Adapted Protocol for Saccharibacteria Cocultivation: Two New Members Join the Club of Candidate Phyla Radiation.

The growing application of metagenomics to different ecological and microbiome niches in recent years has enhanced our knowledge of global microbial biodiversity. Among these abundant and widespread microbes, the candidate phyla radiation (CPR) group has been recognized as representing a large proportion of the microbial kingdom (>26%). CPR are characterized by their obligate symbiotic or exoparasitic activity with other microbial hosts, mainly bacteria. Currently, isolating CPR is still considered challenging for microbiologists. The idea of this study was to develop an adapted protocol for the coculture of CPR with a suitable bacterial host. Based on various sputum samples, we tried to enrich CPR (Saccharibacteria members) and to cocultivate them with pure hosts (Schaalia odontolytica). This protocol was monitored by TaqMan real-time quantitative PCR (qPCR) using a system specific for Saccharibacteria designed in this study, as well as by electron microscopy and sequencing. We succeeded in coculturing and sequencing the complete genomes of two new Saccharibacteria species, "Candidatus Minimicrobia naudis" and "Candidatus Minimicrobia vallesae." In addition, we noticed a decrease in the CT values of Saccharibacteria and a significant multiplication through their physical association with Schaalia odontolytica strains in the enriched medium that we developed. This work may help bridge gaps in the genomic database by providing new CPR members, and in the future, their currently unknown characteristics may be revealed. IMPORTANCE In this study, the first TaqMan real-time quantitative PCR (qPCR) system, targeting Saccharibacteria phylum, has been developed. This technique can specifically quantify Saccharibacteria members in any sample of interest in order to investigate their prevalence. In addition, another easy, specific, and sensitive protocol has been developed to maintain the viability of Saccharibacteria cells in an enriched medium with their bacterial host. The use of this protocol facilitates subsequent studies of the phenotypic characteristics of CPR and their physical interactions with bacterial species, as well as the sequencing of new genomes to improve the current database.

Varibaculum anthropi sp. nov. represented by three genetically different genomovars isolated from clinical material and emended description of the genus Varibaculum.

During the years 1994-2011 five strictly anaerobic, Gram-stain-positive, diphtheroid bacteria (strains CCUG 31793T, CCUG 44221, CCUG 61255, CCUG 45114, and CCUG 44993) were isolated from different clinical samples in Sweden and the United Kingdom. Comparative analysis of 16S rRNA gene sequences showed that the five strains shared 99-100% 16S rRNA gene sequence similarity among each other and 98.3-98.6% sequence similarity to Varibaculum cambriense DSM 15806T. Genomic fingerprint patterns generated with ERIC-, BOX-, and RAPD-PCR, and whole genome sequence (WGS) based comparison by in silico DNA-DNA hybridization (isDDH), average nucleotide identity (ANI) analysis and six housekeeping gene (atpA, rpoB, pgi, metG, gltA and gyrA) based multilocus sequence analysis (MLSA) showed that the strains could be differentiated from V. cambriense DSM 15806T and formed three genomic groups, which could only be differentiated at the species border level. Based on physiological characterizations the five strains could not be clearly distinguished among each other. Based on those data a new Varibaculum species, Varibaculum anthropi (type strain CCUG 31793T=JCM 19104T) is proposed including three genetically distinct genomovars (gv 1: CCUG 31793T, CCUG 44221, CCUG 61255; gv 2: CCUG 45114, and gv 3: CCUG 44993).

Description of strain FC3T as the neotype strain of Actinobaculum massiliense.

Actinobaculum massiliense (Euzéby, 2006) was isolated from the urine of an elderly woman in 2001. Unfortunately, the strain deposited as the type strain was, by error, an Actinobaculum schaalii strain (Yassin et al., 2015). In 2015, we isolated a new strain of A. massiliense, FC3, from the urine of a 12-year-old patient with acute cystitis. We herein present the characteristics of strain FC3 (=CSUR P1982=DSM 100580) and formally propose it as the neotype strain of A. massiliense.

Actinobaculum schaalii bacteremia: A report of two cases.

We report two cases of bacteremia with Actinobaculum schaalii, a rarely reported, anaerobic, Gram-positive bacterium. The first case was a patient with renal cancer who developed pyelonephritis after cryoablation, and the second was a patient who developed sepsis after a urogenital procedure. Bacteremia resolved after administration of empiric antibiotic therapy.

Actinopolyspora biskrensis sp. nov., a novel halophilic actinomycete isolated from Northern Sahara.

A novel halophilic, filamentous actinomycete, designated H254(T), was isolated from a Saharan soil sample collected from Biskra (Northern Sahara), and subjected to a polyphasic taxonomic characterization. The strain is Gram-positive, aerobic, and halophilic, and the optimum NaCl concentration for growth is 15-20 % (w/v). The cell-wall hydrolysate contained meso-diaminopimelic acid, and the diagnostic whole-cell sugars were arabinose and galactose. The diagnostic phospholipid detected was phosphatidylcholine, and MK-9(H4) was the predominant menaquinone. The major fatty acid profiles were anteiso-C17:0 (32.8 %), C15:0 (28 %), and iso-C17:0 (12.3 %). Comparative analysis of the 16S rRNA gene sequences revealed that the strain H254(T) formed a well-separated sub-branch within the radiation of the genus Actinopolyspora, and the microorganism was most closely related to Actinopolyspora saharensis DSM 45459(T) (99.2 %), Actinopolyspora halophila DSM 43834(T) (99.1 %), and Actinopolyspora algeriensis DSM 45476(T) (99.0 %). Nevertheless, the strain had relatively lower mean values for DNA-DNA relatedness with the above strains (57.2, 68.4, and 45.6 %, respectively). Based on phenotypic features and phylogenetic position, we propose that strain H254(T) represents a novel species of the genus Actinopolyspora, for which the name Actinopolyspora biskrensis sp. nov. is proposed. The type strain of A. biskrensis is strain H254(T) (=DSM 46684(T) =CECT 8576(T)).

Dissection of the genus Actinobaculum: Reclassification of Actinobaculum schaalii Lawson et al. 1997 and Actinobaculum urinale Hall et al. 2003 as Actinotignum schaalii gen. nov., comb. nov. and Actinotignum urinale comb. nov., description of Actinotignum sanguinis sp. nov. and emended descriptions of the genus Actinobaculum and Actinobaculum suis; and re-examination of the culture deposited as Actinobaculum massiliense CCUG 47753T ( = DSM 19118T), revealing that it does not represent a strain of this species.

The remarkable host specificity of the species of the genus Actinobaculum led us to recharacterize these species by a polyphasic approach. A comparative chemotaxonomic study including analysis of whole-cell sugars, amino acid composition of the peptidoglycan, fatty acid methyl esters, respiratory quinones and polar lipids revealed significant differences that, in combination with molecular data, support a dissection of the genus Actinobaculum. The proposals of this study include the reclassification of Actinobaculum schaalii and Actinobaculum urinale as Actinotignum schaalii gen. nov., comb. nov. (type strain DSM 15541(T) = CCUG 27420(T)) and Actinotignum urinale comb. nov. (type strain DSM 15805(T) = CCUG 46093(T)), respectively. Emended descriptions of the genus Actinobaculum and Actinomyces suis are also provided. The results of 16S rRNA gene sequence analysis and DNA-DNA hybridization also indicated that the type strain of Actinobaculum massiliense deposited as CCUG 47753(T) ( = DSM 19118(T)) should in fact be considered a member of the species Actinobaculum schaalii. In addition, comparative 16S rRNA gene sequencing and DNA-DNA relatedness studies of four strains recovered from clinical materials demonstrated that three of the isolates belonged to Actinotignum schaalii; the remaining strain represents a novel species, for which the name Actinotignum sanguinis sp. nov. is proposed. The type strain is IMMIB L-2199(T) ( = DSM 26039(T) = CCUG 64068(T)).

Anti-biofilm and antibacterial activities of zinc oxide nanoparticles against the oral opportunistic pathogens Rothia dentocariosa and Rothia mucilaginosa.

Species of the genus Rothia that inhabit the oral cavity have recently been implicated in a number of diseases. To minimize their role in oral infections, it is imperative to reduce and/or control the growth and biofilm formation activity of Rothia spp. In this study, two bacterial isolates, Ora-7 and Ora-16, were obtained from the oral cavity of a healthy male subject and identified as Rothia dentocariosa and Rothia mucilaginosa, respectively, using a polyphasic taxonomic approach. Antimicrobial and anti-biofilm formation activities of zinc oxide nanoparticles (ZnO-NPs), of average size 35 nm, were assessed in in vitro assays using Crystal Violet and live and dead staining techniques. The ZnO-NPs exhibited an IC50 value of 53 and 76 µg ml(-1) against R. dentocariosa and R. mucilaginosa, respectively. Biofilm-formation assays, performed on the surfaces of polystyrene plates, artificial teeth, and dental prostheses, revealed the efficacy of ZnO-NPs as a potential antibacterial agent for controlling the growth of Rothia isolates in both planktonic form and biofilm.

Actinobaculum schaalii causing epididymitis in an elderly patient.

Actinobaculum schaalii is a Gram-positive coccoid rod that causes various infections in humans and is easily overlooked in cultures. A. schaalii has long been thought to be of low prevalence and limited invasive potential, causing benign cystitis in elderly patients with underlying urological conditions. Here, we report the first case of epididymitis caused by this bacterium.

Characterization of oral bacterial diversity of irradiated patients by high-throughput sequencing.

The objective of this study was to investigate the compositional profiles and microbial shifts of oral microbiota during head-and-neck radiotherapy. Bioinformatic analysis based on 16S rRNA gene pyrosequencing was performed to assess the diversity and variation of oral microbiota of irradiated patients. Eight patients with head and neck cancers were involved in this study. For each patient, supragingival plaque samples were collected at seven time points before and during radiotherapy. A total of 147,232 qualified sequences were obtained through pyrosequencing and bioinformatic analysis, representing 3,460 species level operational taxonomic units (OTUs) and 140 genus level taxa. Temporal variations were observed across different time points and supported by cluster analysis based on weighted UniFrac metrics. Moreover, the low evenness of oral microbial communities in relative abundance was revealed by Lorenz curves. This study contributed to a better understanding of the detailed characterization of oral bacterial diversity of irradiated patients.

Lechevalieria fradiae sp. nov., a novel actinomycete isolated from soil in China.

The taxonomic position of a soil isolate, strain Z6(T), was established using a polyphasic approach. The organism showed a range of chemical and morphological properties consistent with its classification in the genus Lechevalieria. An almost complete 16S rRNA gene sequence determined for the strain was aligned with corresponding sequences of representatives of the genus Lechevalieria and related taxa using three tree-making algorithms. The organism formed a distinct phyletic line within the evolutionary radiation occupied by the genus Lechevalieria and was more closely related to the type strain of Lechevalieria aerocolonigenes than to that of Lechevalieria flava. Strain Z6(T) could be distinguished from both these strains using DNA-DNA relatedness data and by a combination of phenotypic properties. The combined genotypic and phenotypic data show that strain Z6(T) should be assigned to the genus Lechevalieria as a representative of a novel species. The name proposed for this new taxon is Lechevalieria fradiae sp. nov. The type strain is Z6(T) (=CGMCC 4.3506(T)=JCM 14205(T)).

"Actinobaculum massiliae," a new species causing chronic urinary tract infection.

We report on a new Actinobaculum species, "Actinobaculum massiliae," isolated from the urine of an elderly woman with recurrent cystitis. Its phenotypic pattern was similar to those of both of the other Actinobaculum species described to date. On 16S rRNA sequencing, the Marseille isolate shared 95% homology with Actinobaculum suis, 92 to 93% homology with Actinobaculum schaalii, 91 to 92% homology with Arcanobacterium spp., and 87 to 90% homology with Actinomyces species. A bootstrap value of 99% supports the node separating the Actinobaculum sp. from its closest neighbor (A. suis). In conclusion, on the basis of phenotypic, genotypic, and phylogenetic assessments, we show that the Marseille isolate is a previously unrecognized organism within the Actinobaculum genus, and we propose placement of the organism in the taxon "Actinobaculum massiliae."

Arcanobacterium hippocoleae sp. nov., from the vagina of a horse.

A polyphasic taxonomic study was performed on a previously unidentified gram-positive, facultatively anaerobic, diphtheroid-shaped organism isolated from a vaginal discharge of a horse. Comparative 16S rRNA gene sequencing demonstrated that the strain was a member of the genus Arcanobacterium, but sequence divergence values of >4% with described species of this genus (viz: Arcanobacterium haemolyticum, Arcanobacterium bernardiae, Arcanobacterium phocae, Arcanobacterium pluranimalium and Arcanobacterium pyogenes) demonstrated that the isolate represented a novel species. The unknown bacterium was readily distinguished from other Arcanobacterium species by biochemical tests. Based on phylogenetic and phenotypic evidence, it is proposed that the unknown bacterium be classified as Arcanobacterium hippocoleae sp. nov. The type strain of A. hippocoleae is CCUG 44697T (= CIP 106850T).

[A definitive identification method: gas-liquid chromatography]. / Metodi di identificazione definitiva: cromatografia gas-liquido.

The definitive identification of many anaerobes requires the use of gas-liquid chromatography to detect alcohol and volatile acids end-products of their metabolism. In this study we have used the gas-liquid chromatography for the identification of 360 non sporing anaerobes isolated from faeces of healthy individuals. These analyses were performed on a Variant-Aerograph mod. 2700 gas chromatography fitted with a column without phosphoric acid. The results showed a good percentage of typing (83%).

Abscess associated with Rothia dentocariosa.

Rothia dentocariosa, an aerobic member of the Actinomycetaceae, was isolated from a pilonidal abscess. The clinical occurrence, bacteriological characteristics, and antimicrobial sensitivity pattern are presented.

[Formation of a new antibiotic, nocamycin, by a culture of Nocardiopsis syringae sp. nov]. / Obrazovanie novogo antibiotika nokamitsina kul'turoi Nocardiopsis syringae sp. nov.

A culture of a new actinomycetous species, Nocardiopsis syrinage was isolated from a soil sample. The antibiotic produced by it was named nocamycin. It accumulated in the culture fluid on cultivation of the organism in a nutrient medium containing soybean meal, glycerin, sodium chloride and calcium carbonate. Crystalline nocamycin had an antitumor effect. In inhibited by 72--73 per cent the development of an intraperitoneally implanted lymphadenosis of strain NK/LI in mice.