Structural variability of CG-rich DNA 18-mers accommodating double T-T mismatches.

Solution and crystal data are reported for DNA 18-mers with sequences related to those of bacterial noncoding single-stranded DNA segments called repetitive extragenic palindromes (REPs). Solution CD and melting data showed that the CG-rich, near-palindromic REPs from various bacterial species exhibit dynamic temperature-dependent and concentration-dependent equilibria, including architectures compatible with not only hairpins, which are expected to be biologically relevant, but also antiparallel duplexes and bimolecular tetraplexes. Three 18-mer oligonucleotides named Hpar-18 (PDB entry 6rou), Chom-18 (PDB entry 6ros) and its brominated variant Chom-18Br (PDB entry 6ror) crystallized as isomorphic right-handed A-like duplexes. The low-resolution crystal structures were solved with the help of experimental phases for Chom-18Br. The center of the duplexes is formed by two successive T-T noncanonical base pairs (mismatches). They do not deform the double-helical geometry. The presence of T-T mismatches prompted an analysis of the geometries of these and other noncanonical pairs in other DNA crystals in terms of their fit to the experimental electron densities (RSCC) and their geometric fit to the NtC (dinucleotide conformational) classes (https://dnatco.datmos.org/). Throughout this work, knowledge of the NtC classes was used to refine and validate the crystal structures, and to analyze the mismatches.

"Double-barrel endocarditis".

We report a case of an 18-year-old woman who presented with infective endocarditis (IE), in two conduits percutaneously delivered in the right ventricle outflow tract ("double-barrel endocarditis"). The patient's clinical presentation, echocardiogram findings, infectious agent, clinical management, surgical approach, and follow-up assessment are described. Percutaneous pulmonary valve implantation has emerged as a viable therapy for conduit dysfunction in the right ventricular outflow tract. Although the percutaneous approach has several advantages, this strategy and the valves used are not complication-free. IE after transcatheter valve deployment has evoked the growing concern, as there is a higher incidence in these patients compared with patients with surgically repaired pulmonary valves. As a result, this type of surgical treatment is especially important.

[PCR diagnosis of infectious diseases]. / PCR diagnostika infekcí.

The practices based on microbe cultivation are the corner stone of laboratory diagnostics of infectious diseases etiology. However, cultivation-independent molecular methods for microorganism detection are increasingly being used in a routine clinical setting as an important complement to the conventional procedures and, in some cases, they have already become the golden diagnostic standard. The PCR-based methods are used the most frequently.Key words: molecular genetic detection of microorganisms - polymerase chain reaction (PCR).

Analysis of the Microbiota of Black Stain in the Primary Dentition.

Black tooth stain is a characteristic extrinsic discoloration commonly seen on the cervical enamel following the contour of the gingiva. To investigate the relationship between black tooth stain and the oral microbiota, we used 16S rRNA gene sequencing to compare the microbial composition of dental plaque and saliva among caries-free children with and without black stain. Dental plaque and saliva, as well as black stain, were sampled from 10 children with and 15 children without black stain. Data were analyzed using the pipeline tool MOTHUR. Student's t-test was used to compare alpha diversities and the Mann-Whitney U test to compare the relative abundances of the microbial taxa. A total of 10 phyla, 19 classes, 32 orders, 61 families and 102 genera were detected in these samples. Shannon and Simpson diversity were found to be significantly lower in saliva samples of children with black stain. Microbial diversity was reduced in the black stain compared to the plaque samples. Actinomyces, Cardiobacterium, Haemophilus, Corynebacterium, Tannerella and Treponema were more abundant and Campylobacter less abundant in plaque samples of children with black stain. Principal component analysis demonstrated clustering among the dental plaque samples from the control group, while the plaque samples from the black stain group were not and appeared to cluster into two subgroups. Alterations in oral microbiota may be associated with the formation of black stain.

Rapid identification of Cardiobacterium hominis by MALDI-TOF mass spectrometry during infective endocarditis.

We report a new case of Cardiobacterium hominis endocarditis identified during an acute coronary syndrome. The positivity of the blood cultures was confirmed rapidly (50 h) as a result of improvements to the automated detection system, whereby it is no longer necessary to incubate the vials for long periods of time when Aggregatibacter-Cardiobacterium-Eikenella-Kingella infections is suspected. The phenotype-based VITEK 2 NH identification system is not able to distinguish between the two species of Cardiobacterium, as it does not contain C. valvarum in its library. The method for 16S rRNA gene sequence analysis is able to separate the two species but is not available in all laboratories. We used MALDI-TOF mass spectrometry, as an alternative, to rapidly distinguish between C. hominis and C. valvarum, because both species are contained in the system library.

Cardiobacterium valvarum infective endocarditis and phenotypic/molecular characterization of 11 Cardiobacterium species strains.

Cardiobacterium valvarum is a newly recognized human pathogen related to infective endocarditis. Cardiobacterium species are, however, only rarely the aetiology of infective endocarditis. An infective endocarditis case is presented and, additionally, phenotypic and phylogenetic comparison of a further 10 collection strains, representing the two species within the genus, was performed. C. valvarum was isolated from the blood and DNA was present in valvular tissue (partial 16S rRNA gene analysis) from a 64-year-old man with infective endocarditis of the mitral valve, rupture of chordae and prolapse of pulmonary valves in addition to a fluttering excrescence. A mechanical mitral valve and neochordae were inserted successfully. Phenotypically, the two species within the genus Cardiobacterium resemble each other greatly. When using the Vitek 2 Neisseria-Haemophilus identification card, the reaction for phenylphosphonate was positive for all Cardiobacterium hominis strains, but negative for all C. valvarum strains, thereby separating the two species. The two species made up two separate clusters by phylogenetic examination using 16S rRNA gene sequence analysis.

Endocarditis caused by Cardiobacterium valvarum.

A fastidious, gram-negative bacterium was isolated from the blood of a 51-year-old man who had acute infectious endocarditis (IE). Characterization of the organism through phenotypic and genotypic analyses revealed the causative role of Cardiobacterium valvarum. This is the third reported case of IE caused by C. valvarum.

Direct detection of Cardiobacterium hominis in serum from a patient with infective endocarditis by broad-range bacterial PCR.

Bacterial DNA was detected directly in the serum of a patient with endocarditis by broad-range 16S rRNA PCR followed by sequencing and analysis of the results by the BLAST search. Using these methods, Cardiobacterium hominis was identified in 2 days from the date of serum collection. The microorganism was also isolated and identified using conventional methods (bacterial culture and biochemical tests) 17 days from the date of sample collection. This is the first report showing the direct detection of C. hominis in a patient's serum using molecular-based methods, emphasizing their potential usefulness as additional and rapid diagnostic tools for the detection and identification of fastidious bacteria.

The role of 16S rRNA gene sequencing in identification of microorganisms misidentified by conventional methods.

Traditional methods for microbial identification require the recognition of differences in morphology, growth, enzymatic activity, and metabolism to define genera and species. Full and partial 16S rRNA gene sequencing methods have emerged as useful tools for identifying phenotypically aberrant microorganisms. We report on three bacterial blood isolates from three different College of American Pathologists-certified laboratories that were referred to ARUP Laboratories for definitive identification. Because phenotypic identification suggested unusual organisms not typically associated with the submitted clinical diagnosis, consultation with the Medical Director was sought and further testing was performed including partial 16S rRNA gene sequencing. All three patients had endocarditis, and conventional methods identified isolates from patients A, B, and C as a Facklamia sp., Eubacterium tenue, and a Bifidobacterium sp. 16S rRNA gene sequencing identified the isolates as Enterococcus faecalis, Cardiobacterium valvarum, and Streptococcus mutans, respectively. We conclude that the initial identifications of these three isolates were erroneous, may have misled clinicians, and potentially impacted patient care. 16S rRNA gene sequencing is a more objective identification tool, unaffected by phenotypic variation or technologist bias, and has the potential to reduce laboratory errors.

Characterization of oral strains of Cardiobacterium valvarum and emended description of the organism.

The description of the new species Cardiobacterium valvarum prompted a search for additional strains of the organism. Here we report characterization of four oral Cardiobacterium strains from the Culture Collection of the University of Goteborg. The 16S rRNA gene sequences of the organisms exhibited 99.6% to 99.3% homology with Cardiobacterium valvarum. The cellular fatty acid profiles, electrophoretic patterns of whole-cell proteins, growth rate and nutritional requirement, colonial and cellular morphology, and biochemical reactions were also similar to those of C. valvarum. These results thus classify these organisms as oral strains of C. valvarum. All strains were susceptible to many antibiotics tested. The description of the species was emended. C. valvarum is a rare cause of endocarditis, and its relationship with periodontal diseases may need investigation.

[Detection of bacterial molecular markers in the tissue of cardiac valves in normal and pathological states by gas chromatography and mass spectrometry]. / Detektsiia molekuliarnykh markerov bakterii v tkani klapanov serdtsa v norme i pri patologii s primeneniem metoda gazovoi khromatografii i mass-spektrometrii.

Samples from cardiac valves of 31 patients were analyzed by gas chromatography and mass spectrometry. The algorithm of mass spectrometric parameters was developed, which permitted the determination of about 200 known microbial fatty acids, aldehydes and sterols, sufficient for the detection and quantitative determination of more that 170 taxons of clinically significant microorganisms on the genus or species levels. The quantitative and qualitative differences in the composition of microbial markers of endocardial valves in normal and pathological states, particularly in cases of infectious endocarditis, were detected. The participation of 37 microbial taxons in the process was confirmed. The level of endocardium colonization in infectious endocarditis reached from 2 to 7 x 10(9) microbial cells/g of valvular tissue (which exceeded twofold the equivalent concentrations of the marker in the normal state). In terms of quantity, the leading role was played by Cardiobacterium hominis.

Endocarditis with ruptured cerebral aneurysm caused by Cardiobacterium valvarum sp. nov.

A fastidious gram-negative bacterium was isolated from the blood of a 37-year-old man who had insidious endocarditis with a sudden rupture of a cerebral aneurysm. Characterization of the organism through phylogenetic and phenotypic analyses revealed a novel species of Cardiobacterium, for which the name Cardiobacterium valvarum sp. nov. is proposed. C. valvarum will supplement the current sole species Cardiobacterium hominis, a known cause of endocarditis. Surgeries and antibiotic treatment cured the patient's infection and associated complications. During cardiac surgery, a congenital bicuspid aortic valve was found to be the predisposing factor for his endocarditis.