Actual distribution of bacteriocytes in the trophosome of a beard worm (Oligobrachia mashikoi, Siboglinidae, Annelida): clarification using whole-mount in situ hybridization

Beard worms (Siboglinidae, Polychaeta) lack a mouth and a digestive tract and harbour chaemosynthetic bacteria in the bacteriocytes of the trophosome. Since beard worms depend on the organic compounds produced by the bacteria for nourishment, the bacteriocytes should be efficient in exchanging various substances with body fluids. For this reason, it is important to determine how the bacteriocytes are organized in the trophosome. As the first step of the present study, the appearance of bacteriocytes was examined in routinely stained paraffin sections. Second, visualization of the actual distribution of the bacteriocytes was attempted using whole‐mount in situ hybridization with a probe of the 16S rRNA nucleotide sequence of the bacterium. After routine haematoxylin & eosin staining, the bacteriocytes appeared to be aligned in cell cords accompanied with nutrient‐deposit cells that extended from both sides of the trophosome toward the dorsal side and folded up in the coelomic spaces. In whole‐mount preparations, however, bacteriocytes with intense signals of 16S rRNA were seen three‐dimensionally as many irregular leaves arranged from both sides of the ventral vessel toward the dorsal vessel. We will discuss the physiological significance of this characteristic distribution of the bacteriocytes in the present species.     

荧光原位杂交技术在医学微生物学中的应用

以16S rRNA 为靶序列的寡核苷酸探针荧光原位杂交技术已广泛应用于分析复杂环境中的微生物群落构成,包括监测和鉴定病原微生物以及未被培养微生物．通过对临床样品中微生物细胞的检测能提供微生物在人体中的种类、数量和空间分布等信息．其结果快速准确,较之传统的病原微生物诊断方法具有明显的优越性,在临床应用中有广泛的前景．     

Seven-hour fluorescence in situ hybridization technique for enumeration of Enterobacteriaceae in food and environmental water sample

AIMS: A fluorescent in situ hybridization (FISH) technique using an Enterobacteriaceae-specific probe (probe D) to target 16S rRNA was improved in order to enumerate, within a single working day, Enterobacteriaceae present in food and environmental water samples. METHODS AND RESULTS: In order to minimize the time required for the FISH procedure, each step of FISH with probe D was re-evaluated using cultured Escherichia coli. Five minutes of ethanol treatment for cell fixation and hybridization were sufficient to visualize cultured E. coli, and FISH could be performed within 1 h. Because of the difficulties in detecting low levels of bacterial cells by FISH without cultivation, a FISH technique for detecting microcolonies on membrane filters was investigated to improve the bacterial detection limit. FISH with probe D following 6 h of cultivation to grow microcolonies on a 13 mm diameter membrane filter was performed, and whole Enterobacteriaceae microcolonies on the filter were then detected and enumerated by manual epifluorescence microscopic scanning at magnification of x100 in ca 5 min. The total time for FISH with probe D following cultivation (FISHFC) was reduced to within 7 h. FISHFC can be applied to enumerate cultivable Enterobacteriaceae in food (above 100 cells g-1) and environmental water samples (above 1 cell ml-1). CONCLUSIONS: Cultivable Enterobacteriaceae in food and water samples were enumerated accurately within 7 h using the FISHFC method. SIGNIFICANCE AND IMPACT OF THE STUDY: A FISHFC method capable of evaluating Enterobacteriaceae contamination in food and environmental water within a single working day was developed.     

Design of species-specific oligonucleotide probes for the detection of Bacteroides and Parabacteroides by fluorescence in situ hybridization and their application to the analysis of mouse caecal Bacteroides-Parabacteroides microbiota

Aims: To develop species‐specific monitoring techniques for rapid detection of Bacteroides and Parabacteroides inhabiting the mouse intestine by fluorescence in situ hybridization. Methods and Results: The specificity of oligonucleotide probes was evaluated by fluorescence whole‐cell hybridization. Oligonucleotide probes specific for each species hybridized only with the target bacteria. Using these probes, caecal Bacteroides–Parabacteroides microbiota of conventional mice and specific pathogen‐free (SPF) mice from three different breeders were analysed. It was shown that Bacteroides acidifaciens Group‐1, Group‐2 and Group‐3 were dominant in conventional mice and SPF mice from two out of three breeders. Bacteroides vulgatus and Parabacteroides distasonis were detected in one of these two SPF breeding colonies in addition to Bact. acidifaciens. SPF mice of the remaining breeder harboured characteristic Bacteroides–Parabacteroides microbiota, consisting of Bacteroides sp. ASF519 and Bacteroides caccae. Conclusions: Bacteroides acidifaciens is the dominant and most typical species in the mouse Bacteroides–Parabacteroides microbiota. The Group‐3 was identified as a novel group and revealed to occupy a major niche together with Bact. acidifaciens Group‐1 and Group‐2. Significance and Impact of the Study: The species‐specific probe set developed in this study was the efficient tool for rapid detection of target bacterial groups inhabiting the mouse intestine. The results of this study provide important new information on the mouse Bacteroides–Parabacteroides community.     

Quantification of Leuconostoc populations in mixed dairy starter cultures using fluorescence in situ hybridization

AIMS: Development of a rapid method to identify and quantify Leuconostoc populations in mesophilic starter cultures. METHODS AND RESULTS: 16S rRNA-targeted oligonucleotide probes were used in a whole cell in situ hybridization assay for the identification of the genus Leuconostoc and an undescribed Leuconostoc ribospecies. The probes were fluorescently labelled and used to quantify the Leuconostoc populations in five different mixed starter cultures. CONCLUSIONS: There was a good correlation between the results obtained using fluorescence in situ hybridization (FISH) with that of standard plate counting methods. SIGNIFICANCE AND IMPACT OF THE STUDY: To develop a FISH method capable of identifying and quantifying the Leuconostoc population in starter cultures within 1 day.     

Spatial distribution of bacterial phylotypes in the gut of the termite Reticulitermes speratus and the bacterial community colonizing the gut epithelium

The bacterial community colonizing the gut wall of the termite Reticulitermes speratus was characterized without cultivation. Analysis of 16S rRNA genes after fractionation of the gut revealed that the bacterial composition on the gut wall was diverse and significantly different from that able to move unconfined in the gut fluid or physically associated with the gut protists. Actinobacteria, Firmicutes and Bacteroidetes were dominant on the gut wall, but Spirochaetes and the Termite group 1 phylum, abundant in the gut lumen, were relatively rare. A sequence-specific probe enabled the in situ detection of a rod-shaped Actinobacteria member, abundantly colonizing the gut paunch epithelium.     

Specific detection of Pseudomonas spp. in milk by fluorescence in situ hybridization using ribosomal RNA directed probes

AIMS: Pseudomonas spp. are considered the most important milk spoilage organisms. Here we describe development of a fluorescence in situ hybridization (FISH) probe specific for detection and enumeration of Pseudomonas spp. in milk. METHODS AND RESULTS: 16S rRNA sequences were analysed to develop specific oligonucleotide probe for the genus Pseudomonas. Twenty different Pseudomonas spp. and 23 bacterial species from genera other than Pseudomonas (as negative controls) were tested. All tested Pseudomonas spp. yielded a positive FISH reaction, whereas negative controls showed no FISH reaction except for Burkholderia cepacia that showed a relatively weak FISH reaction. The FISH assay specifically stains Pseudomonas in milk when the milk contains a mixture of other bacterial species. The FISH assay takes 2 h and compares favourably with current culturing methods, which take a minimum of 48 h. Specificity of the probe was validated using polymerase chain reaction to selectively amplifying the Pseudomonas rDNA gene and sequencing the gene products. CONCLUSIONS: The method presented in this study allows simultaneously detection, identification and enumeration of Pseudomonas spp. in milk. SIGNIFICANCE AND IMPACT OF THE STUDY: Rapid and accurate enumeration of Pseudomonas facilitates the identification of specific contamination sources in dairy plants, the accurate validation of pasteurization treatments and the prediction of shelf life of processed milk.     

FISH技术在微生物生态学中的研究及进展

分子生物学技术在微生物生态学研究中具有灵敏、精确和快速的优势,但不能提供微生物的形态学、数量性状、空间分布等信息。荧光原位杂交技术结合了分子生物学的精确性和显微镜的可视性信息,可以在自然生境中监测和鉴定不同的微生物个体,尤其是对难培养和未被培养的微生物进行检测。荧光原位杂交技术被广泛用于微生物群落结构诊断和评价,现已成为微生物分子生态学研究中的热点技术。对荧光原位杂交技术的发展和在微生物分子生态学中的应用进行了综述,探讨了该技术应用中存在的问题和发展前景。     

Application of cation-exchange membranes for characterisation and imaging ammonia-oxidising bacteria in soils

A new approach, in which ammonia-oxidizing bacteria (AOB) are entrapped from soil onto cation-exchange membranes, was applied to identify terrestrial AOB by fluorescence in situ hybridization (FISH). An experimental hot spot of ammonia oxidation was developed by establishing a gradient of ammonium substrate (200 to <20 mg NH4+-N l(-1)) diffused through the cation-exchange membranes incubated in soil for 6 months. By this approach we were able to characterise and image indigenous AOB populations growing in heavily oil-polluted soil using FISH and sequence analysis of PCR-amplified 16S rRNA genes, respectively. The FISH results revealed that Nitrosospira-like AOB were dominant on the ammonium-enriched membranes incubated in the soil. Fourteen unique Nitrosospira-like 16S rRNA gene sequences belonging to clusters 2 and 3 were recovered from the soil-incubated membranes and from the soil, suggesting the importance of Nitrosospira-like AOB in the oil-polluted landfarming soil.     

用电镜原位杂交技术对玉米中期染色体中RNA的研究

用生物素标记的玉米１８ＳｒＲＮＡ、小麦５ＳｒＲＮＡ 及ｔＲＮＡ 的ｃＤＮＡ 作为探针,在Ｋ４Ｍ 树脂包埋的玉米（Ｚｅａ ｍ ａｙｓ）根尖超薄切片上进行原位杂交。杂交后,用与１０ ｎｍ 金颗粒相连的亲和素对杂交子在电镜下进行检测。结果发现,在玉米中期染色体中存在有１８ＳｒＲＮＡ、５ＳｒＲＮＡ 和ｔＲＮＡ 分子,这些ＲＮＡ分子在染色体中的分布是随机的,即这些ＲＮＡ 分子在染色体的内部和周边均有分布。５ＳｒＲＮＡ 和ｔＲ－ＮＡ 在染色体中和细胞质中的含量基本相等,而１８ＳｒＲＮＡ 在染色体中的含量则明显高于细胞质。这一结果表明染色体中的ＲＮＡ 一部分来自于核仁,而另一部分则来自于核质     

Identification of bacterial diversity in the oyster Crassostrea gigas by fluorescent in situ hybridization and polymerase chain reaction

AIMS: To carry out a rapid and reliable identification of bacterial diversity in the oyster Crassostrea gigas from Todos Santos Bay, México, in the current study we applied the molecular techniques of fluorescent in situ hybridization (FISH) and polymerase chain reaction (PCR). In order to reach this goal, genus and group-specific oligonucleotides targeted to 16S rDNA/rRNA were used. METHODS AND RESULTS: Oysters were collected and different tissues were analysed by means of culture-independent methodologies. In the digestive glands and gonads gamma-Proteobacteria and Gram-positive bacteria with a low G+C content, were identified as metabolically active by FISH. In the oyster gills a higher active diversity was observed, including Gram-positive bacteria with a low and high G+C content, members of the Cytophaga/Flavobacterium cluster and gamma-Proteobacteria. Consistent with FISH analysis, the amplification of 16S rDNA genes fragments with genus and group-specific oligonucleotides confirmed the presence of the same groups, as well as members of the alpha- and beta-Proteobacterias, Pseudomonas spp. and Bacillus spp. CONCLUSIONS: The combination of accurate and very easy-to-apply molecular methods allowed us to carry out a rapid screening of high bacterial diversity in oysters. SIGNIFICANCE AND IMPACT OF THE STUDY: This work is the first report about bacterial diversity in oyster tissues analysed by FISH and PCR, without using culture-dependent methods and allowed us to determine the phylogenetic diversity of the bacterial communities present in oyster cultures, including bacteria with and without metabolic activity, as well as uncultivable cells, which are generally underestimated by traditional identification.