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.

Studies of cervicovaginal smears for the presence of actinomycetes.

Cytologic and direct fluorescent antibody techniques were used to detect the presence of Actinomyces israelii and Arachnia propionica in cervicovaginal smears collected from 100 women, 94 of whom did not use intrauterine contraceptive devices. In no case were these organisms found. The possible significance of these results is discussed.

PIP: A total of 100 women who went to an Atlanta, Georgia family planning clinic for Papanicolaou (PAP) tests were the subjects of this study of cervicovaginal smears to detect the presence of actinomycetes. Of the 100 women, 73 were clinically normal; of the 27 women with abnormal vaginal tracts, 15 had Trichomonas vaginalis or Candida sp. infections, and 12 had miscellaneous problems. Paired PAP smears were studied; 1 smear was viewed under a light microscope for organisms morphologically consistent with actinomycetes, and the other was divided into 3 sectors and stained with fluorescein isothiocyanate (FITC)-labeled globulins. The 3 sectors were variously stained for serotypes of actinomyces israelii or acrachnia propionica. No actinomycetes were seen in either the cytologic or direct fluorescent antibody viewings of the PAP smears, even though 6 of the 100 women did use an IUD. Therefore, earlier reports of actinomycetes commonly occurring in cervicovaginal smears in IUD users especially were not confirmed. However, a case-control study of IUD users with better age variation should be performed.

Quantitative procedure for enumeration of bifidobacteria.

A membrane filter technique has been developed for the enumeration of bifidobacteria in natural aquatic environments. The technique is quantitative, selective, and differential. The medium (YN-6) contains: yeast extract, 2.0 g; agar, 1.5 g; polypeptone peptone, 1.0 g; vitamin-free Casamino Acids, 0.8 g; sodium chloride, 0.32 g; and L-cysteine hydrochloride, 0.003 g; in 100 ml of deionized water. The medium is adjusted to pH 7.0 before autoclaving. Nalidixic acid (80 micrograms/ml), neomycin sulfate (2.5 micrograms/ml), and bromcresol green (300 micrograms/ml) are included as selective and differential agents. After incubation for 48 h at 37 degrees C in an anaerobic environment, Gram-stained smears from green, glistening, smooth entire colonies are examined microscopically for typical bifidobacterial morphology. No significant difference in recoveries was observed when YN-6 was compared with reinforced clostridial agar, using bifidobacteria freshly isolated from feces and raw sewage. Using this technique with aquatic and fecal samples, less than 9% false-positive and 8% false-negative isolates were observed. These results indicated that the medium was able to satisfactorily recover organisms from a variety of situations.

Selective localization and growth of Bifidobacterium bifidum in mouse tumors following intravenous administration.

A strain of domestic bacteria, Bifidobacterium bifidum (Lac B), which is nonpathogenic and anerobic, selectively localized and proliferated in several types of mouse tumors following i.v. administration. None of the same bacilli could be detected in the tissues of healthy organs such as the liver, spleen, kidney, lung, blood, bone marrow, and muscle 48 or 96 hr after i.v. administration into tumor-bearing mice. Proliferation of Lac B in the tumor was artifically stimulated by i.p. administration of DDD-H-2s mice of a synthesized disaccharide, lactulose (4-O-beta-D-galactopyranosyl-D-fructofuranose), a sugar which is not metabolized by mammalian tissue cells. Lac B, which survices and proliferates selectively in the tumor following i.v. administration into the tumor-bearing host, should aid in diagnosis and selective therapy for cancer.