Species Composition of Bacteroidales Order Bacteria in the Feces of

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(phylum Bacteroidetes, class Bacteroidia). ... result of mucosal injury or immune dysfunction can ... the Bacteroidales order belong to the Bacteroides genus,.

Biosci. Biotechnol. Biochem., 76 (1), 169–171, 2012

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Species Composition of Bacteroidales Order Bacteria in the Feces of Healthy People of Various Ages Elena V. K ULAGINA, Boris A. E FIMOV, Phillipp Y. M AXIMOV, Lyudmila I. K AFARSKAIA, Andrei V. C HAPLIN, and Andrei N. S HKOPOROVy Department of Microbiology and Virology, Russian State Medical University, Ostrovitjanova Street 1, 117997 Moscow, Russia Received June 3, 2011; Accepted September 27, 2011; Online Publication, January 7, 2012 [doi:10.1271/bbb.110434]

A study of species distribution of numerically predominant Bacteroidales order isolates in feces of healthy people aged 1–33 years was accomplished using a combination of amplified ribosomal DNA restriction analysis (ARDRA) and 16S ribosomal DNA (rDNA) sequencing. It was found that the majority of isolates in all age groups belonged to species B. xylanisolvens, B. vulgatus, and B. uniformis. Members of genera Alistipes, Parabacteroides, Odoribacter, Barnesiella, and Prevotella were also detected frequently. Key words:

number of new species where described, while some of those which earlier belonged to the genus Bacteroides were re-classified into several novel genera, including

Bacteroidales; Bacteroides; intestinal microbiota; 16S rRNA

The human colon is inhabited by a large and diverse community of microorganisms, with bacterial counts exceeding 1011 organisms per g of wet weight. The vast majority of these bacteria are obligate anaerobes belonging to the orders Clostridiales (phylum Firmicutes, class Clostridia), Bifidobacteriales (phylum Actinobacteria, class Actinobacteria), and Bacteroidales (phylum Bacteroidetes, class Bacteroidia). This latter order consists of anaerobic, bile-resistant, non-sporeforming, gram-negative rods. In the human large intestine, Bacteroidales order species maintain symbiotic relationships with the host, taking part in carbohydrate utilization, influencing the local immunity, and preventing gut colonization by food-borne pathogens. However, translocation of these commensal bacteria from the intestinal lumen to ectopic sites as a result of mucosal injury or immune dysfunction can cause various inflammatory conditions, including abscess formation.1) Commonly detected species of the Bacteroidales order isolated from human feces include Bacteroides fragilis, B. thetaiotaomicron, B. ovatus, B. caccae, B. vulgatus, B. uniformis, Parabacteroides merdae, Parabacteroides distasonis, Prevotella tannerae, Odoribacter splanchnicus, and members of the genus Alistipes.2–4) The most studied representative species of the Bacteroidales order belong to the Bacteroides genus, and less is known about the biology and significance for human health and disease of the other genera. During the past two decades the Bacteroidales order underwent extensive taxonomic rearrangements. A

Fig. 1. Phylogram Showing Phylogenetic Relationships of 16S rDNA Sequences of Bacteroidales Type Strains and Isolates from This Study ( The latter are designated EBA). This tree was constructed with MEGA v. 5.05 by the neighborjoining method and the Tamura-Nei model. The Chlorobium vibrioforme sequence was used as outgroup in rooting the tree. Bootstrapping was performed using 1,000 resamplings. Only bootstrap percentages greater than 50 are shown. The scale bar represents 0.1 substitution per nucleotide position.

y To whom correspondence should be addressed. Fax: +7-495-434-1766; E-mail: [email protected] Abbreviations: rDNA, ribosomal DNA; ARDRA, amplified ribosomal DNA restriction analysis; CFU, colony-forming units

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E. V. KULAGINA et al. Table 1. Occurrence Rates and Viable Counts of Bacteroidales Order Species in Fecal Samples Group I (1–5 y. o., n ¼ 9)

order Bacteroidales Bacteroides vulgatus Bacteroides uniformis Bacteroides xylanisolvens Bacteroides caccae Bacteroides dorei Bacteroides coprocola Bacteroides ovatus Bacteroides thetaiotaomicron Bacteroides fragilis Bacteroides finegoldii Bacteroides intestinalis Bacteroides stercoris Parabacteroides merdae Parabacteroides distasonis Alistipes finegoldii Alistipes shahii Alistipes onderdonkii Alistipes putredinis genus Alistipes Prevotella copri Odoribacter splanchnicus Barnesiella intestinihominis

a

9/100 4/44.4 3/33.3 4/44.4 3/33.3 4/44.4 1/11.1 2/22.2 1/11.1 2/22.2 1/11.1 ND ND ND 01.11.01 ND ND ND ND ND ND ND ND

b

10:4  0:2 9:7  0:4 9:7  0:4 9:8  0:5 9:3  0:7 9:5  0:3 8.5 9:5  0:9 9.6 9:9  0:2 9.3 ND ND ND 9.6 ND ND ND ND ND ND ND ND

Group II (6–14 y. o., n ¼ 17) 17/100 9/53 6/35.3 8/47 6/35.3 2/11.7 2/11.7 1/5.9 1/5.9 2/11.7 2/11.7 1/5.9 ND 1/5.9 3/17.7 3/17.7 2/11.7 ND 1/5.9 6/35.3 1/5.9 1/5.9 4/23.5

10:1  0:4 9:6  0:4 9:4  0:7 9:3  0:7 9:2  0:2 9.8 9:1  0:3 8.3 9.5 9:7  0:5 9.0 8.7 ND 9.0 9:2  0:2 9:8  0:1 9:5  0:2 ND 9.4 9:6  0:2 9.9 8.5 9:6  0:3

Group III (18–33 y. o., n ¼ 10) 10/100 6/60 5/50 4/40 ND ND 1/10 ND 1/10 ND ND ND 1/10 3/30 3/30 ND 1/10 4/40 2/20 7/70 ND ND ND

10  0:6 9:6  0:6 9:6  0:4 8:8  0:8 ND ND 9.7 ND 9.3 ND ND ND 9.0 9:3  0:6 8:3  0:6 ND 9.0 9:1  0:9 9:9  0:6 9:3  0:7 ND ND ND

a

Frequency of occurrence (absolute number of subjects/percentage of subjects) Mean  SD of log 10 number of viable microorganism/g of feces  p < 0:05 in group III vs. group I ND, not detected b

Parabacteroides, Tannerella, Alistipes, and Odoribacter. Among the newly described species were Bacteroides finegoldii, Bacteroides dorei,5) Bacteroides coprocola, Barnesiella intestinihominis,6) Alistipes onderdonkii, and Alistipes shahii.7) Today little is known about age differences in the species composition of Bacteroidales order bacteria in the human intestine. It has been found that total counts and the diversity of Bacteroidales species increase during childhood,4,8) reaching adult levels at approximately 17 years.9) From that point fecal counts of Bacteroides remain more or less stable throughout life, but decrease with aging.10) The goal of the present study was to compare species composition and viable counts of dominant cultivable Bacteroidales order bacteria in the feces of young people of ages ranging from 1 to 33 years. Feces were collected from 36 clinically healthy subjects (12 males and 24 females). The volunteers participating in the study were divided into three groups. Group I consisted of nine children aged 1–5 years, group II included 17 children aged 6–14 years, and group III included 10 adults aged 18–33 years. Bacteroidales order bacteria were isolated and identified essentially as described previously.3) Briefly, fecal homogenates were serially diluted 10-fold, plated onto Columbia-blood agar (Himedia, Mumbai, India) and cultured under anaerobic conditions. Representative colonies of each distinct type were re-plated on the same medium under both aerobic and anaerobic conditions and examined microscopically with Gram staining. Isolates of gramnegative anaerobic non-spore-forming rods were preliminary classified as Bacteroidales order members and subjected to further analysis. DNA from bacterial isolates was extracted by boiling. Templates for sequencing and amplified ribosomal DNA restriction analysis (ARDRA) were obtained using 16S rDNA-

targeted universal primers (Bact8F and Bact1391R).3) Restriction analysis of 16S rRNA gene amplicons was performed using AluI, Kzo9I, and TaqI restriction endonucleases, as previously described.3) Nucleotide sequences were analyzed by means of the Megablast on-line tool and deposited in GenBank database under accession nos. JF298871-JF298897. Multiple sequence alignment was performed using ClustalW v. 2.01. Statistical analyses were accomplished using Student’s t test and the 2 test. Differences were considered significant at p < 0:05. During the present investigation, a total of 172 strains of Bacteroides-like bacteria were isolated from the feces of 36 subjects. The occurrence rate of Bacteroidales order strains was 100%, and the average viable counts were 10:4  0:2, 10:1  0:4, and 10  0:6 (log CFU/g SD) in age groups I, II, and III respectively (p > 0:05). A comparison of ARDRA profiles generated from 16S rDNA with a previously established ARDRA dataset3) allowed us to identify unambiguously the majority of isolates to the species level. The remaining strains were identified by partial 16S rDNA sequencing. Notably, 15 strains initially characterized as Bacteroides-like bacteria on the basis of morphological and cultural properties turned out to belong to completely different phylogenetic groups. Among these were Clostridium, Eubacterium, Desulfovibrio, Veillonella, Bifidobacterium, Coriobacterium, Ruminococcus, Megamonas, Sutterella genera, and Lachnospiraceae-family members. The remaining strains belonged to a total of 21 species of the Bacteroidales order. The species diversity of the Bacteroidales bacteria in the three age groups was as follows: group I harbored 11 species, group II 19 species, and group III 11 species. The viable counts and occurrence rates of the detected Bacteroidales species are shown in Table 1. Among the

Bacteroidales Order Species in Human Gut

dominating species in age group I were Bacteroides xylanisolvens, Bacteroides vulgatus, Bacteroides dorei, Bacteroides uniformis, and Bacteroides caccae. In group II Bacteroides vulgatus, Bacteroides xylanisolvens, Bacteroides uniformis, and Bacteroides caccae were detected most frequently. Fecal samples from group III were principally colonized by Bacteroides vulgatus, Bacteroides uniformis, Bacteroides xylanisolvens, and also Alistipes onderdonkii. The presence of B. xylanisolvens as a predominant species is intriguing, since it was only recently described as a separate species.11) It is important to note that A. onderdonkii, found in a large proportion of the group III samples, is also among the newly described taxa.7) Several other recently described and re-classified species, including Parabacteroides distasonis12) and Prevotella copri,13) were detected. A dendrogram revealing the phylogenetic relationships of the 16S rRNA gene sequences of the isolated strains and the type strain for each species is shown in Fig. 1. The average number of different Bacteroidales order species per sample was 3:0  1:4, 3:2  1:8, and 3:1  1:3 for groups I-III respectively (p > 0:05). The viable counts of the various Bacteroidales species varied more than an order of magnitude, from 8.3 to 9.9 (lg CFU/g). Although there were some variations in the occurrence rates and viable counts of Bacteroidales species among the three age groups, the only significant difference was associated with the Alistipes genus, whose incidence increased with age from 0% in group I to 35.3% in group II and 70% in group III (p < 0:05). Our data indicate that Bacteroides genus members prevail among Bacteroidales order bacteria in the human gut. At least in some subjects, the genera Alistipes, Parabacteroides, and Barnesiella make up a

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significant part of anaerobic bacterial consortium. These observations are concordant with previously published results.2–4) However, taking into account the complexity of intestinal Bacteroidales populations and their undoubted significance for human physiology and pathology, studies of diversity and age dynamics of this taxonomic group should be continued.

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