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Enumeration , Isolation , and Identification of Bifidobacteria from Infant Feces
| Content Provider | Semantic Scholar |
|---|---|
| Author | Buj, Dufournet Christel Áb, Á. K. O. V. |
| Copyright Year | 2004 |
| Abstract | Thirty-three fully breast-fed infants aged between 1 and 12 weeks were screened for bifidobacteria in feces. Bifidobacteria counts in most fecal samples determined both by TPY agar and FISH procedure ranged from 108 to 1011 CFU/g. Three infants did not contain any bifidobacteria in their fecal samples. One child was delivered by caesarean section and the other two by normal vaginal delivery. All bifidobacteria-free infants possessed Gram-positive regular rods as a major group of their fecal flora. These bacteria were identified as clostridia using genus-specific FISH probe. In bifidobacteria-positive samples, B. longum (57.9 % of the samples) was the most frequently found species, followed by B. adolescentis (31.6 %), B. biidum (21.0 %), B. breve (10.5 %), B. pseudocatenulatum (5.3 %), and B. dentium (5.3 %). f The gastrointestinal microflora is a very complex ecosystem. More than 400 species within the intestinal microflora can be identified and may attain population levels nearly as high as 1012 per g in the colon (Mitsuoka 1992; Tannock 1999). Bacteroides, bifidobacteria, eubacteria and clostridia dominate the anaerobic flora. Entrobacteria, mainly E. coli, and enterococci predominate among aerobic bacteria (Orrhage and Nord 2000). Initial colonization of the aseptic intestine of the newborn happens during birth when bacteria from the mother's intestinal and vaginal flora inoculate the gastro-intestinal tract of the fetus. This leads to an inoculation with a diverse flora of bifidobacteria, enterobacteria, Bacteriodes, clostridia, and Gram-positive cocci (Sakata et al. 1985). After this first inoculation, the flora changes rapidly presumably under the influence of the diet. In breast-fed infants, bifidobacteria dominate among intestinal bacteria since they are supported by bifidogenic factors presented in human milk. While in breast-fed infants bifidobacteria account more than 90 % of the total intestinal bacteria, formula-fed infants' flora is more diverse (Stark and Lee 1982). Bifidobacteria are Gram-positive pleomorphic nonsporeforming nonmotile anaerobic rods (Scardovi 1986). These bacteria play an important role in maintaining human health. They suppress harmful bacteria by controlling the pH of the large intestine (Gibson and Wang 1994). Bifidobacteria have anticancerogenic (Reddy and Rivenson 1993), anticholesterolemic (Pereira and Gibson 2002), and immune system activation effects (Mitsuoka 1992). Other effects that have been ascribed to this genus are alleviation of lactose intolerance and vitamin production (Fooks et al. 1992). Genus Bifidobacterium can be distinguished from other bacterial groups by exhibiting the activity of fructose-6-phosphate phosphoketolase (EC 4.1.2.22; F6PPK), a key enzyme in bifidobacterial metabolic pathway (Scardovi 1986). Biochemical tests for the identification of bifidobacteria are now largely superseded by the use of the genus-specific (Kok et al. 1996) and species-specific PCR primers (Matsuki et al. 1999; Dong et al. 2000; Ventura et al. 2001). Genus-specific hybridization probes have proved useful in the enumeration of the total population of bifidobacteria in faecal samples (Langendijk et al. 1995). The aim of this work was to enumerate, isolate and identify bifidobacteria from infant feces. MATERIAL AND METHODS Thirty-three infants aged between 1 and 12 weeks were investigated; six of them were delivered by caesarean section. All infants were born in Czechia and were fully breast-fed. Fluorescence in situ hybridization (FISH) kit for Bifidobacterium sp. (RiboTechnologies, The Netherlands) was used for quantitative 210 E. VLKOVÁ et al. Vol. 49 detection of bifidobacteria in infant feces. A component of the kit is a genus-specific oligonucleotide DNA probe labeled by fluorescein isothiocyanate (FITC), which is bound to bifidobacterial rRNA. Universal fluorescent DNA probe reactive towards virtually all bacterial RNAs was used as a positive control. After the hybridization, the samples were analyzed with a Nikon E-800 epifluorescence microscope. Bifidobacteria in fecal samples were also enumerated and isolated using TPY agar (Sharlau, Spain) modified by the addition of mupirocin (100 mg/L) and concentrated acetic acid (1 ml/L) according to Rada and Petr (2000). Typical colonies were counted to determine bifidobacterial counts. Pure cultures were isolated and enriched in TPY broth. Isolates were identified as members of the genus Bifidobacterium by exhibiting F6PPK activity (Orban and Patterson 2000). Bifidobacterial cultures were characterized using API 50 CHL and API ID 32 A Rapid tests (bioMérieux, France). In addition, ability to grow at 46 °C was tested (Gavini et al. 1991). Subsequently, all strains were identified to the species level using computer program Bacter http://kounou.lille.inra.fr (INRA, France). Identification on a species level was also done by PCR using species-specific primers. The genomic DNA of the strains used was extracted by heating at 100 °C for 10 min in 1 % Triton X-100 (Sigma, USA) (Wang et al. 1996). Amplifications were performed with a thermal cycler (Techne Techgene, UK) with solutions, species-specific primers and temperature profiles described by Matsuki et al. (1999). PCR amplified products were analyzed by 1 % agarose gel electrophoresis at a constant voltage of 7 V/cm and visualized with ethidium bromide (0.5 μg/mL) under UV light (λ = 260 nm). FISH kit for Clostridium sp. (RiboTechnologies, The Netherlands) was used for identification of nonbifidobacterial isolates that were preliminarily considered as clostridia. RESULTS AND DISCUSSION Bifidobacterial counts were determined in twenty infants using culturing on selective media, and were from 108 to 1011 CFU/g. These counts correspond to the counts reported by Harmsen et al. (2000) and Matsuki et al. (1999). Thirteen infants were investigated using both, FISH and culturing. Bifidobacteria counts in fecal samples are shown in Table I. There were no significant differences among the data obtained using modified TPY agar and FISH. Our study showed that FISH procedure could be the method of reference for studies designed to enumerating bifidobacteria fecal samples. High selectivity was seen for modified TPY agar from which all of the isolates were F6PPK positive and were identified as bifidobacteria. Modified TPY agar could be used for the routine enumeration and isolation of bifidobacteria from feces. Three infants did not contain any bifidobacteria in their fecal samples. One of them was delivered by caesarean section and two by normal vaginal delivery. All bifidobacteria-free infants possessed Gram-positive regular rods as a major group of their fecal flora. These bacteria were identified as clostridia using genus specific FISH probe. The distribution of bifidobacterial species in breast-fed infants and their frequencies are shown in Table II. In bifidobacteria-positive samples, B. longum (57.9 % of the samples) was the most frequently found species, followed by B. adolescentis (31.6 %), B. bifidum (21.0 %), B. breve (10.5 %), B. pseudocatenulatum (5.3 %), and B. dentium (5.3 %). Table I. Bifidobacterial counts (log CFU/g) determined in infant fecesa using FISH and modified TPY agarb No. FISH method TPY agar Clostridia 1 10.1 10.7 – 2 10.4 10.6 – 3 10.5 10.6 – 4 10.6 10.8 – |
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| Language | English |
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| Content Type | Text |
| Resource Type | Article |
