Recovery of Previously Uncultured Bacterial Genera from Three Mediterranean Sponges

Sponges often harbour a dense and diverse microbial community. Presently, a large discrepancy exists between the cultivable bacterial fraction from sponges and the community in its natural environment. Here, we aimed to acquire additional insights into cultivability of (previously uncultured) bacteria from three sponge species, namely Aplysina aerophoba, Corticium candelabrum and Petrosia ficiformis, by studying bacterial growth on five media in the form of 60 communities scraped from plates without antibiotics, as well as in the form of individual isolates that were grown on these media supplemented with antibiotics. We applied (double-)barcoded 16S ribosomal RNA (rRNA) gene amplicon sequencing for species identification. We show that previously uncultured bacteria can be cultivated using conventional plating and that application of antibiotics in the media can serve to capture a greater bacterial diversity. Moreover, we present criteria to address an important caveat of the plate scraping method whereby bacteria may be detected that did not actually grow. Fourteen out of 27 cultivated novel taxa (<95% identity of the 16S rRNA gene amplicon to reported species) belong to Actinobacteria, which indicates the presence of a large untapped reservoir of bioactive compounds. Three Flavobacteriaceae spp. were isolated that potentially constitute two new genera and one new species.     

In Vitro Culture of Previously Uncultured Oral Bacterial Phylotypes

Around a third of oral bacteria cannot be grown using conventional bacteriological culture media. Community profiling targeting 16S rRNA and shotgun metagenomics methods have proved valuable in revealing the complexity of the oral bacterial community. Studies investigating the role of oral bacteria in health and disease require phenotypic characterizations that are possible only with live cultures. The aim of this study was to develop novel culture media and use an in vitro biofilm model to culture previously uncultured oral bacteria. Subgingival plaque samples collected from subjects with periodontitis were cultured on complex mucin-containing agar plates supplemented with proteose peptone (PPA), beef extract (BEA), or Gelysate (GA) as well as on fastidious anaerobe agar plus 5% horse blood (FAA). In vitro biofilms inoculated with the subgingival plaque samples and proteose peptone broth (PPB) as the growth medium were established using the Calgary biofilm device. Specific PCR primers were designed and validated for the previously uncultivated oral taxa Bacteroidetes bacteria HOT 365 and HOT 281, Lachnospiraceae bacteria HOT 100 and HOT 500, and Clostridiales bacterium HOT 093. All agar media were able to support the growth of 10 reference strains of oral bacteria. One previously uncultivated phylotype, Actinomyces sp. HOT 525, was cultivated on FAA. Of 93 previously uncultivated phylotypes found in the inocula, 26 were detected in in vitro-cultivated biofilms. Lachnospiraceae bacterium HOT 500 was successfully cultured from biofilm material harvested from PPA plates in coculture with Parvimonas micra or Veillonella dispar/parvula after colony hybridization-directed enrichment. The establishment of in vitro biofilms from oral inocula enables the cultivation of previously uncultured oral bacteria and provides source material for isolation in coculture.     

2株瘤胃纤维降解细菌的分离鉴定

从日粮精粗比为3:7的小尾寒羊×蒙古羊杂交一代绵羊的瘤胃内容物中分离到2株严格厌氧细菌,一株球菌WQ-1,1株弧菌WH-2,二者对滤纸有很好的降解能力。通过酶活力试验测得WQ-1的滤纸酶活、羧甲基纤维素酶活和β-葡萄糖苷酶活分别为0.66,7.0 U/mL和15.3 U/mL;WH-2的滤纸酶活、羧甲基纤维素酶活和β-葡萄糖苷酶活分别为0.52,6.9 U/mL和17.2 U/mL。经形态学、生理生化反应、生态特性和遗传型的鉴定,WQ-1归类为瘤胃球菌属(Ruminococcus)的黄色瘤胃球菌(Ruminococcus flavefaciens)。WH-2归类为丁酸弧菌属(Butyrivibrio)的溶纤维丁酸弧菌(Butyrivibrio fibrisolvens)。     

Enumeration and Isolation of Lactate-Utilizing Bacteria from the Rumen of Sheep

A highly specific medium was developed for the enumeration of lactate-utilizing bacteria in the rumen of sheep. This medium, which contained 2.0% lactate, 2.0% Trypticase, 0.2% yeast extract, and volatile fatty acids, hemin, and trace elements in place of rumen fluid, enabled high counts (42 × 10⁷ to 190 × 10⁷/g of ingesta) of lactate-utilizing bacteria to be made with a high degree of specificity (96%). The medium also supported the growth of all species of predominant lactate-utilizing bacteria reported to occur in the rumen and thus is of importance for ecological studies where the incidence and influence of the different species on lactate metabolism under changing conditions in the rumen cannot be predicted. The survival rate of isolates was increased from 60 to 96% by addition to the modified maintenance medium of 40% rumen fluid in place of the volatile fatty acids, hemin, and trace elements used in the counting medium. These results, together with the slow growth of colonies in roll bottles, showed that, although highly selective, the counting medium was not optimal for the types selected.     

The Isolation and Characterization of Strains of Lipolytic Bacteria from the Ovine Rumen

The first anaerobic lipolytic bacterium isolated from the rumen was Anaerovibrio lipolytica . In this study strains of anaerobic lipolytic bacteria were isolated from a sheep rumen. All the new isolates were Gram negative curved rods with flagella. The bacteria, which produced propionic acid as a major fermentation product, could ferment only a small range of substrates. The new isolates are thought to belong to the same genus as Anaerovibrio lipolytica .     

Isolation and Metabolic Characteristics of Previously Uncultured Members of the Order Aquificales in a Subsurface Gold Mine

Culture-dependent and -independent techniques were combined to characterize the physiological properties and the ecological impacts of culture-resistant phylotypes of thermophiles within the order Aquificales from a subsurface hot aquifer of a Japanese gold mine. Thermophilic bacteria phylogenetically associated with previously uncultured phylotypes of Aquificales were successfully isolated. 16S ribosomal DNA clone analysis of the entire microbial DNA assemblage and fluorescence in situ whole-cell hybridization analysis indicated that the isolates dominated the microbial population in the subsurface aquifer. The isolates were facultatively anaerobic, hydrogen- or sulfur/thiosulfate-oxidizing, thermophilic chemolithoautotrophs utilizing molecular oxygen, nitrate, ferric iron, arsenate, selenate, and selenite as electron acceptors. Their versatile energy-generating systems may reflect the geochemical conditions of their habitat in the geothermally active subsurface gold mine.     

Isolation and Characterization of the Strains Producing Bacterial Cellulose

Microbiology - Two new strains producing bacterial cellulose were isolated from the Kombucha and Tibicos symbiotic communities. Based on the 16S rRNA gene analysis and the morphological,...     

New Strategies for Cultivation and Detection of Previously Uncultured Microbes

An integrative approach was used to obtain pure cultures of previously uncultivated members of the divisions Acidobacteria and Verrucomicrobia from agricultural soil and from the guts of wood-feeding termites. Some elements of the cultivation procedure included the following: the use of agar media with little or no added nutrients; relatively long periods of incubation (more than 30 days); protection of cells from exogenous peroxides; and inclusion of humic acids or a humic acid analogue (anthraquinone disulfonate) and quorum-signaling compounds (acyl homoserine lactones) in growth media. The bacteria were incubated in the presence of air and in hypoxic (1 to 2% O₂ [vol/vol]) and anoxic atmospheres. Some bacteria were incubated with elevated concentrations of CO₂ (5% [vol/vol]). Significantly more Acidobacteria were found on isolation plates that had been incubated with 5% CO₂. A simple, high-throughput, PCR-based surveillance method (plate wash PCR) was developed. This method greatly facilitated detection and ultimate isolation of target bacteria from as many as 1,000 colonies of nontarget microbes growing on the same agar plates. Results illustrate the power of integrating culture methods with molecular techniques to isolate bacteria from phylogenetic groups underrepresented in culture.     

Bacterial Population Adherent to the Epithelium on the Roo of the Dorsal Rumen of Sheep

By anaerobic procedures, the total number of adherent bacteria was determined on tissue samples obtained from the roof of the dorsal rumen of three sheep. After four washings, 1.91 × 10⁷, 0.34 × 10⁷, and 1.23 × 10⁷ bacteria per cm² were still attached to the rumen epithelium in sheep 1, 2, and 3, respectively. A total of 95 strains of bacteria were isolated from these three samples. Based on morphology, Gram stain, anaerobiosis, motility, and fermentation end products, they were presumptively identified as follows: Butyrivibrio fibrisolvens, 30 strains; atypical Butyrivibrio, 5 strains; Bacteroides ruminicola, 22 strains; Lactobacillus, 1 strain; and unknown Bacteroides species, 37 strains. For sheep 3, washing the rumen epithelium a total of 10 times reduced the adherent bacterial population by 93% (8.4 × 10⁵ bacteria per cm²). Of 30 strains isolated from this sample, 22 were presumptively identified as Butyrivibrio and Bacteroides types. These results suggest that the epithelium on the roof of the dorsal rumen is primarily colonized by two genera of bacteria, Butyrivibrio and Bacteroides. Most Butyrivibrio and Bacteroides ruminicola strains appeared to be similar to previously isolated rumen strains. However, the unknown Bacteroides species differed considerably from the three species of this genus which are commonly isolated from rumen contents.     

两株具有抗癌活性内生细菌的分离及分类

从西双版纳植物样品爵床[Rostellularia procumbens(L.) Nees]中分离到2株具有较强抗癌活性的内生细菌YIM 56077和YIM 56081。通过对其进行表型特征、生长及生理生化特性、细胞化学组份以及系统进化分析,发现这2株菌与B.flexus IFO15715T的亲缘关系最近,但它们在生长、生理生化等特征上表现明显差异,是芽胞杆菌属的2个具有开发潜力的新菌株。     

Isolation and Characterization of Bacterial Strains with Inulinase Activity

Thirty-two bacterial strains growing on inulin as the sole carbon and energy source were isolated from soil samples by enrichment culture on a mineral medium. Twenty of the strains were identified as Flavobacterium multivorum. All the bacteria contained a β-fructosidase that was active on both inulin and sucrose. The enzyme activity was cell bound and was produced at the end of the growth phase. These enzymes have potential uses in the preparation of fructose syrups from inulin and invert sugar from sucrose.     

Bacterial Content in Samples from Different Sites in the Rumen of Sheep and Cows as Determined in Two Culture Media

Samples of material from the rumen of cattle and of sheep were cultured in both Gall Broth (GB) and Reinforced Clostridium Medium (RCM). Samples were taken, at monthly intervals, from five sites in cattle and two sites in sheep in two series and, at weekly intervals, from two sites in cattle and in sheep in two series. Comparisons were made of the growth of organisms in the media, using the number of fertile tubes per set of six dilutions as a measure of the viable count. RCM gave a greater number of fertile tubes than GB with samples from cattle, but the reverse was the case with samples from sheep. Real differences in the number of fertile tubes from different sites in cattle were found at different times, but there was no consistent pattern. Simultaneous sampling from different sites in the rumen is, therefore, essential to provide an estimate of sampling error within animals. Although no site to site differences were detectable with rumen material from sheep, it is advisable to take more than one sample at a given time to assess sampling error.     

山羊瘤胃微生物宏基因组BAC文库的构建与分析

从山羊瘤胃液中提取混合微生物DNA,经BamHI部分酶切得到50kb～800kb的DNA片段后,将其连接到pCCIBAC载体上,转化E．coliEPI300,建立山羊瘤胃微生物BAC文库。经RFLP鉴定分析,该文库12672个克隆,平均插入片段为6lkb。该文库的构建为后续新型基因的筛选提供了材料,为进一步研究山羊瘤胃微生物奠定了基础。     

A Simple Method for Isolation of Lipopolysaccharides from Pseudomonas aeruginosa and Some Other Bacterial Strains

A simple procedure for the isolation of bacterial lipopolysaccharides was developed. The method consists of the following three steps; recovery of lipopolysaccharides in an insoluble form together with denatured proteins in a hot MgCl₂-Triton X-100 solution (step 1), solubilization with EDTA-Triton X-100 (step 2) and precipitation with MgCl₂ (step 3). A good yield of lipopolysaccharides was obtained with most of the Pseudomonas strains tested. Although the yield was lower with strains belonging to Salmonella and Escherichia, irrespective of whether they are smooth-type or rough-type strains, the amounts obtained from 100 mg of cells as dry weight were sufficient for detailed chemical characterization. The gel-electrophoretic profiles of the lipopolysaccharides prepared by the present method were essentially the same as those of the lipopolysaccharides prepared by the conventional phenol-water method. The method allowed us to prepare lipopolysac-charides from a large number of bacterial strains in one day.     

Rumen Anaerobic Fungi of Cattle and Sheep

A system for the large-scale production and purification of mouse mammary tumor virus in the absence of detectable endogenous murine leukemia virus is described. By utilizing the Mm5mt/c1 cell line established from an adenocarcinoma of a C3H mouse, the continuous production of over 25,000 liters of mouse mammary tumor virus-containing tissue culture fluids has been achieved. By the strict adherence to well-defined tissue culture conditions, mammary tumor virus production was accomplished without the expression of murine leukemia virus. Various biochemical and immunological systems were established for the rapid and precise detection of the endogenous leukemia virus, the expression of which could be enhanced under conditions of culture stress.     

Bacterial and Protozoal Communities and Fatty Acid Profile in the Rumen of Sheep Fed a Diet Containing Added Tannins

This study evaluated the effects of tannins on ruminal biohydrogenation (BH) due to shifts in the ruminal microbial environment in sheep. Thirteen lambs (45 days of age) were assigned to two dietary treatments: seven lambs were fed a barley-based concentrate (control group) while the other six lambs received the same concentrate with supplemental quebracho tannins (9.57% of dry matter). At 122 days of age, the lambs were slaughtered, and the ruminal contents were subjected to fatty acid analysis and sampled to quantify populations of Butyrivibrio fibrisolvens, which converts C_18:2 c9-c12 (linoleic acid [LA]) to C_18:2 c9-t11 (rumenic acid [RA]) and then RA to C_18:1 t11 (vaccenic acid [VA]); we also sampled for Butyrivibrio proteoclasticus, which converts VA to C_18:0 (stearic acid [SA]). Tannins increased (P < 0.005) VA in the rumen compared to the tannin-free diet. The concentration of SA was not affected by tannins. The SA/VA ratio was lower (P < 0.005) for the tannin-fed lambs than for the controls, suggesting that the last step of the BH process was inhibited by tannins. The B. proteoclasticus population was lower (−30.6%; P < 0.1), and B. fibrisolvens and protozoan populations were higher (+107% and +56.1%, respectively; P < 0.05) in the rumen of lambs fed the tannin-supplemented diet than in controls. These results suggest that quebracho tannins altered BH by changing ruminal microbial populations.The fatty acid profile of the meat and milk of ruminants is strongly affected by diet (2, 15). When ingested, the dietary polyunsaturated fatty acids (PUFA) undergo a process known as biohydrogenation (BH) carried out by ruminal microorganisms (20). During the BH of C_18:2(n-6) (linoleic acid [LA]) and C_18:3(n-3) (linolenic acid [LNA]) a number of C_18:1 and C_18:2 isomers are formed (6). The last step in the BH process leads to the formation of C_18:0 (stearic acid [SA]). Among the intermediate products formed during this process, the isomer C_18:2 c9t11 (rumenic acid [RA]) is active in preventing cancer in mammals (17). Only a small amount of the RA found in meat and milk originates during BH. It is produced to a larger extent in muscle and mammary glands from the desaturation of C_18:1 t11 (vaccenic acid [VA], another intermediate of ruminal BH) by the action of Δ⁹-desaturase enzyme (41, 43).Ruminal BH is carried out mostly by bacteria belonging to the Butyrivibrio genus (38). Butyrivibrio fibrisolvens has the capacity to convert LA to RA and RA to VA, while Butyrivibrio proteoclasticus (previously classified as Clostridium proteoclasticum [35]) hydrogenates VA to SA (38, 39). According to Or-Rashid et al. (37), ruminal protozoa also play a role in BH by converting LA to RA. However, this issue is still controversial, as Devillard et al. (11) have reported that protozoa do not have the capability of hydrogenating LA. The proportion of BH intermediates in the rumen can vary depending on changes in ruminal microbial populations (7, 51). Changes in ruminal fatty acid profiles are also reflected in intramuscular fatty acid composition (48, 52).Tannins are phenolic compounds that are widespread in plants. When ingested by ruminants in large amounts, tannins can reduce the activity and the proliferation of ruminal microorganisms (34). Tannins from Lotus corniculatus (33) or from Acacia spp. (12) reduce the proliferation of B. proteoclasticus B316^T and B. proteoclasticus P18, respectively. Durmic et al. (12) reported that VA increased and SA decreased when extracts from Acacia iteaphylla, which contains condensed tannins (1), were incubated in vitro with sheep ruminal fluid inoculated with B. fibrisolvens JW11 and B. proteoclasticus P18 strains. In two recent in vitro studies, the inclusion of tannins in fermentor systems containing bovine ruminal fluid inhibited the conversion of VA to SA, while no effect was detected on RA production (21, 47). These results have been also confirmed in vivo in the rumen of sheep fed a diet with 4.0% dry matter (DM) quebracho tannin (48). However, to date there is no in vivo study focusing on the effects of dietary tannins on the proliferation of the microorganisms involved in ruminal BH.We assessed whether dietary tannins may affect the BH pathway via changes in bacterial and protozoal ruminal populations. We gave particular emphasis to B. fibrisolvens and B. proteoclasticus. We also assayed the production of conjugated linoleic acids (CLAs) by linoleic acid isomerase (LA-I) enzyme.     

Sulfate Reduction by a Desulfovibrio Species Isolated from Sheep Rumen

Several dissimilatory, sulfate-reducing bacteria were isolated from the rumen fluid of sheep fed purified diets containing sulfate. One isolate, strain D, was selected for characterization. This organism is a nonsporeforming, obligately anaerobic, mesophilic, nonmotile, gram-negative, straight rod. Cell-free extracts show absorption maxima for cytochrome c(3) and desulfoviridin, characteristic of Desulfovibrio. Carbohydrates, as a sole carbon source, will support growth. Lactate supports growth in the presence of sulfate, not in its absence, whereas glucose or pyruvate support growth either in the presence or absence of sulfate. The isolate has a deoxyribonucleic acid base composition of 61.2% guanine plus cytosine, which is similar to that of several other species of Desulfovibrio; however, it differs from previously described species in morphology, motility, and carbon source utilization. Cell-free extracts of this bacterium exhibit adenosine 5'-triphosphate-sulfurylase, adenosine-5'-phosphosulfate-reductase, and hydrogenase activity. After incubation of cell-free extracts with adenine 5'-triphosphate and (35)SO(4) (2-), adenosine-5'-phosphosulfate rather than 3'-phosphoadenosine-5'-phosphosulfate was shown to be labeled, indicating that the pathway of sulfate reduction in this organism is similar to that of other dissimilatory sulfate reducers. This is the first report of a Desulfovibrio sp. isolated from the rumen.     

Isolation, Culture, and Fermentation Characteristics of Selenomonas ruminantium var. bryanti var. n. from the Rumen of Sheep

Large forms of Selenomonas sp. were isolated from the sheep rumen on a rumen fluid-glucose-agar medium by using a differential centrifugation technique to purify the inoculum. The cells from the six isolated strains were curved, gram-negative, strictly anaerobic crescents, and rapidly motile by flagella attached to the concave side of the cell. One or more of the volatile fatty acids were essential for growth. None of the strains produced indole or reduced nitrate. All strains grew on fructose, glucose, mannose, cellobiose, maltose, sucrose, and salicin. Fermentation end products from glucose were mainly lactate, acetate, propionate, and formate. Small amounts of succinate were formed. The final pH in a glucose medium ranged between 4.3 and 4.5. On the basis of the sugar fermentation characteristics and the capacity to form hydrogen sulfide from cysteine, it is suggested that one of the strains is a large form of Selenomonas ruminantium. The other five strains are designated S. ruminantium var. bryanti, var. n.