Comparison of six methods of DNA extraction for the diagnosis of bovine brucellosis by real-time PCR Abstract Brucellosis is an infectious disease caused by bacteria of the genus Brucella, which affects domestic animals and is transmissible to humans. The objective of this study was to evaluate six methods of DNA extraction directly from bovine tissue to detect Brucella spp. The Cq values for all samples were above 30 and varied according to the extraction kit used, but four kits showed no statistical difference in sensitivity. This work demonstrates the importance of choosing the best extraction kit before validation of a molecular diagnostic technique.

Euonymus japonicus phyllosphere microbiome is significantly changed by powdery mildew Abstract Euonymus japonicus Thunb. is a woody and ornamental plant popular in China, Europe and North America. Powdery mildew is one of the most serious diseases that affect E. japonicus growth. In this study, the diseased and apparently healthy leaves were collected from E. japonicus planted in a greenbelt in Beijing, and the effect of powdery mildew on the epiphytic microbial community was investigated by using Illumina sequencing. The results showed that the healthy leaves (HL) harbored greater bacterial and fungal diversity than diseased leaves (DL). Furthermore, both bacterial and fungal communities in DL exhibited significantly different structures from those in HL. The relative abundance of several bacterial phyla (Proteobacteria and Firmicutes) and fungal phyla (Ascomycota and Basidiomycota) were altered by powdery mildew. At the genus level, most genera decreased as powdery mildew pathogen Erysiphe increased, while the genera Kocuria and Exiguobacterium markedly increased. Leaf properties, especially protein content was found to significantly affect beta-diversity of the bacterial and fungal community. Network analysis revealed that positive bacterial interactions in DL were stronger than those in HL samples. Insights into the underlying the indigenous microbial phyllosphere populations of E. japonicus response to powdery mildew will help in the development of methods for controlling plant diseases.

Amniculibacterium aquaticum gen. nov., sp. nov., a new member of the family Flavobacteriaceae isolated from a stream Abstract Strain KYPW7T, isolated from the Funglin Stream in Taiwan, was characterized using a polyphasic taxonomy approach. Cells of strain KYPW7T were Gram-stain-negative, aerobic, non-spore forming, non-motile rods and formed white colonies. Growth occurred at 15–30 °C (optimum 25 °C), at pH 6–8 (optimum pH 6.5) and with 0–1% NaCl (optimum 0%). Phylogenetic analyses based on 16S rRNA and coding sequences of 92 protein clusters showed that strain KYPW7T represents a novel genus in the family Flavobacteriaceae. The 16S rRNA gene sequence of strain KYPW7T was related to the species of the genera Chryseobacterium (91.8–96.0% sequence similarity), Bergeyella (95.1–95.8%), Cloacibacterium (94.5–95.7%), Daejeonia (95.6%) and Riemerella (94.0–95.0%). Strain KYPW7T showed less than 72% average nucleotide identity and less than 24% digital DNA–DNA hybridization identity compared to the type strains of related genera within the family Flavobacteriaceae. The predominant fatty acids were iso-C15:0 and iso-C17:0 3-OH. The major isoprenoid quinone was MK-6 and the DNA G + C content was 36.8 mol%. The polar lipids had phosphatidylethanolamine, three uncharacterized aminophospholipids and an uncharacterized phospholipid. The polyamines contained homospermidine, putrescine and spermidine. On the basis of the genotypic and phenotypic data, strain KYPW7T represents a novel species of a new genus in the family Flavobacteriaceae, for which the name Amniculibacterium aquaticum gen. nov., sp. nov. is proposed. The type strain is KYPW7T (= BCRC 81123T = LMG 30598T = KCTC 62512T).

Neptunomonas marina sp. nov., isolated from seawater Abstract Strain HPM-16T, isolated from seawater, was characterized using a polyphasic taxonomy approach. Phylogenetic analyses based on 16S rRNA gene sequences and coding sequences of an up-to-date bacterial core gene set (92 protein clusters) indicated that strain HPM-16T formed a phylogenetic lineage in the genus Neptunomonas. Strain HPM-16T was most closely related to Neptunomonas concharum LHW37T with 16S rRNA gene sequence similarity of 96.7%. Cells were Gram-stain negative, facultatively anaerobic, motile by means of a single polar flagellum, rod-shaped and formed white colonies. Optimal growth occurred at 30–35 °C, pH 6.5–8, and in the presence of 2–5% NaCl. C18:1ω7c and summed feature 3 (C16:1ω7c and/or C16:1ω6c) were the predominant fatty acids. The only isoprenoid quinone was Q-8. The polar lipid profile revealed the presence of phosphatidylethanolamine, phosphatidylglycerol and several uncharacterized lipids. The major polyamines were putrescine and spermidine. The draft genome was approximately 3.68 Mb in size with a G + C content of 50.5 mol%. Differential phenotypic properties, together with the phylogenetic inference, demonstrate that strain HPM-16T should be classified as a novel species of the genus Neptunomonas, for which the name Neptunomonas marina sp. nov. is presented. The type strain is HPM-16T (= BCRC 80980T = LMG 29560T = KCTC 52235T).

Identification of clonal complexes of Mycobacterium bovis in Brazil Abstract Bovine tuberculosis is a disease that is widely distributed around the world. Its causative agent, Mycobacterium bovis, has characteristics of a microorganism with clonal multiplication in populations with no evidence of genetic exchange between strains, and, consequently, a group of strains can be identified as descending from a common ancestor. The aim of this study was to investigate the clonal complexes of M. bovis isolated from samples of lesions suggestive of bovine tuberculosis collected from slaughterhouses in various states of Brazil between 2006 and 2012. Ninety samples were analyzed, and it was found that 14.4% belonged to the clonal complex European1 and 81.1% to the clonal complex European2, while 4.65% were not identified as any of the four known complexes.

Bacterial communities associated with anthracnose symptomatic and asymptomatic leaves of guarana, an endogenous tropical crop, and their pathogen antagonistic effects Abstract Plants are colonized by diverse microorganisms that can substantially impact their health and growth. Understanding bacterial diversity and the relationships between bacteria and phytopathogens may be key to finding effective biocontrol agents. We evaluated the bacterial community associated with anthracnose symptomatic and asymptomatic leaves of guarana, a typical tropical crop. Bacterial communities were assessed through culture-independent techniques based on extensive 16S rRNA sequencing, and cultured bacterial strains were evaluated for their ability to inhibit the growth of Colletotrichum sp. as well as for enzyme and siderophore production. The culture-independent method revealed that Proteobacteria was the most abundant phylum, but many sequences were unclassified. The emergence of anthracnose disease did not significantly affect the bacterial community, but the abundance of the genera Acinetobacter, Pseudomonas and Klebsiella were significantly higher in the symptomatic leaves. In vitro growth of Colletotrichum sp. was inhibited by 11.38% of the cultured bacterial strains, and bacteria with the highest inhibition rates were isolated from symptomatic leaves, while asymptomatic leaves hosted significantly more bacteria that produced amylase and polygalacturonase. The bacterial isolate Bacillus sp. EpD2-5 demonstrated the highest inhibition rate against Colletotrichum sp., whereas the isolates EpD2-12 and FD5-12 from the same genus also had high inhibition rates. These isolates were also able to produce several hydrolytic enzymes and siderophores, indicating that they may be good candidates for the biocontrol of anthracnose. Our work demonstrated the importance of using a polyphasic approach to study microbial communities from plant diseases, and future work should focus on elucidating the roles of culture-independent bacterial communities in guarana anthracnose disease.

Lactobacillus intestinalis efficiently produces equol from daidzein and chungkookjang, short-term fermented soybeans Abstract Equol improves menopausal symptoms and it is synthesized from daidzein, one of the isoflavonoids in soybeans, by the bacteria in the large intestines of some people. The purpose of this study was to isolate equol-producing bacteria using daidzein from the intestinal microflora and to produce equol-containing chungkookjang (short-term fermented soybean). Equol-producing bacteria from the feces of Sprague–Dawley female rats were isolated using media containing daidzein. The isolated bacteria were cultured in thioglycollate media and equol production was identified through thin-layer chromatography and ultraperformance liquid chromatography–mass spectrometry. The bacteria were identified by 16S rRNA sequencing. The rate of equol production in different concentrations of daidzein was assessed. The expression of genes that code for enzymes associated with the production of equol from daidzein was detected through reverse transcription quantitative PCR. The bacterium we isolated was Lactobacillus intestinalis (LC096206.1, 99%). L. intestinalis was found to express daidzein reductase, dihydrodaidzein reductase, and tetrahydrodaidzein reductase, the enzymes involved in producing equol from daidzein. The conversion rate of equol from daidzein was highest (29.5%) using 200 μM daidzein for 48 h of incubation. When chungkookjang fermented with Bacillus amyloquencies SRCM100001 was incubated with L. intestinalis, 0.32 ± 0.04 mg equol/g chungkookjang was produced. In conclusion, L. intestinalis efficiently produces equol from not only daidzein but also in chungkookjang.

In vitro lytic activity and antifungal susceptibility of infrequently isolated yeasts Abstract Non-albicans Candida species have acquired relevance in the last decades as a cause of serious disease. The virulence factors and antifungal susceptibility of these rare pathogens remain largely unrecognized. We examined a total of 50 yeast isolates corresponding to 11 different infrequently isolated yeast species for their in vitro enzymatic profile and susceptibility pattern as first-line antifungals. We found aspartyl protease activity for 100% of the isolates tested as well as variable DNAse, hemolysin, phospholipase and esterase activities. All strains had low MICs for amphotericin B and showed a variable response to fluconazole (0.125–32 µg/mL) and the echinocandins tested (0.25–> 8 µg/mL).

Phenotypic and genotypic characterization of endophytic bacteria associated with transgenic and non-transgenic soybean plants Abstract Endophytic bacteria isolated from non-transgenic and transgenic Roundup Ready® glyphosate-resistant (GR) soybean plants were investigated to analyze the correspondence between phenotypic and genotypic characteristics and to determine whether or not the strains could be grouped based on the source of isolation in transgenic or non-transgenic plants, respectively. Most of the strains recovered from GR plants have shown the ability for plant growth promotion (PGP) by means of IAA production and inorganic phosphate solubilization, and 100% of the strains showed great motility (swarm or swim); in addition, 90% of the strains were able to metabolize the majority of carbon sources tested. GR soybean fields showed higher endophytes abundance than non-transgenic; however, analyzing the phylogenetic trees constructed using the partial 16SrRNA gene sequences, higher diversity was observed in non-transgenic soybean fields. Overall the majority of isolated endophytes could utilize multiple patterns of carbon sources and express resistance to antibiotics, while isolates varied widely in the PGP ability. The greater pattern and frequency of utilization of carbon sources and frequency and intensity of antibiotic resistance compared with PGP ability within the soybean endophytes community suggest that carbon sources metabolism and antibiotic resistance confer a greater relative fitness benefit than PGP ability. In conclusion, cluster analysis of the phenotypes and 16SrRNA gene sequences reveals lack of correspondence between the pattern of bacterial isolates and the transgenic character of plants, and the heterogeneity of clustering suggested that various adaptive processes, such as stress response, could have contributed to generate phenotypic variability to enhance endophytes overall fitness.

Description of Gelidibacter japonicus sp. nov., isolated from the Inland Sea (Setonaikai) in Japan Abstract A novel Gelidibacter strain, JCM 31967T, was isolated from seawater collected from the Inland Sea (Setonaikai) in Japan. It was characterized as a Gram-negative, halophilic, oxidase-negative, catalase-positive, aerobic, nonmotile, but gliding, rod-shaped bacterium without flagella. Based on 16S rDNA gene identity, strain JCM 31967T is closely related to Gelidibacter mesophilus (DSM 14095T, 96.6% identity), G. gilvus (IC158T, 96.4%), G. algens (DSM 12408T, 96.1%), G. sediminis (S11-41T, 94.7%), and G. salicanalis (IC162T, 94.7%). The G + C content of strain JCM 31967T DNA was found to be 39.1%. The average nucleotide identity (ANI) values of JCM 31967T against G. algens DSM 12408T and G. mesophilus DSM 14095T were 79.1% and 80.9%, respectively. Strain JCM 31967T phenotypically differed from the closest related Gelidibacter species in its utilization of methyl α-d-mannopyranoside, methyl α-d-glucopyranoside, and d-ribose and in its lack of utilization of l-arginine and d-arabinose. It was further differentiated based on its fatty acid composition, specifically properties of C18:0 and C20:2 ω6c, 9c, which were significantly different from those of G. algens, G. gilvus, G. mesophilus, G. salicanalis, and G. sediminis type strains. Overall, the results of DNA–DNA hybridization and physiological and biochemical analyses differentiated strain JCM 31967T from a previously described species of Gelidibacter. Based on these polyphasic taxonomic findings, it was concluded that strain JCM 31967T is a novel Gelidibacter species, for which the name Gelidibacter japonicus sp. nov. is proposed, with JCM 31967T (= LMG 30063T) as the type strain.