding in saliva from periodontally healthy children and young adults but a frequent one in saliva from adult periodontitis patients.