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No sample of animal feces or sewage showed a positive result when hybridized with the BDE probe. We have studied the Gram-stained vaginal smears from pregnant women since 1991 [5] [6] [7] and during that time we began to question whether women with an intermediate vaginal flora, especially a Nugent Score of 4, should all be treated regardless of the types of bacteria observed in their vaginal smear. 3.2. The amplified 16S rRNA gene was fixed onto nylon membranes, and hybridization with the appropriate probe was performed according to the protocol described above. (29) also developed an identification methodology for five Bifidobacterium species present in the human intestine based on the use of 16S rRNA-targeted oligonucleotide probes.

In addition, the BAN probe was able to detect B. globosum when it was in a proportion of 1:102 with B. dentium (Fig. Pregnant women with the Bifidobacterium type were judged to require no treatment of their vaginal flora because Bifidobacterium spp. This work and the related PDF file are licensed under a Creative Commons Attribution 4.0 International License. Figure 4. The detection of B. dentium in urban sewage by the procedure described here demonstrates that this species is present in such samples. It is a question whether the Bifidobacterium spp. Moreover, none of these samples presented positive hybridization for the BAN probe. The subjects were 566 pregnant women who visited the Kitami Red Cross Hospital (KRCH) in Hokkaido. (26). Journal of Microbiology & Biology Education, Microbiology and Molecular Biology Reviews. The distribution of four bacterial morphotypes for 58 cases with a Nugent score of 4. results of the bacterial morphotypes and culture, prescribed antibiotics and tocolytics, medical history and pregnancy outcome.

All of the seven pregnant women of non-bacterial type (Figure 4) were transferred to our hospital because they were suspected to be preterm PROM; Six of them were actually preterm PROM, and five of them experienced preterm birth despite prophylactic antibiotic therapy recommended in the 2017 Guidelines for Obstetrical Practice in Japan and obstetrics textbooks [11] [12]. Benzyl chloride (150 μl) was added to each extraction, and then the extractions were incubated at 50°C for 30 min, with vigorous agitation every 5 min. Her vaginal bacterial flora was totally eradicated, because she had received antibiotic treatment as a potential preterm PROM patient at the previous hospital.

Whereas the animal strains were able to grow at 45°C, most of the human strains could not. This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Despite efforts to minimize fecal input into the water cycle, the problem persists because of inefficient sewage treatment plants, leaking septic systems, agricultural runoff, and wildlife (2). When the detection limits were evaluated with mixed bacterial suspensions, one target cell in 100 nontarget cells was detected in the case of the BAN probe and one target cell in 10,000 nontarget cells was detected in the case of the BDE probe. 1 0 obj
��=$4l�B�E��,uv�3w�w��ǰ��� ��y`;����[��b밹�l#� s��a��Q� �1�X�[�b��9Y�m��rpZFlq�+ຍ12{��:^�o�d�Y��Ө ��l&�x�D٨m�G��ut��L[?��9k�}�s�,\9�=�`�24wx All authors meet the ICMJE authorship criteria. Interfering species were B. globosum DSM 20092T for the BDE probe and B. dentium DSM 20084 for the BAN probe. All of the urban sewage samples presented positive hybridization with the BDE probe, except for one sample of hospital sewage and one sample of urban sewage. Clone j, related to B. dentium, was found in only one sample, as was clone g, Bifidobacterium spp., which did not reach the 98% threshold but had 97% similarity to B. adolescentis (Fig. This threshold is similar to that obtained in studies of other bacteria (7). Previous research has proposed that the genus Bifidobacterium be used to indicate the origin of fecal pollution (14, 15). The other probe, BAN, was able to detect a group of Bifidobacterium species isolated exclusively from blood-warm animals and honeybees (B. animalis, B. asteroides, B. coryneforme, B. cuniculi, and B. globosum), as well as two species isolated from sewage of uncertain origin (B. minimum and B. subtile). Figure 11. �⢂r-S��P��ɮ�wn~�lg!�mS��-k�����2���z�ka #=B�?��7����;Շ� &6��\i�+��*�~�ۀ& .� ʯܷ6��i쌿F߽�z���zE��� "c��s�Uf�e����aB�

DNA-DNA hybridization with the BDE probe showed it to be specific for B. dentium DSM 20084 (Table 1) when the hybridization temperature was 60°C. When PCR with primers lm26 and lm3 was used to amplify the 16S rRNA gene from human feces (infants and adults) all of the samples gave the expected amplimer of 1.35 kb.

Figure 6. We emphasize the importance of the Gram stain in urine samples and the addition of enriched media and enhanced atmosphere over time for urine cultures with pyuria. Finally, the DNA was suspended in 100 μl of TE (10 mM Tris-HCl, 1 mM EDTA; pH 8.0). The cotton swab was rolled on glass slides, and the specimen was stained by the Gram stain according to the Bartholomew and Mittwer method. In our study, there were five cases of Bifidobacterium type that resulted in term delivery with no therapy.

All animal samples were negative for the BDE probe except for one sample from a cattle slaughterhouse. Bifidobacterium spp. After centrifugation at 16,000 × g for 10 min, the supernatant was collected and DNA was obtained by isopropanol precipitation. A sterile cotton transfer swab (by COPAN ITALIA S. p. Two 16S rDNA-targeted probes were designed. Then, 150 μl of 3 M sodium acetate was added, and the mixtures were cooled on ice for 15 min. Additionally, 12 samples of animal sewage were taken from collectors of pig, cattle, and chicken slaughterhouses. Cell debris was removed by centrifugation at 16,000 × g, and 2 μl of the supernatant was used for PCR analysis. Anaerobic culture is not performed in routine practice, based on Hillier’s statement that “Cultures for G. vaginalis or other individual microbes have little utility for diagnosis of BV.” [13] Thus, in this study, confirmation of Bifidobacterium spp., which are anaerobic bacteria, is based on the bacterial morphotype instead of anaerobic culture, except for the diagnosis in case 7 (Figure 3), in whom Bifidobacterium spp. First, the pellets were suspended in 250 μl of extraction buffer (100 mM Tris-HCl, 40 mM EDTA; pH 9.0) and 50 μl of 10% sodium dodecyl sulfate (SDS) and then freeze-thawed. Adhesion to the intestinal mucus layer is considered a prerequisite for colonization by mucosa-associated bacteria and represents a selection criterion for probiotic microbes ().Given the importance of intestinal mucus at the microbe-mammal interface, we sought to identify whether Bifidobacterium dentium could adhere to and modulate intestinal mucins. Several microbial and biochemical approaches to fecal source identification have been investigated. These strains were isolated mainly from human samples. We adopt the Nugent scoring system of gram-stained vaginal smears for the diagnosis of BV in our daily practice, with additional aerobic culture for the confirmation of bacteria including GBS and E. coli. She recovered fully and was retransferred to the previous hospital.

Each Gram-stained smear was observed under a microscope at a magnification of X1000, and BV was diagnosed according to Nugent scoring system [8]. Number of positive amplifications and DNA-DNA hybridizations with Bif, BAN, and BDE probes. At least 22.4% of them are the gram-positive cocci type and Bifidobacterium type which should not be involved in BV. The use of molecular rather than culture-based methods to detect them could overcome the problems associated with growing strict anaerobes. Therefore, this finding supports the potential use of this probe in detecting fecal pollution of human origin. Among these, there were 38 cases (65.5%) of mixed-type, 3 cases (5.2%) of gram-positive cocci type, 10 cases (17.2%) of Bifidobacterium type, and 7 cases (12.1%) of non-bacterial type (Figure 9).

In order to increase the sensibility of the BAN and BDE probes, a PCR amplification step was added before hybridization.

Some Bifidobacterium species are of human origin, whereas others are exclusively found in animals (3, 24).

The most widely used fecal indicator microorganisms (coliforms, fecal coliforms, Escherichia coli, and enterococci) are found in both human and animal feces and thus do not allow source differentiation. Also, in two of the cases with the GBS vaginal flora, the patients suffered threatened preterm labor; they were prescribed penicillin and tocolytics, and their clinical course resulted in term delivery. DNA extraction from pure cultures.DNA extraction from bifidobacteria was performed by using a previously described protocol (11). A total of 58 patients of the 566 cases had a Nugent score of 4 (Figure 8).

Another characteristic of this genus is the different ecological distribution of its species. The use of primers or probes based on the ribosomal DNA (rDNA) sequence has been useful in the detection and identification of certain species of Bifidobacterium in mixed populations, which could be difficult and not always feasible by phenotypic characterization (5, 20, 23, 27). Gram-stained vaginal smear from the pregnant woman of case 1 (shown in Table 1). All of the three cases resulted in term deliveries. Therefore, this probe cannot be used to detect fecal pollution of animal origin. In particular, while pregnant patients with GBS and preterm PROM have been treated according to obstetrical textbooks, therapeutic approaches for Bifidobacterium spp.

Three experimental temperatures (Tm, Tm −5°C, and Tm, +5°C) were used to hybridize dot blot membranes with the target species and possible interfering species. All samples were transported at 4°C to the laboratory. Preparation of purified bacterial DNA from the Gram-stained vaginal smear.

This smear is called “mixed-type” as it consists of 4+ Lactobacillus morphotypes and 4+ Gardnerella spp.

DNA-DNA hybridization with the BAN probe was only positive for samples from cattle and goats. The BDE target species, B. dentium, was detected up to a concentration of ca.

The detection of human-related Bifidobacterium species in a polluted sample could therefore indicate the human origin of the fecal pollution. Bifidobacterium species are difficult to identify and may be underreported or not recovered by many laboratories because of their slow growth. Supposedly, the bacteria had been washed out by amniotic fluid during the preterm PROM. One of the most widely used molecular approaches in ecological and taxonomic studies is the use of the rRNA molecule and its gene as a target (4, 9, 13, 31). The distribution of Nugent scores for 566 cases. or Bacteroides spp. Mara and Oragui (15) described a new selective medium, human bifid-sorbitol agar, which was able to isolate sorbitol-fermenting strains.

Of the 29 samples of human sewage amplified by PCR, 27 gave a positive signal for DNA-DNA hybridization with the BDE probe, whereas none did so for hybridization with the BAN probe. In three gram-positive cocci type cases, two cases of GBS (cases 1, 2) and one case of enterococcus spp. Bacterial strains and growth conditions.Bacterial strains used in the present study are listed in Table 1.

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