Bifidobacteria from the Faeces of Piglets

S ummary . Ninety-five strains of bifidobacteria isolated from 52 specimens of piglet faeces collected at 19 farms were studied. The main phenotypic characters of the strains were determined; however their assignment to known species of the genus Bifidobacterium was based primarily on their deoxyribonucleic acid homology relationships following DNA-DNA hybridization tests. The majority of the strains were recognized as Bifidobacterium suis Matteuzzi et al. Some strains could not be assigned to any known species of the genus so they were allotted provisionally to 2 unassigned bacterial groups.     

Enumeration of Escherichia coli in Frozen Samples After Recovery from Injury

More than 90% of the surviving cells of Escherichia coli NCSM were injured after freezing in water at -78 C. Injury was determined by the ability of cells to form colonies on Trypticase soy agar with yeast extract but not on violet red-bile agar and deoxycholate-lactose agar. Exposure of the injured cells to Brilliant Green-bile broth and lauryl sulfate broth prevented subsequent colony formation on Trypticase soy agar with yeast extract. The freeze-injury could be repaired rapidly in a medium such as Trypticase soy broth with yeast extract (TSYB). The repaired cells formed colonies on violet red-bile agar and deoxycholate-lactose agar and were not inhibited by Brilliant Green-bile broth and lauryl sulfate broth. At least 90% of the cells repaired in TSYB within 30 min at 20 to 45 C and began multiplication within 2 h at 25 C. When the cells were frozen in different foods, 60 to 90% of the survivors were injured. Repair of the injured cells occurred in foods during 1 h at 25 C, but generally repair was greater and more reproducible when the foods were incubated in TSYB. The study indicated that the repair of freeze-injured coliform bacteria should be accomplished before such cells are exposed to selective media for their enumeration.     

Development of an Enumeration Method for Arsenic Methylating Bacteria from Mixed Culture Samples

Bacterial methylation of arsenic converts inorganic arsenic into volatile and non-volatile methylated species. It plays an important role in the arsenic cycle in the environment. Despite the potential environmental significance of AsMB, an assessment of their population size and activity remains unknown. This study has now established a protocol for enumeration of AsMB by means of the anaerobic-culture-tube, most probable number (MPN) method. Direct detection of volatile arsenic species is then done by GC-MS. This method is advantageous as it can simultaneously enumerate AsMB and acetate and formate-utilizing methanogens. The incubation time for this method was determined to be 6 weeks, sufficient time for AsMB growth.     

New Real-Time Quantitative PCR Procedure for Quantification of Bifidobacteria in Human Fecal Samples

The application of a real-time quantitative PCR method (5′ nuclease assay), based on the use of a probe labeled at its 5′ end with a stable, fluorescent lanthanide chelate, for the quantification of human fecal bifidobacteria was evaluated. The specificities of the primers and the primer-probe combination were evaluated by conventional PCR and real-time PCR, respectively. The results obtained by real-time PCR were compared with those obtained by fluorescent in situ hybridization, the current gold standard for intestinal microbiota quantification. In general, a good correlation between the two methods was observed. In order to determine the detection limit and the accuracy of the real-time PCR procedure, germfree rat feces were spiked with known amounts of bifidobacteria and analyzed by both methods. The detection limit of the method used in this study was found to be about 5 × 10⁴ cells per g of feces. Both methods, real-time PCR and fluorescent in situ hybridization, led to an accurate quantification of the spiked samples with high levels of bifidobacteria, but real-time PCR was more accurate for samples with low levels. We conclude that the real-time PCR procedure described here is a specific, accurate, rapid, and easy method for the quantification of bifidobacteria in feces.     

Restriction endonucleases from Bifidobacteria

The sitespecific restriction endonucleases were found in four strains among the twelve strains of anaerobic bacteria of generum Bifidobacterium. Two of the restriction endonucleases studied, BadI from B. adolescentis LVA1 and BbfI from B. bifidum LVA3, are isoshizomers of XhoI and recognize the nucleotide sequence CTCGAG. The restriction endonucleases Bbf7411I from B. bifidum 7411 and Bla7920I from B. lactentis 7920 recognize and hydrolize the nucleotide sequence TCCGGA having the specifity analogous to the one of restriction endonuclease CauB3I. Like CauB3I, these restriction endonucleases are unable to hydrolyize DNA if the adenine residues in the recognition site are methylated.     

Enumeration of Marine Viruses in Culture and Natural Samples by Flow Cytometry

Flow cytometry (FCM) was successfully used to enumerate viruses in seawater after staining with the nucleic acid-specific dye SYBR Green-I. The technique was first optimized by using the Phaeocystis lytic virus PpV-01. Then it was used to analyze natural samples from different oceanic locations. Virus samples were fixed with 0.5% glutaraldehyde and deep frozen for delayed analysis. The samples were then diluted in Tris-EDTA buffer and analyzed in the presence of SYBR Green-I. A duplicate sample was heated at 80°C in the presence of detergent before analysis. Virus counts obtained by FCM were highly correlated to, although slightly higher than, those obtained by epifluorescence microscopy or by transmission electron microscopy (r = 0.937, n = 14, and r = 0.96, n = 8, respectively). Analysis of a depth profile from the Mediterranean Sea revealed that the abundance of viruses displayed the same vertical trend as that of planktonic cells. FCM permits us to distinguish between at least two and sometimes three virus populations in natural samples. Because of its speed and accuracy, FCM should prove very useful for studies of virus infection in cultures and should allow us to better understand the structure and dynamics of virus populations in natural waters.     

Mining the Microbiota of the Neonatal Gastrointestinal Tract for Conjugated Linoleic Acid-Producing Bifidobacteria

This study was designed to isolate different strains of the genus Bifidobacterium from the fecal material of neonates and to assess their ability to produce the cis-9, trans-11 conjugated linoleic acid (CLA) isomer from free linoleic acid. Fecal material was collected from 24 neonates aged between 3 days and 2 months in a neonatal unit (Erinville Hospital, Cork, Ireland). A total of 46 isolates from six neonates were confirmed to be Bifidobacterium species based on a combination of the fructose-6-phosphate phosphoketolase assay, RAPD [random(ly) amplified polymorphic DNA] PCR, pulsed-field gel electrophoresis (PFGE), and partial 16S ribosomal DNA sequencing. Interestingly, only 1 of the 11 neonates that had received antibiotic treatment produced bifidobacteria. PFGE after genomic digestion with the restriction enzyme XbaI demonstrated that the bifidobacteria population displayed considerable genomic diversity among the neonates, with each containing between one and five dominant strains, whereas 11 different macro restriction patterns were obtained. In only one case did a single strain appear in two neonates. All genetically distinct strains were then screened for CLA production after 72 h of incubation with 0.5 mg of free linoleic acid ml⁻¹ by using gas-liquid chromatography. The most efficient producers belonged to the species Bifidobacterium breve, of which two different strains converted 29 and 27% of the free linoleic acid to the cis-9, trans-11 isomer per microgram of dry cells, respectively. In addition, a strain of Bifidobacterium bifidum showed a conversion rate of 18%/μg dry cells. The ability of some Bifidobacterium strains to produce CLA could be another human health-promoting property linked to members of the genus, given that this metabolite has demonstrated anticarcinogenic activity in vitro and in vivo.     

Biological Activity of Polar Lipids from Bifidobacteria

Fractions of polar lipids have been isolated from bifidobacteria, and the immunoreactivity and serological specificity of glycolipids and phospholipids have been studied. Enzyme immunoassay (dot-EIA) of polar lipids demonstrates that the fractions of glycolipids and phospholipids of bifidobacteria are highly immunoreactive. Pronounced reactions of major glycolipids and phospholipids with a homologous polyvalent antiserum against B. adolescentis 94-BIM have been observed at antigen concentrations of approximately 100 ng. The antiserum contained high titers of specific antibodies against glycolipids and phospholipids of bifidobacteria, as demonstrated by heterogeneous solid-phase enzyme immunoassay (ELISA). Experimental data confirming the presence of subpopulations of specific antibodies (antiglycolipid and antiphospholipid) in the blood sera of immunized animals lead to the conclusion that the major glycolipids and phospholipids of bifidobacteria are specific markers appropriate for serological diagnostics.     

Isolation of Salmonellae from Foods Samples: V. Determination of the Method of Choice for Enumeration of Salmonella

Comparison of three methods by which salmonellae may be isolated and enumerated from dried albumen, direct inoculation of enrichment media, centrifugation of samples, and pre-enrichment in noninhibitory media, reveals pre-enrichment to be the method of choice.

The superiority of pre-enrichment manifests itself in replicate aliquots of the same sample by producing a statistically significant increase in numbers of isolations of salmonellae and in empirical use with various albumen samples by consistently higher values of most probable numbers (MPN).

The primary factor involved in this superiority appears to be the greater ability of small numbers of salmonellae to initiate growth in the nonselective mannitol purple sugar broth than in the inhibitory enrichment media.

The method of analysis recommended entails inoculation of mannitol broth pre-enrichment medium, transfer of 24-hr culture aliquots to tetrathionate broth, and streaking on brilliant green agar for isolation of salmonellae.

     

Enumeration of Salmonella and Campylobacter spp. in Environmental Farm Samples and Processing Plant Carcass Rinses from Commercial Broiler Chicken Flocks

A prospective cohort study was performed to evaluate the prevalences and loads of Salmonella and Campylobacter spp. in farm and processing plant samples collected from 55 commercial broiler chicken flocks. Environmental samples were collected from broiler houses within 48 h before slaughter, and carcass rinses were performed on birds from the same flocks at 4 different stages of processing. Salmonella was detected in farm samples of 50 (90.9%) flocks and in processing samples of 52 (94.5%) flocks. Campylobacter was detected in farm samples of 35 (63.6%) flocks and in processing samples of 48 (87.3%) flocks. There was a significant positive relationship between environmental farm samples and processing plant carcass rinses with respect to both Salmonella and Campylobacter prevalences and loads. Campylobacter loads were significantly higher than Salmonella loads, and the correlations between samples collected from the same flocks were higher for Campylobacter than they were for Salmonella. Boot socks were the most sensitive sample type for detection of Salmonella on the farm, whereas litter samples had the strongest association with Salmonella loads in pre- and postchill carcass rinses. Boot socks, drag swabs, and fecal samples all had similar sensitivities for detecting Campylobacter on the farm, and all were more strongly associated with Campylobacter loads in carcass rinses than were litter samples. Farm samples explained a greater proportion of the variability in carcass rinse prevalences and loads for Campylobacter than they did for Salmonella. Salmonella and Campylobacter prevalences and loads both decreased significantly as birds progressed through the processing plant.     

Carbohydrate metabolism in Bifidobacteria

Members of the genus Bifidobacterium can be found as components of the gastrointestinal microbiota, and are believed to play an important role in maintaining and promoting human health by eliciting a number of beneficial properties. Bifidobacteria can utilize a diverse range of dietary carbohydrates that escape degradation in the upper parts of the intestine, many of which are plant-derived oligo- and polysaccharides. The gene content of a bifidobacterial genome reflects this apparent metabolic adaptation to a complex carbohydrate-rich gastrointestinal tract environment as it encodes a large number of predicted carbohydrate-modifying enzymes. Different bifidobacterial strains may possess different carbohydrate utilizing abilities, as established by a number of studies reviewed here. Carbohydrate-degrading activities described for bifidobacteria and their relevance to the deliberate enhancement of number and/or activity of bifidobacteria in the gut are also discussed in this review.     

Enumeration of bifidobacteria in gastrointestinal samples from piglets

The population of Bifidobacterium spp. in fecal samples from suckling piglets was investigated, and Beerens, raffinose-bifidobacterium (RB), and modified Wilkins-Chalgren (MW) agar media were evaluated with regard to the enumeration of bifidobacteria in porcine intestinal samples. The results demonstrated that the population of bifidobacteria in the feces of suckling piglets is numerically low, and a phylogenetic analysis of the 16S rRNA gene from bifidobacterial isolates suggested that a possibly new Bifidobacterium species was isolated. Beerens, RB, and MW agar media were not selective for bifidobacteria in the fecal samples. The highest recovery and diversity of bifidobacteria were obtained for MW agar. Nonbifidobacterial isolates from the three agar media were identified and may contribute to the future formulation of improved selective media for the enumeration of bifidobacteria.     

Exploring Vertical Transmission of Bifidobacteria from Mother to Child

Passage through the birth canal and consequent exposure to the mother''s microbiota is considered to represent the initiating event for microbial colonization of the gastrointestinal tract of the newborn. However, a precise evaluation of such suspected vertical microbiota transmission has yet to be performed. Here, we evaluated the microbiomes of four sample sets, each consisting of a mother''s fecal and milk samples and the corresponding infant''s fecal sample, by means of amplicon-based profiling supported by shotgun metagenomics data for two key samples. Notably, targeted genome reconstruction from microbiome data revealed vertical transmission of a Bifidobacterium breve strain and a Bifidobacterium longum subsp. longum strain from mother to infant, a notion confirmed by strain isolation and genome sequencing. Furthermore, PCR analyses targeting unique genes from these two strains highlighted their persistence in the infant gut at 6 months. Thus, this study demonstrates the existence of specific bifidobacterial strains that are common to mother and child and thus indicative of vertical transmission and that are maintained in the infant for at least relatively short time spans.     

鸡源双歧杆菌定向选育和发酵参数研究

目的从肉仔鸡肠道中筛选出耐酸、耐胆盐和耐消化酶的优良双歧杆菌,研究其生长特性,并优化其发酵参数,为转化生产力提供理论依据。方法通过无菌采样并分离得到多株双歧杆菌,对分离获得的双歧杆菌进行形态学、生化特性研究,然后采用牛津杯法,测定90株双歧杆菌对大肠埃希菌和沙门菌的抑制作用,采用改良MRS培养基,模拟鸡胃肠道逆环境,对其耐消化道特性进行研究,筛选出优良双歧杆菌,再进行生长特性研究及发酵参数优化。结果从肉仔鸡肠道分离出90株双歧杆菌,初步挑选出23株作为候选菌株,抑菌试验测得双歧杆菌B1、B2和B3具有良好的抑菌效果,然后经过耐受消化道逆环境试验,发现B2菌株的耐受能力最好,初步鉴定双歧杆菌B2为小鸡双歧杆菌,并将其定名为Bifidobacterium pullorum B2,对其生长特性的研究发现经18 h发酵细菌总数可以从8.3×105CFU/mL升高到1.3×109CFU/mL,运用优化的发酵培养基进行中试试验,发酵后的活菌数可达1.41×1010CFU/mL。结论本实验从肉仔鸡肠道中分离筛选并初步鉴定了Bifidobacterium pullorum B2,优化了制备Bifidobacterium pullorum B2发酵液的发酵条件,降低了生产成本。     

Influence of Dietary Zinc Oxide and Copper Sulfate on the Gastrointestinal Ecosystem in Newly Weaned Piglets

Dietary doses of 2,500 ppm ZnO-Zn reduced bacterial activity (ATP accumulation) in digesta from the gastrointestinal tracts of newly weaned piglets compared to that in animals receiving 100 ppm ZnO-Zn. The amounts of lactic acid bacteria (MRS counts) and lactobacilli (Rogosa counts) were reduced, whereas coliforms (MacConkey counts) and enterococci (Slanetz counts, red colonies) were more numerous in animals receiving the high ZnO dose. Based on 16S rRNA gene sequencing, the colonies on MRS were dominated by three phylotypes, tentatively identified as Lactobacillus amylovorus (OTU171), Lactobacillus reuteri (OTU173), and Streptococcus alactolyticus (OTU180). The colonies on Rogosa plates were dominated by the two Lactobacillus phylotypes only. Terminal restriction fragment length polymorphism analysis supported the observations of three phylotypes of lactic acid bacteria dominating in piglets receiving the low ZnO dose and of coliforms and enterococci dominating in piglets receiving the high ZnO dose. Dietary doses of 175 ppm CuSO₄-Cu also reduced MRS and Rogosa counts of stomach contents, but for these animals, the numbers of coliforms were reduced in the cecum and the colon. The influence of ZnO on the gastrointestinal microbiota resembles the working mechanism suggested for some growth-promoting antibiotics, namely, the suppression of gram-positive commensals rather than potentially pathogenic gram-negative organisms. Reduced fermentation of digestible nutrients in the proximal part of the gastrointestinal tract may render more energy available for the host animal and contribute to the growth-promoting effect of high dietary ZnO doses. Dietary CuSO₄ inhibited the coliforms and thus potential pathogens as well, but overall the observed effect of CuSO₄ was limited compared to that of ZnO.     

Comparative Evaluation of Quantitative Polymerase Chain Reaction Methods for Routine Enumeration of Specific Bacterial DNA in Aquatic Samples

Summary Three quantitative polymerase chain reaction (PCR) methods, the internal standard method (IS-PCR), competitive PCR (cPCR) and most probable number-PCR (MPN-PCR), were compared in terms of their ability to quantify specific bacterial DNA in environmental samples. Serially diluted Pseudomonas putida BH, the target bacterium, was inoculated into sterilized potassium phosphate buffer (PPB), river water and activated sludge, total DNA was extracted, and the number of pheB genes carried by P. putida BH in each sample was enumerated. IS-PCR and cPCR could not quantify the pheB gene at low concentrations (1.0 × 10³ copies ml^-1 in all samples and 1.0 × 10⁴ copies ml^--1 in some samples) and tended to give overestimations because of differences in amplification efficiencies between pheB gene and the internal standard/competitor in a reaction tube. Although reproducibility of MPN-PCR was slightly lower than that of the other two methods, MPN-PCR was the most sensitive, enabling us to quantify the pheB gene at 1.0 × 10³ copies ml^--1, and it had a good correlation with the inoculum size of P. putida BH. These results suggest that MPN-PCR is the best suited for routine microbial monitoring in natural environmental samples because of the simple handling, the ease of modification as occasion demands and the wide detection range, especially at low cell densities of the target microbe.