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.

Determination of the expression of three fimbrial subunit proteins in cultured Trueperella pyogenes.

BACKGROUND: Trueperella pyogenes is a commensal and a significant opportunistic pathogen in animals. A variety of identified or putative virulence factors are considered to significantly contribute to the occurrence of T. pyogenes infection in different species. However, these virulence factors are not fully understood. RESULTS: In the current study, the genes encoding putative fimbrial proteins, i.e. Fim A, Fim C, and Fim E, were cloned. Recombinant Fim A (rFim A), Fim C (rFim C), and Fim E (rFim E) were prepared and used to generate rabbit anti-rFim A, anti-rFim C, and anti-rFim E serum, respectively. Using these sera, we found that only Fim E was constitutively expressed in T. pyogenes. The expression level of Fim E in T. pyogenes peaked within 6-10 h of culture period in pH 7.5. Fim E protein expression was unaffected by anaerobic condition, but was inhibited by the microaerophilic condition. Tube agglutination tests indicated that Fim E was exhibited on the surface of T. pyogenes cells because anti-rFim E serum caused strong agglutination. Additionally, the blots for Fim A detection showed nonspecific reactions. Furthermore, the tube agglutination tests showed that anti-Fim A serum failed to cause agglutination of T. pyogenes cells, which indicated that Fim A was not, or poorly, expressed in cultured T. pyogenes. Anti-rFim C serum caused strong agglutination. However, the blots for Fim C detection showed a strong nonspecific reaction. Thus, the expression of Fim C was difficult to be determined using the current method. CONCLUSIONS: Fim E was expressed in cultured T. pyogenes. However, Fim A was either not or poorly expressed in cultured T. pyogenes. Moreover, Fim C expression was not determined using the current strategy.

Significant increase in cultivation of Gardnerella vaginalis, Alloscardovia omnicolens, Actinotignum schaalii, and Actinomyces spp. in urine samples with total laboratory automation.

While total laboratory automation (TLA) is well established in laboratory medicine, only a few microbiological laboratories are using TLA systems. Especially in terms of speed and accuracy, working with TLA is expected to be superior to conventional microbiology. We compared in total 35,564 microbiological urine cultures with and without incubation and processing with BD Kiestra TLA for a 6-month period each retrospectively. Sixteen thousand three hundred thirty-eight urine samples were analyzed in the pre-TLA period and 19,226 with TLA. Sixty-two percent (n = 10,101/16338) of the cultures processed without TLA and 68% (n = 13,102/19226) of the cultures processed with TLA showed growth. There were significantly more samples with two or more species per sample and with low numbers of colony forming units (CFU) after incubation with TLA. Regarding the type of bacteria, there were comparable amounts of Enterobacteriaceae in the samples, slightly less non-fermenting Gram-negative bacteria, but significantly more Gram-positive cocci, and Gram-positive rods. Especially Alloscardivia omnicolens, Gardnerella vaginalis, Actinomyces spp., and Actinotignum schaalii were significantly more abundant in the samples incubated and processed with TLA. The time to report was significantly lower in the TLA processed samples by 1.5 h. We provide the first report in Europe of a large number of urine samples processed with TLA. TLA showed enhanced growth of non-classical and rarely cultured bacteria from urine samples. Our findings suggest that previously underestimated bacteria may be relevant pathogens for urinary tract infections. Further studies are needed to confirm our findings.

Cultivation of Peptidiphaga gingivicola from subgingival plaque: The first representative of a novel genus of Actinomycetaceae.

A novel bacterium was isolated from the subgingival plaque of a patient with periodontal disease. Bacterial strain BA112T is a facultative Gram-positive coccus. It metabolizes alanine, arginine, glycine, histidine, leucine, proline, serine and tyrosine, but does not appear to use carbohydrates. Urease, esculin, indole, catalase and nitrate reduction tests were all negative. Major cellular fatty acids were C18:0 , C12:0 , C16:0 , C18:1 w9c and C20:0 . The genome was sequenced and is 2.4 Mbp in length and has 64% GC content. Based on phylogenetics of the 16S rRNA sequence and concatenated alignments of 37 conserved proteins, BA112T belongs to the family Actinomycetaceae but is located on a branch of the tree without currently named members. Based on our phenotypic and phylogenetic studies, we propose that BA112T is the first known representative of a new genus, for which the name Peptidiphaga gingivicola gen. nov., sp. nov. is proposed. The type strain is BA112T .

Comparative transcriptome analysis of Trueperella pyogenes reveals a novel antimicrobial strategy.

Trueperella pyogenes is a prevalent opportunistic bacterium that normally causes diverse suppurative lesions, endometritis and pneumonia in various economically important animals. Although the genomic information of this species has been announced, little is known about its functional profiles. In this study, by performing a comparative transcriptome analysis between the highly and moderately virulent T. pyogenes isolates, we found the expression of a LuxR-type DNA-binding response regulator, PloR, was significantly up-regulated in the highly virulent T. pyogenes. Protein crystal structure prediction and primary functional assessment suggested that, the quorum-sensing signal molecules of Gram-negative bacteria such as Pseudomonas aeruginosa and Escherichia coli could significantly inhibit the growth, biofilm production and hemolysis of T. pyogenes by binding to the upstream sensor histidine kinase, PloS. Therefore, the PloS/PlosR two-component regulatory system might dominate the virulence of T. pyogenes. Our findings provide a major advance in understanding the pathogenesis of T. pyogenes, and may shed new light on the development of novel therapeutic strategies to control T. pyogenes infection.

Multiple genetic elements carry the tetracycline resistance gene tet(W) in the animal pathogen Arcanobacterium pyogenes.

The tet(W) gene is associated with tetracycline resistance in a wide range of bacterial species, including obligately anaerobic rumen bacteria and isolates from the human gut and oral mucosa. However, little is known about how this gene is disseminated and the types of genetic elements it is carried on. We examined tetracycline-resistant isolates of the animal commensal and opportunistic pathogen Arcanobacterium pyogenes, all of which carried tet(W), and identified three genetic elements designated ATE-1, ATE-2, and ATE-3. These elements were found in 25%, 35%, and 60% of tetracycline-resistant isolates, respectively, with some strains carrying both ATE-2 and ATE-3. ATE-1 shows characteristics of a mobilizable transposon, and the tet(W) genes from strains carrying this element can be transferred at low frequencies between A. pyogenes strains. ATE-2 has characteristics of a simple transposon, carrying only the resistance gene and a transposase, while in ATE-3, the tet(W) gene is associated with a streptomycin resistance gene that is 100% identical at the DNA level with the aadE gene from the Campylobacter jejuni plasmid pCG8245. Both ATE-2 and ATE-3 show evidence of being carried on larger genetic elements, but conjugation to other strains was not observed under the conditions tested. ATE-1 was preferentially associated with A. pyogenes strains of bovine origin, while ATE-2 and ATE-3 elements were primarily found in porcine isolates, suggesting that these elements may circulate in different environments. In addition, four alleles of the tet(W) gene, primarily associated with different elements, were detected among A. pyogenes isolates.

Clinical evaluation of postpartum vaginal mucus reflects uterine bacterial infection and the immune response in cattle.

Bacteria contaminate the uterus of most dairy cattle after parturition and endometritis causes infertility. An endometritis score can be ascribed based on the vaginal mucus character and odour but it is not clear if the clinical score reflects the number of uterine bacteria or the inflammatory response. The present study tested the hypothesis that clinical evaluation of endometritis reflects the number of bacteria present in the uterus, and the acute phase protein response. Swabs (n = 328) were collected from the uterine lumen of dairy cattle, 21 and 28 days postpartum, vaginal mucus was scored for character and odour, and blood samples collected for acute phase protein measurement. Bacteria were identified following aerobic and anaerobic culture, and the bacterial growth density was scored semi-quantitatively. When bacteria were categorised by their expected pathogenic potential in the uterus, purulent or fetid odour vaginal mucus was associated with the growth density of pathogenic bacteria but not opportunist contaminants. When bacteria were analysed independently, Arcanobacterium pyogenes, Proteus and Fusobacterium necrophorum growth densities were associated with mucopurulent or purulent vaginal mucus. The bacterial growth densities for A. pyogenes, Escherichia coli, non-hemolytic Streptococci, and Mannheimia haemolytica were associated with a fetid mucus odour. Peripheral plasma concentrations of alpha(1)-acid glycoprotein were higher if there was a fetid compared with a normal vaginal mucus odour (1.50 +/- 0.09 mg/mL versus 1.05 +/- 0.02 mg/mL, P < 0.001), but did not differ significantly between vaginal mucus character scores. The evaluation of the character and odour of vaginal mucus reflects the number of bacteria in the uterus, and the acute phase protein response.

Minimum inhibitory concentrations of some antimicrobial drugs against bacteria causing uterine infections in cattle.

The minimum inhibitory concentrations (MICs) of oxytetracycline, cephapirin, cephapirin/mecillinam, cefquinome, ceftiofur and enrofloxacin, candidate antibiotics for the principal bacteria associated with uterine infections: Escherichia coli, Arcanobacterium pyogenes and the anaerobic bacteria Fusobacterium necrophorum and Prevotella melaninogenicus, were determined by the agar dilution method. The bacteria were isolated from animals with clinical metritis and/or endometritis. For E coli, cefquinome and enrofloxacin had the lowest MIC90 and MIC50 values (< 0.06 microg/ml), and oxytetracycline and cephapirin had the highest values. For A pyogenes, oxytetracycline had the highest MIC50 value (16 microg/ml), but all the cephalosporins had values below 0.06 microg/ml. For the anaerobic bacteria, enrofloxacin and oxytetracycline had the highest MIC50 values but all the cephalosporins had values of 0.06 microg/ml or below.

Molecular cloning and nucleotide sequence of the pyruvate kinase gene of an actinomycete Microbispora thermodiastatica.

The gene for the thermostable pyruvate kinase of Microbispora thermodiastatica IFO 14046, a moderate thermophilic actinomycete, was cloned in Escherichia coli. This gene consists of an open reading frame of 1422 nucleotides and encodes a protein of 474 amino acids with molecular mass of 50,805 Da. The open reading frame was confirmed as the pyruvate kinase gene by comparison with the N-terminal amino acid sequence of the purified pyruvate kinase from M. thermodiastatica.

Fatty acid characterization of rapidly growing pathogenic aerobic actinomycetes as a means of identification.

The fatty acid compositions of 39 type strains and 529 clinical or reference strains of pathogenic aerobic actinomycetes were analyzed after standardized culture by using the Microbial Identification System (MIS). Library entries for each type strain were created by using the MIS Library Generation Software, and the fatty acid profiles of clinical and reference strains were compared to these library entries. The bacteria separated into two large groups based upon major amounts of branched-chain or of saturated or monounsaturated straight-chain fatty acids. Identification of isolates was possible by using only the type strains for comparison, but fatty acid heterogeneity occurred within most species.

Branched Escherichia coli cells.

We report that the normally rod-shaped bacterium Escherichia coli can form branched cells. These were found in strains in which chromosome replication or nucleoid segregation was disturbed, e.g. in minB mutants, intR1 strains, and in strains exhibiting stable DNA replication. Often chromosome DNA was found to be located in the branch point of the cells. The branching frequency was dependent upon the growth medium: in rich medium no branched cells were found, whereas in minimal medium containing acetate and casamino acids the frequency of branched cells was increased. The genetic background of the strains also affected the tendency to branch. Furthermore, electron microscopy of thin-sectioned branched cells revealed additional membrane-like structures, which were not observed in wild-type cells. Finally, the branched cells are compared with bacteria that normally branch, and probable causes for branching in E. coli are discussed.

Starvation-survival of a p-nitrophenol-degrading bacterium.

An environmental actinomycetes, capable of utilizing p-nitrophenol as its sole carbon and nitrogen source, was starved for an 8-week period and showed no reduction in its ability to biodegrade p-nitrophenol. Microscopic examination revealed that starvation of the bacterium resulted in the fragmentation of filaments into individual cells.

[Preservation of fungi and actinomycetes of medical importance in distilled water]. / Preservação de fungos e actinomicetos de interesse médico em água destilada.

Several methods have been used for the preservation of fungi, all of them presenting advantages and disadvantages. The choice of the methods depends upon the laboratory availabilities, time of preservation, genetic stability of the cultures and other factors. In this work the results obtained through the utilization of Castellani's method (preservation in distilled water) for the maintenance of 174 strains belonging to the "Micoteca do Instituto de Medicina Tropical de São Paulo" are presented. These strains were analyzed after 6, 12, 18 and 24 months, with regard to the percentage of viability taking into consideration the rates of growth and contamination. The smallest percentage of viability occurred in the group of the actinomycetes (50 to 100%) and the largest one in the group of the yeasts (near 100%). According to other authors, the Castellani's method, besides being simple and economically feasible for small size laboratories, yields good results.

[Bacterial regrowth in drinking water. IV. Bacterial flora in fresh and stagnant water in drinking water purification and in the drinking water distribution system]. / Bakterielle Wieder-Verkeimung im Trinkwasser. IV. Mitteilung: Bakterienflora in Frisch- und Stagnationswasser bei der Trinkwasseraufbereitung und im Trinkwasser-Verteilersystem.

Six dead end water pipes were installed inside a Zurich drinking water plant and five others over a distance of 12 km along the distribution system and the water was left stagnating in there for 2 weeks. A total of 1508 bacteria from fresh and stagnating water were isolated and identified. Of these, 241 bacterial isolates from the distribution system were examined using the nutrient-tolerance test, i.e. testing the ability to grow in tap water and in media with low and very high nutrient content. In the fresh water of the treatment plant specific bacterial populations were obtained, these occurring particularly after the filters. According to different chlorine dosage and chlorine demand, they were finally washed into the distribution system in varying amounts and compositions. It was shown that in the fresh water of the distribution system the genera of Pseudomonas, Azotobacter and Actinobacteria were each present at a level of approximately 30%. After two weeks stagnation non-fluorescing pseudomonads were dominating in the treatment plant as well as in the fresh water of the distribution system. All isolated Actinobacteria and Azotobacter and almost half of the Pseudomonads proved to be oligotrophic oligocarbotolerants or oligocarbophilic organisms in the nutrient-tolerance test. The other half of the Pseudomonads plus the Flexibacter species were mesotrophic oligocarbotolerants, since they could grow in tap water and in culture media with very high nutrient content. Attention is drawn to the unrecognized danger of recontamination of mesotrophic bacteria growing rapidly in stagnating drinking water, which is used as rinsing water for cleaning food processing equipment.

High performance liquid chromatographic determination of Bifidobacterium bifidum growth factors in human milk.

An isocratic high performance liquid chromatography (HPLC) method has been developed for the determination of Bifidobacterium bifidum growth factors in human milk. The method involves the gradual addition of 3 volumes of ethanol to the milk sample, filtration, and analysis of the growth factors in the filtrate by HPLC. The HPLC system consisted of a carbohydrate analysis column, a water-acetonitrile (70 + 30) solvent system, a flow rate of 1.0 mL/min, and a refractive index detector. The method is simpler and requires less time than the present microbiological method. Moreover, it revealed for the first time the presence of 2 separable growth factors in all human milk samples tested. The HPLC method developed is sensitive and can be used to monitor the type and the amount of growth factors in mothers' milk during lactation.

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.