Modulation of rat cecal microbiota by administration of raffinose and encapsulated Bifidobacterium breve

To investigate the effects of administration of raffinose and encapsulated Bifidobacterium breve JCM 1192T cells on the rat cecal microbiota, in a preclinical synbiotic study groups of male WKAH/Hkm Slc rats were fed for 3 weeks with four different test diets: basal diet (group BD), basal diet supplemented with raffinose (group RAF), basal diet supplemented with encapsulated B. breve (group CB), and basal diet supplemented with both raffinose and encapsulated B. breve (group RCB). The bacterial populations in cecal samples were determined by fluorescence in situ hybridization (FISH) and terminal restriction fragment length polymorphism (T-RFLP). B. breve cells were detected only in the RCB group and accounted for about 6.3% of the total cells as determined by FISH analysis. B. breve was also detected only in the RCB group by T-RFLP analysis. This was in contrast to the CB group, in which no B. breve signals were detected by either FISH or T-RFLP. Increases in the sizes of the populations of Bifidobacterium animalis, a Bifidobacterium indigenous to the rat, were observed in the RAF and RCB groups. Principal-component analysis of T-RFLP results revealed significant alterations in the bacterial populations of rats in the RAF and RCB groups; the population in the CB group was similar to that in the control group (group BD). To the best of our knowledge, these results provide the first clear picture of the changes in the rat cecal microbiota in response to synbiotic administration.     

A novel multi‐strain probiotic and synbiotic supplement for prevention of Clostridium difficile infection in a murine model

The protective effect of a multi‐strain probiotic and synbiotic formulation was evaluated in C57BL/6 mice infected with Clostridium difficile (CD) NAP1/027. Antibiotic‐treated mice were divided into the following four groups: Group 1, fed with a synbiotic formulation consisting of Lactobacillus plantarum F44, L. paracasei F8, Bifidobacterium breve 46, B. lactis 8:8, galacto‐oligosaccharides, isomalto‐oligosaccharides, and resistant starch; Group 2, fed with the same four probiotic strains as Group 1; Group 3, fed with the same prebiotic supplements as Group 1 for 7 days before CD infection; and Group 4 (control group) antibiotic treated and infected with NAP1/027 strain. Feces and cecal contents were collected for microbial cell viability, quantitative PCR (qPCR), toxin analyses and histopathology. Synbiotics‐ and probiotics‐fed mice showed a significant increase in total bifidobacteria (P < 0.05). The total lactobacilli count was increased in Group 1. Tests for cecal toxins were negative in Group 2 mice, whereas one sample each from Group 1 and 3 was positive. qPCR of cecal contents showed significant reduction in NAP1/027 DNA copies in Groups 1 and 2 and significantly higher numbers of B. breve 46, L. plantarum F44, and L. paracasei F8 in Groups 1 and 2 (P < 0.05); these changes were much less pronounced in Groups 3 and 4. Our findings indicate that the newly developed synbiotic or multi‐strain probiotic formulation confers protection against NAP1/027 infection in C57BL/6 mice. This holds promise for performing human studies.     

Population Dynamics of Bifidobacterium Species in Human Feces during Raffinose Administration Monitored by Fluorescence In Situ Hybridization-Flow Cytometry

The population dynamics of bifidobacteria in human feces during raffinose administration were investigated at the species level by using fluorescence in situ hybridization (FISH) coupled with flow cytometry (FCM) analysis. Although double-staining FISH-FCM using both fluorescein isothiocyanate (FITC) and indodicarbocyanine (Cy5) as labeling dyes for fecal samples has been reported, the analysis was interfered with by strong autofluorescence at the FITC fluorescence region because of the presence of autofluorescence particles/debris in the fecal samples. We circumvented this problem by using only Cy5 fluorescent dye in the FISH-FCM analysis. Thirteen subjects received 2 g of raffinose twice a day for 4 weeks. Fecal samples were collected, and the bifidobacterial populations were monitored using the established FISH-FCM method. The results showed an increase in bifidobacteria from about 12.5% of total bacteria in the prefeeding period to about 28.7 and 37.2% after the 2-week and 4-week feeding periods, respectively. Bifidobacterium adolescentis, the Bifidobacterium catenulatum group, and Bifidobacterium longum were the major species, in that order, at the prefeeding period, and these bacteria were found to increase nearly in parallel during the raffinose administration. During the feeding periods, indigenous bifidobacterial populations became more diverse, such that minor species in human adults, such as Bifidobacterium breve, Bifidobacterium bifidum, Bifidobacterium dentium, and Bifidobacterium angulatum, proliferated. Four weeks after raffinose administration was stopped, the proportion of each major bifidobacterial species, as well as that of total bifidobacteria, returned to approximately the original values for the prefeeding period, whereas that of each minor species appeared to differ considerably from its original value. To the best of our knowledge, these results provide the first clear demonstration of the population dynamics of indigenous bifidobacteria at the species level in response to raffinose administration.     

Antiobesity Effects of Bifidobacterium breve Strain B-3 Supplementation in a Mouse Model with High-Fat Diet-Induced Obesity

The aim of the present study was to evaluate the anti-obesity activity of a probiotic bifidobacterial strain in a mouse model with obesity induced by a high-fat diet. The mice were fed a high-fat diet supplemented with Bifidobacterium breve B-3 at 10⁸ or 10⁹ CFU/d for 8 weeks. B. breve B-3 supplementation dose-dependently suppressed the accumulation of body weight and epididymal fat, and improved the serum levels of total cholesterol, fasting glucose and insulin. The bifidobacterial counts in the caecal contents and feces were significantly increased with the B. breve B-3 administration. The expression of genes related to fat metabolism and insulin sensitivity in the gut and epididymal fat tissue was up-regulated by this administration. These results suggest that the use of B. breve B-3 would be effective in reducing the risk of obesity.     

Anti‐infectious activity of synbiotics in a novel mouse model of methicillin‐resistant Staphylococcus aureus infection

The anti‐infectious activity of synbiotics against methicillin‐resistant Staphylococcus aureus (MRSA) infection was evaluated using a novel lethal mouse model. Groups of 12 mice treated with multiple antibiotics were infected orally with a clinical isolate of MRSA at an inoculum of 10⁸ CFU on day 7 after starting the antibiotics. A dose of 400 mg/kg 5‐fluorouracil (5‐FU) was injected intraperitoneally on day 7 after the infection. A dose of 10⁸ CFU Bifidobacterium breve strain Yakult and 10 mg of galactooligosaccharides (GOS) were given orally to mice daily with the antibiotic treatment until day 28. The intestinal population levels of MRSA in the mice on multiple antibiotics were maintained stably at 10⁸ CFU/g of intestinal contents after oral MRSA infection and the subsequent 5‐FU treatment killed all the mice in the group within 14 days. B. breve administration saved most of the mice, but the synbiotic treatment saved all of the mice from lethal MRSA infection. The synbiotic treatment was effective for the treatment of intestinal infection caused by four MRSA strains with different toxin productions. There was a large difference among the six Bifidobacteria strains that were naturally resistant to the antibacterial drugs used. B. breve in combination with GOS is demonstrated to have valuable preventive and curative effects against even fatal MRSA infections.     

Variation in Consumption of Human Milk Oligosaccharides by Infant Gut-Associated Strains of Bifidobacterium breve

Human milk contains a high concentration of complex oligosaccharides that influence the composition of the intestinal microbiota in breast-fed infants. Previous studies have indicated that select species such as Bifidobacterium longum subsp. infantis and Bifidobacterium bifidum can utilize human milk oligosaccharides (HMO) in vitro as the sole carbon source, while the relatively few B. longum subsp. longum and Bifidobacterium breve isolates tested appear less adapted to these substrates. Considering the high frequency at which B. breve is isolated from breast-fed infant feces, we postulated that some B. breve strains can more vigorously consume HMO and thus are enriched in the breast-fed infant gastrointestinal tract. To examine this, a number of B. breve isolates from breast-fed infant feces were characterized for the presence of different glycosyl hydrolases that participate in HMO utilization, as well as by their ability to grow on HMO or specific HMO species such as lacto-N-tetraose (LNT) and fucosyllactose. All B. breve strains showed high levels of growth on LNT and lacto-N-neotetraose (LNnT), and, in general, growth on total HMO was moderate for most of the strains, with several strain differences. Growth and consumption of fucosylated HMO were strain dependent, mostly in isolates possessing a glycosyl hydrolase family 29 α-fucosidase. Glycoprofiling of the spent supernatant after HMO fermentation by select strains revealed that all B. breve strains can utilize sialylated HMO to a certain extent, especially sialyl-lacto-N-tetraose. Interestingly, this specific oligosaccharide was depleted before neutral LNT by strain SC95. In aggregate, this work indicates that the HMO consumption phenotype in B. breve is variable; however, some strains display specific adaptations to these substrates, enabling more vigorous consumption of fucosylated and sialylated HMO. These results provide a rationale for the predominance of this species in breast-fed infant feces and contribute to a more accurate picture of the ecology of the developing infant intestinal microbiota.     

High-yield production and characterization of α-galactosidase from Bifidobacterium breve grown on raffinose

Bifidobacteria assimilated raffinose about 4-fold more effectively than other intestinal bacteria, and -galactosidase was active in all strains of Bifidobacteria tested. The enzyme activity of Bifidobacterium breve grown on raffinose was highly and specifically increased. Its activity was 30-fold higher than that of B. breve grown on glucose. Melibiose was also effective for production of the enzyme. The enzyme was purified to homogeneity from B. breve. It is a homodimer with Mr of about 160 kDa, and its optimum pH for activity of 5.5–6.5. The enzyme showed strict substrate specificity for -galactoside although it had slight activity for -glucoside. It hydrolysed stachyose, melibiose (Km = 2 mM) and raffinose (Km = 0.7 mM).     

Specific growth rate of bifidobacteria cultured on different sugars

The ability of six bifidobacterial strains (3 of human origin and 3 isolates from fermented milk products) to utilize glucose, lactose, melezitose, sucrose, raffinose, and stachyose was determined. Dairy-related bifidobacterial strains were identified asBifidobacterium animalis (2 strains) or asB. pseudolongum (1 strain). Human strains includedB. longum (2 strains) andB. breve (1 strain). All strains fermented lactose, sucrose, raffinose, and stachyose. Melezitose was utilized only byB. longum. B. pseudolongum did not ferment either glucose or melezitose. All isolates had a higher specific growth rate on raffinose and stachyose than on glucose. Dairy strains grew slowly on glucose compared to human strains.     

Effects of dietary Bacillus subtilis and inulin supplementation on performance,eggshell quality,intestinal morphology and microflora composition of laying hens in the late phase of production

Eighty Lohmann White laying hens were used to investigate the effect of dietary inclusion of Bacillus subtilis and inulin, individually or in combination, on egg production, eggshell quality, tibia traits, Ca retention, and small intestine morphology and microflora composition from 64 to 75 weeks of age. Hens were randomly distributed into 4 treatment groups, with 5 replicates per treatment and 4 hens per replicate. Treatment groups were fed basal diet (control), basal diet plus 1 g/kg B. subtilis (2.3 × 10⁸ cfu/g), basal diet plus 1 g/kg inulin, or basal diet plus a synbiotic combination of 1 g/kg B. subtilis (2.3 × 10⁸ cfu/g) and 1 g/kg inulin. Dietary supplementation of B. subtilis, inulin or synbiotic improved (P<0.05) feed conversion, egg performance, eggshell quality and calcium retention compared with the control. B. subtilis and synbiotic groups exhibited the highest (P<0.05) increase in egg production and egg weight. Inulin and synbiotic groups exhibited the highest (P<0.05) increase in eggshell thickness and eggshell calcium content, and the lowest (P<0.05) eggshell deformations. Unmarketable eggs were 8.4% (P<0.05) of the total eggs produced by the control group compared to 3.5%, 1.7%, and 1.5% for the B. subtilis, inulin and synbiotic groups, respectively. Tibia density, ash, and Ca content increased (P<0.05) by inulin and synbiotic inclusions, compared with the control. B. subtilis, inulin, and their synbiotic combination increased (P<0.05) villus height and crypt depth in all intestinal segments, compared with the control. B. subtilis and inulin modulated the ileal and caecal microflora composition by decreasing (P<0.05) numbers of Clostridium and Coliforms and increasing (P<0.05) numbers of bifidobacteria and lactobacilli, compared with the control. Colonization of the beneficial microflora along with increasing the villi–crypts absorptive area were directly associated with the improvements in performance and eggshell quality. It can be concluded that egg production and eggshell quality of laying hens can be improved (P<0.05) in the late phase of production by dietary inclusion of B. subtilis and inulin.     

Prebiotic-non-digestible oligosaccharides preference of probiotic bifidobacteria and antimicrobial activity against Clostridium difficile

Bifidobacterium breve 46, Bifidobacterium lactis 8:8 and Bifidobacterium longum 6:18 and three reference strains B. breve CCUG 24611, B. lactis JCM 10602, and Bifidobacterium pseudocatenulatum JCM 1200 were examined for acid and bile tolerance, prebiotic utilization and antimicrobial activity against four Clostridium difficile (CD) strains including the hypervirulent strain, PCR ribotype NAP1/027. B. lactis 8:8 and B. lactis JCM 10602 exhibited a high tolerance in MRSC broth with pH 2.5 for 30 min. B. breve 46 and B. lactis 8:8 remained 100% viable in MRSC broth with 5% porcine bile after 4 h. All six strains showed a high prebiotic degrading ability (prebiotic score) with galactooligosaccharides (GOS), isomaltooligosaccharides (IMOS) and lactulose as carbon sources and moderate degradation of fructooligosaccharides (FOS). Xylooligosaccharides (XOS) was metabolized to a greater extent by B. lactis 8:8, B. lactis JCM 10602, B. pseudocatenulatum JCM 1200 and B. longum 6:18 (prebiotic score >50%). All strains exhibited extracellular antimicrobial activity (AMA) against four CD strains including the CD NAP1/027. AMA of B. breve 46, B. lactis 8:8 and B. lactis JCM 10602 strains was mainly ascribed to a combined action of organic acids and heat stable, protease sensitive antimicrobial peptides when cells were grown in MRSC broth with glucose and by acids when grown with five different prebiotic-non-digestible oligosaccharides (NDOs). None of C. difficile strains degraded five prebiotic-NDOs. Whole cells of B. breve 46 and B. lactis 8:8 and their supernatants inhibited the growth and toxin production of the CD NAP1/027 strain.     

Cholesterol-lowering probiotics: in vitro selection and in vivo testing of bifidobacteria

Thirty-four strains of bifidobacteria belonging to Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium longum, and Bifidobacterium pseu-docatenulatum were assayed in vitro for the ability to assimilate cholesterol and for bile salt hydrolase (BSH) against glycocholic and taurodeoxycholic acids (GCA and TDCA). Cholesterol assimilation was peculiar characteristic of two strains belonging to the species B. bifidum (B. bifidum MB 107 and B. bifidum MB 109), which removed 81 and 50 mg of cholesterol per gram of biomass, being the median of specific cholesterol absorption by bifidobacteria 19 mg/g. Significant differences in BSH activities were not established among bifidobacterial species. However, the screening resulted in the selection of promising strains able to efficiently deconjugate GCA and TDCA. No relationship was recognized between BSH phenotype and the extent of cholesterol assimilation. On the basis of cholesterol assimilation or BSH_GCA and BSH_TDCA activities, B. bifidum MB 109 (DSMZ 23731), B. breve MB 113 (DSMZ 23732), and B. animalis subsp. lactis MB 2409 (DSMZ 23733) were combined in a probiotic mixture to be fed to hypercholesterolemic rats. The administration of this probiotic formulation resulted in a significant reduction of total cholesterol and low-density cholesterol (LDL-C), whereas it did not affect high-density cholesterol (HDL-C) and HDL-C/LDL-C ratio.     

Differential Induction of Antimicrobial REGIII by the Intestinal Microbiota and Bifidobacterium breve NCC2950

The intestinal microbiota is a key determinant of gut homeostasis, which is achieved, in part, through regulation of antimicrobial peptide secretion. The aim of this study was to determine the efficiency by which members of the intestinal microbiota induce the antimicrobial peptide REGIII and to elucidate the underlying pathways. We showed that germfree mice have low levels of REGIII-γ in their ileum and colon compared to mice with different intestinal microbiota backgrounds. Colonization with a microbiota of low diversity (altered Schaedler flora) did not induce the expression of REGIII-γ as effectively as a complex community (specific pathogen free). Monocolonization with the probiotic Bifidobacterium breve, but not with the nonprobiotic commensal Escherichia coli JM83, upregulated REGIII-γ expression. Induction of REGIII-γ by B. breve was abrogated in mice lacking MyD88 and Ticam1 signaling. Both live and heat-inactivated B. breve but not spent culture medium from B. breve induced the expression of REGIII-α, the human ortholog and homolog of REGIII-γ, in human colonic epithelial cells (Caco-2). Taken together, the results suggest that REGIII-γ expression in the intestine correlates with the richness of microbiota composition. Also, specific bacteria such as Bifidobacterium breve NCC2950 effectively induce REGIII production in the intestine via the MyD88-Ticam1 pathway. Treatment with this probiotic may enhance the mucosal barrier and protect the host from infection and inflammation.     

Isolation of a Bifidogenic Peptide from the Pepsin Hydrolysate of Bovine Lactoferrin

Lactoferrin is an iron-binding glycoprotein found in the milk of most mammals for which various biological functions have been reported, such as antimicrobial activity and bifidogenic activity. In this study, we compared the bifidogenic activity of bovine lactoferrin (bLF) and pepsin hydrolysate of bLF (bLFH), isolated bifidogenic peptide from bLFH, and investigated the bifidogenic spectra of bLF, bLFH, and its active peptide against 42 bifidobacterial strains comprising nine species. Against Bifidobacterium breve ATCC 15700^T, minimal effective concentrations of bLF and bLFH were 300 and 10 μg/ml. Against Bifidobacterium longum subsp. infantis ATCC 15697^T, the minimal effective concentration of bLFH was 30 μg/ml, and bLF did not show bifidogenic activity within 300 μg/ml. As an active peptide, a heterodimer of A¹-W¹⁶ and L⁴³-A⁴⁸ linked by a disulfide bond was isolated. Previously, this peptide was identified as having antibacterial activity. An amino acid mixture with the same composition as this peptide showed no bifidogenic activity. The strains of each species whose growth was highly promoted (>150%) by this peptide at 3.75 μM were as follows: B. breve (7 out of 7 strains [7/7]), B. longum subsp. infantis (5/5), Bifidobacterium bifidum (2/5), B. longum subsp. longum (1/3), Bifidobacterium adolescentis (3/6), Bifidobacterium catenulatum (1/4), Bifidobacterium pseudocatenulatum (0/4), Bifidobacterium dentium (0/5), and Bifidobacterium angulatum (0/3). Growth of none of the strains was highly promoted by bLF at 3.75 μM. We demonstrated that bLFH showed stronger bifidogenic activity than natural bLF, especially against infant-representative species, B. breve and B. longum subsp. infantis; furthermore, we isolated its active peptide. This is the first report about a bifidogenic peptide derived from bLF.     

Nutritional impacts of dietary oregano and Enviva essential oils on the performance,gut microbiota and blood biochemicals of growing ducks

Nowadays, there is much legislation in the world devoted to restrict the use of synthetic antibiotics in the poultry industry, which could reduce performance rate and production profits. Various phyto-biotic growth promoters have been proposed to serve as antibiotic alternatives with emphasis on plant extracts and essential oils. This study was conducted to assess the impacts of using the oregano essential oil (OEO) (comprised of 5% thymol and 65% carvacrol) and Enviva essential oil (EEO) (4.5% cinnamaldehyde and 13.5% thymol) as phytobiotic feed additives (PFA) on growth performance, cecal microbiota and serum biochemicals of growing ducks. In total, 800 11-day-old ducklings, housed in 20 floor pens, were allotted randomly into five dietary treatments: (i) A basal diet (BD) (control), (ii.) BD+50 mg EEO/kg, (iii.) BD+100 mg EEO/kg, (iv.) BD+150 mg OEO/kg and (v.) BD+300 mg OEO/kg diet. The growth performance traits were studied between 11 and 42 days of age. At the experiment end, 40 ducks were slaughtered (eight/ treatment) and cecal digesta and blood samples were collected to estimate the cecal bacterial populations and serum blood biochemicals. The results indicated that the tested levels of OEO and EEO did not display any significant effect (P>0.05) on the duck’s final BW, BW gain, growth rate, feed intake, feed conversion ratio or survivability rate. Besides, the different levels of EEO and OEO decreased the cecal populations of Coliforms (P<0.01), total aerobes (P<0.01) and lactose-negative Enterobacteria (P<0.05) in comparison with those of the control group. Finally, the tested EEO and OEO levels did not show any significant effect on the serum variables; in terms of total protein, albumin, globulin, total cholesterol, triglycerides, alanine aminotransferase and aspartate aminotransferase. In conclusion, the antimicrobial effect of the OEO and EEO against the cecal microbiota has been proven, while they did not display significant effects on the growth performance or blood variables of growing ducks.     

Quantitative Real-Time PCR Assays To Identify and Quantify Fecal Bifidobacterium Species in Infants Receiving a Prebiotic Infant Formula

A healthy intestinal microbiota is considered to be important for priming of the infants' mucosal and systemic immunity. Breast-fed infants typically have an intestinal microbiota dominated by different Bifidobacterium species. It has been described that allergic infants have different levels of specific Bifidobacterium species than healthy infants. For the accurate quantification of Bifidobacterium adolescentis, Bifidobacterium angulatum, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium catenulatum, Bifidobacterium dentium, Bifidobacterium infantis, and Bifidobacterium longum in fecal samples, duplex 5′ nuclease assays were developed. The assays, targeting rRNA gene intergenic spacer regions, were validated and compared with conventional PCR and fluorescent in situ hybridization methods. The 5′ nuclease assays were subsequently used to determine the relative amounts of different Bifidobacterium species in fecal samples from infants receiving a standard formula or a standard formula supplemented with galacto- and fructo-oligosaccharides (OSF). A breast-fed group was studied in parallel as a reference. The results showed a significant increase in the total amount of fecal bifidobacteria (54.8% ± 9.8% to 73.4% ± 4.0%) in infants receiving the prebiotic formula (OSF), with a diversity of Bifidobacterium species similar to breast-fed infants. The intestinal microbiota of infants who received a standard formula seems to resemble a more adult-like distribution of bifidobacteria and contains relatively more B. catenulatum and B. adolescentis (2.71% ± 1.92% and 8.11% ± 4.12%, respectively, versus 0.15% ± 0.11% and 1.38% ± 0.98% for the OSF group). In conclusion, the specific prebiotic infant formula used induces a fecal microbiota that closely resembles the microbiota of breast-fed infants also at the level of the different Bifidobacterium species.     

Discovery of a Conjugative Megaplasmid in Bifidobacterium breve

Bifidobacterium breve is a common and sometimes very abundant inhabitant of the human gut. Genome sequencing of B. breve JCM 7017 revealed the presence of an extrachromosomal element, designated pMP7017 consisting of >190 kb, thus representing the first reported bifidobacterial megaplasmid. In silico characterization of this element revealed several genomic features supporting a stable establishment of the megaplasmid in its host, illustrated by predicted CRISPR-Cas functions that are known to protect the host against intrusion of foreign DNA. Interestingly, pMP7017 is also predicted to encode a conjugative DNA transfer apparatus and, consistent with this notion, we demonstrate here the conjugal transfer of pMP7017 to representative strains of B. breve and B. longum subsp. longum. We also demonstrate the presence of a megaplasmid with homology to pMP7017 in three B. longum subsp. longum strains.     

Characterization of Bifidobacterium Strains Using Box Primers

Twenty-five Bifidobacterium strains isolated from infants' faeces were identified by Rep-PCR. Using BOX-PCR, characteristic bands of Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium infantis and Bifidobacterium adolescentis were found in 40 strains of bidfidobacteria. These bands were not found in lactobacilli. By computerized numerical analysis strains were grouped in two major clusters. Strains of B. bifidum fell into a well-differentiated cluster that joined the cluster of the remaining species at 0.771 of similarity. The predominant species among the isolated strains were Bifidobacterium bifidum, Bifidobacterium longum andBifidobacterium breve . In another set of experiments, DNA was extracted from bacteria harvested from fermented milks to which different concentrations of bifidobacteria had been added. In all cases characteristic bands in the agarose gel belonging to lactobacilli and streptococci were detected. Bifidobacterium was detected only when 10⁸CFU/ml were added to the fermented milks. On the basis of our results, we propose this methodology as another tool in the polyphasic taxonomy.