Uncultivable Bacterium Research Articles

Identification of the bacteria associated to the phycosphere of the Chlorella-like strain SEC_LI_ChL_1

The associations and the co-evolution of bacteria and eukaryotic microalgae are raising great interest in the last years, especially in the “phycosphere”, that is the area around the algal cells, where extracellular products of the algae are used by bacteria; here, different interactions between microalgae and bacteria might occur. The Chlorella-like strain SEC_LI_ChL_1 was previously characterized with a multidisciplinary integrated approach based on phylogenetic reconstructions, morphological-ultrastructural analysis and physiological characterization in presence of different trophic conditions. In this work, the isolation and characterization of fifteen cultivable and one uncultivable bacterial strains strictly associated to the phycosphere of strain SEC_LI_ChL_1 was carried out. The molecular identification followed by the taxonomic reconstruction allowed to highlight the presence of a “primary” culturable associated bacterium taxonomically related to Stenotrophomonas rhizophila, and other culturable bacterial strains taxonomically related to Actinobacteria (Microbacterium), Gammaproteobacteria (Pseudomonas, Stenotrophomonas and Pseudoxanthomonas), Alphaproteobacteria (Bosea, and Brevundimonas), Bacteroidetes and Firmicutes (Priestia and Bacillus). Since the microalgal strain was tolerant to different metal concentrations, the cultivable bacterial strains were exposed to the same metals at the same concentrations, highlighting similar tolerance patterns to the microalgae. The IAA production ability was tested as well in order to highlight this PGP trait in the isolated strains. An uncultivable bacterium taxonomically related to Shinella sp. was also molecularly characterized from the DNA of washed algal cells thus suggesting, in this case, an even more strict connection with the algae. A possible role in the microalgal growth promotion and defence against environmental pollutants is here discussed for all the bacterial strains associated to the SEC_LI_ChL_1 phycosphere.

Sequencing of Treponema pallidum subsp. pallidum from isolate UZ1974 using Anti-Treponemal Antibodies Enrichment: First complete whole genome sequence obtained directly from human clinical material.

Treponema pallidum subsp. pallidum (TPA) is the infectious agent of syphilis, a disease that infects more than 5 million people annually. Since TPA is an uncultivable bacterium, most of the information on TPA genetics comes from genome sequencing and molecular typing studies. This study presents the first complete TPA genome (without sequencing gaps) of clinical isolate (UZ1974), which was obtained directly from clinical material, without multiplication in rabbits. Whole genome sequencing was performed using a newly developed Anti-Treponemal Antibody Enrichment technique combined with previously reported Pooled Segment Genome Sequencing. We identified the UW074B genome, isolated from a sample previously propagated in rabbits, to be the closest relative of the UZ1974 genome and calculated the TPA mutation rate as 2.8 x 10−10 per site per generation.

Draft Genome Sequence of a Dyella-Like Bacterium from the Planthopper Hyalesthes obsoletus.

We report here the draft genome sequence of a Dyella-like bacterium (DLB) isolated from Hyalesthes obsoletus, the insect vector of the uncultivable mollicute bacterium “Candidatus Phytoplasma.” This isolate inhibits Spiroplasma melliferum, a cultivable mollicute. The draft genome of DLB consists of 4,196,214 bp, with a 68.6% G+C content, and 3,757 genes were predicted.

Anaplasma platys Immunoblot Test Using Major Surface Antigens.

Anaplasma platys is an uncultivable tick-borne obligatory intracellular bacterium, which is known to infect platelets of dogs. A. platys causes infectious canine cyclic thrombocytopenia in subtropical and tropical regions throughout the world. Several cases of human infection with A. platys infection have also been reported. However, seroprevalence of A. platys exposure and infection has not been determined in most of the regions, in part, due to lack of a simple and reliable assay method. Furthermore, A. platys antigens recognized by dogs are unknown. We previously sequenced gene encoding A. platys major outer membrane proteins P44 and Omp-1X. In the present study, we obtained purified recombinant A. platys P44 and Omp-1X proteins, and using them as antigens in immunoblotting examined seroreactivity in dogs. Of 34 specimens from Venezuela where A. platys infection was previously reported, 25 specimens (73.5%) reacted to rAplP44 and/or rAplOMP-1X. Neither Anaplasma phagocytophilum-seropositive (N = 10) nor A. phagocytophilum-seronegative canine specimens (N = 10) from the geographic regions where A. platys infection has never been reported, reacted rAplP44 or rAplOMP-1X. The result indicates a high A. platys seroprevalence rate in tested dogs from Venezuela and suggests that the immunoblot analysis based on recombinant A. platys major outer membrane proteins can provide a simple and defined tool to enlighten the prevalence of A. platys infection.

Quantitative PCR analysis of Mycoplasma suis shedding patterns during experimental infection

The uncultivable hemotrophic bacterium Mycoplasma suis causes infectious anemia in pigs worldwide. The mechanisms by which M. suis is transmitted from pig to pig are largely unknown. Thus, the present study aimed at investigating urine, feces, saliva, nasal and vaginal secrets as well as environmental samples for the presence of M. suis DNA to get insights into potential transmission routes. Seven pigs were experimentally infected with M. suis KI3806. Samples were taken for 8 days post infection (p.i.). A quantitative LightCycler msg1 PCR was used to detect and quantify M. suis. Shedding was found in saliva as well as nasal and vaginal secrets from day 6 p.i. on with a quantity of 3.4×102 to 2.7×105M. suis/swab. In urine M. suis DNA could be detected in 100.0% of the samples from day 6 p.i. on with a quantity of 4.7×102 to 6.3×105M. suis per mL. When shedding patterns were correlated to the median bacterial blood loads shedding was observed at loads of 2.0×109–7.0×1010M. suis per mL blood. No M. suis DNA could be amplified from feces. Dust and water samples of the pig drinking troughs were positive for M. suis on days 2 and 6 post infection, air samples were M. suis-negative throughout the experiment. Our results indicate that blood independent direct transmission as well as indirect transmission via environmental contamination could play a role in the epidemiology of M. suis infections.

Occurrence of Mycoplasma suis in wild boars ( Sus scrofa L.)

Porcine infectious anemia is a well-known disease that occurs worldwide and is caused by the uncultivable hemotrophic bacterium Mycoplasma suis. In this study the occurrence of M. suis in wild boars was investigated by employing a quantitative real-time LightCycler PCR. M. suis infections were detected in 36 out of 359 wild boars (10.03%). Sequencing of the 16S rRNA gene and subsequent phylogenetic analysis revealed the existence of two genetically distinct M. suis subtypes in the wild boar population: one subtype was >99.0% identical to known American and European M. suis isolates, and the second subtype showed the highest homology to known Chinese isolates. In summary, this is the first detection of M. suis in wild boars. The role of M. suis as pathogen in wild boars has yet to be established, but the present findings revealed a possible wildlife reservoir for these bacteria.

Phylogeny of a novel “Helicobacter heilmannii” organism from a Japanese patient with chronic gastritis based on DNA sequence analysis of 16S rRNA and urease genes

"Helicobacter heilmannii" is an uncultivable spiral-shaped bacterium inhabiting the human gastric mucosa. It is larger and more tightly-coiled than H. pylori. We encountered a patient with chronic gastritis infected a "H. heilmannii"-like organism (HHLO), designated as SH6. Gastric mucosa derived from the patient was orally ingested by specific pathogen free mice. Colonization of the mice by SH6 was confirmed by electron microscopy of gastric tissue specimens. In an attempt to characterize SH6, 16S rRNA and urease genes were sequenced. The 16S rRNA gene sequence was most similar (99.4%; 1,437/1,445 bp) to HHLO C4E from a cheetah. However, the urease gene sequence displayed low similarity (81.7%; 1,240/1,516 bp) with HHLO C4E. Taxonomic analysis disclosed that SH6 represents a novel strain and should constitute a novel taxon in the phylogenetic trees, being discriminated from any other taxon, with the ability of infecting human gastric mucosa.

Ehrlichia ewingiiInfection Delays Spontaneous Neutrophil Apoptosis through Stabilization of Mitochondria

The uncultivable obligate intracellular bacterium Ehrlichia ewingii, previously known only as a canine pathogen, is the most recently recognized agent of human ehrlichiosis. E. ewingii is the only Ehrlichia species known to infect neutrophils. In the blood or in ex vivo culture, neutrophils generally have a short life span. In the present study, we investigated the effect of E. ewingii infection on spontaneous apoptosis of neutrophils. E. ewingii infection significantly delayed dog neutrophil apoptosis during ex vivo culture. The inhibitory effect on neutrophil apoptosis by E. ewingii was reversible on clearance of the organism. By using the fluorescent mitochondrial dyes Mitotracker Red 580 and JC-1, we found that E. ewingii infection stabilized mitochondrial integrity by maintaining mitochondrial membrane potential in neutrophils. These results suggest that E. ewingii delays spontaneous apoptosis of neutrophils via stabilization of host cell mitochondria.

Genome sequence of Symbiobacterium thermophilum, an uncultivable bacterium that depends on microbial commensalism.

Symbiobacterium thermophilum is an uncultivable bacterium isolated from compost that depends on microbial commensalism. The 16S ribosomal DNA-based phylogeny suggests that this bacterium belongs to an unknown taxon in the Gram-positive bacterial cluster. Here, we describe the 3.57 Mb genome sequence of S.thermophilum. The genome consists of 3338 protein-coding sequences, out of which 2082 have functional assignments. Despite the high G + C content (68.7%), the genome is closest to that of Firmicutes, a phylum consisting of low G + C Gram-positive bacteria. This provides evidence for the presence of an undefined category in the Gram-positive bacterial group. The presence of both spo and related genes and microscopic observation indicate that S.thermophilum is the first high G + C organism that forms endospores. The S.thermophilum genome is also characterized by the widespread insertion of class C group II introns, which are oriented in the same direction as chromosomal replication. The genome has many membrane transporters, a number of which are involved in the uptake of peptides and amino acids. The genes involved in primary metabolism are largely identified, except those that code several biosynthetic enzymes and carbonic anhydrase. The organism also has a variety of respiratory systems including Nap nitrate reductase, which has been found only in Gram-negative bacteria. Overall, these features suggest that S.thermophilum is adaptable to and thus lives in various environments, such that its growth requirement could be a substance or a physiological condition that is generally available in the natural environment rather than a highly specific substance that is present only in a limited niche. The genomic information from S.thermophilum offers new insights into microbial diversity and evolutionary sciences, and provides a framework for characterizing the molecular basis underlying microbial commensalism.

Taxonomy of Campylobacter, Arcobacter, Helicobacter and related bacteria: current status, future prospects and immediate concerns.

The taxonomy of the genus Campylobacter has changed dramatically since its inception in 1963. At that time the genus comprised just two species. At present, taxa that were once assigned to Campylobacter may belong to one of over 50 species distributed among six genera. Most of these taxa belong to a phylogenetically distinct group referred to as either ribosomal RNA (rRNA) superfamily VI or the epsilon division of the class Proteobacteria. The taxonomic diversity of the group is matched by the diverse habitats in which they may be found, and by the wide range of diseases that they are associated with. Recognition of their clinical and economic importance has resulted in intense interest in the group, and the application of increasingly sophisticated isolation, detection and chemotaxonomic methods continues to elucidate new aspects of their biodiversity. However, despite the advances in new bacterial systematics, there remain a number of important issues concerning the classification of various campylobacterial taxa that require careful consideration. Ultimately, these issues are relevant to many working in the field of applied microbiology, including clinicians, veterinarians, epidemiologists and taxonomists. The purpose of this article is briefly to review the major developments in the taxonomy of Campylobacter from its inception to the present day; summarize the most recent changes in the field; analyse current topical issues of special relevance to applied microbiologists, including identification of the bacteria; and speculate on future prospects for campylobacterial taxonomy.