Simple method for quantification of metal-based particles in biopsy samples of patients with long bone implants - Pilot study.

The presence of particles fixed in tissue samples due to implant degradation or disintegration plays an important role in post-operative complications. The ability to determine the size, shape, chemical composition and, above all, the number of these particles can be used in many areas of medicine. This study presents a novel, simple metal-based particle detection method using scanning electron microscopy with energy dispersive spectrometer (SEM-EDS). The presence of metal particles in biopsy specimens from long bone nail-fixated implants (10 patients with titanium steel nails and 10 patients with stainless steel nails) was studied. The samples were analysed using automated area analysis based on image binarization and brightness to 255 grayscale. The results were supplemented with histological data and statistically analysed. The method based on the software used was found to be accurate and easy to use and, thus, appears to be very suitable for particle detection in similar samples.

Settled iron-based road dust and its characteristics and possible association with detection in human tissues.

Settled road dust was examined to detect the presence of non-airborne submicron and nano-sized iron-based particles and to characterize these particles. Samples were collected from a road surface near a busy road junction in the city of Ostrava, Czech Republic, once a month from March to October. The eight collected samples were subjected to a combination of experimental techniques including elemental analysis, Raman microspectroscopy, scanning electron microscopy (SEM) analysis, and magnetometry. The data thereby obtained confirmed the presence of non-agglomerated spherical nano-sized iron-based particles, with average sizes ranging from 2 down to 490 nm. There are several sources in road traffic which generate road dust particles, including exhaust and non-exhaust processes. Some of them (e.g., brake wear) produce iron as the dominant metallic element. Raman microspectroscopy revealed forms of iron (mainly as oxides, Fe2O3, and mixtures of Fe2O3 and Fe3O4). Moreover, Fe3O4 was also detected in samples of human tissues from the upper and lower respiratory tract. In view of the fact that no agglomeration of those particles was found by SEM, it is supposed that these particles may be easily resuspended and represent a risk to human health due to inhalation exposure, as proved by the detection of particles with similar morphology and phase composition in human tissues.

Macrolide treatment failure in a case of secondary syphilis: a novel A2059G mutation in the 23S rRNA gene of Treponema pallidum subsp. pallidum.

We report an occurrence of treatment failure after oral spiramycin therapy in a man with secondary syphilis and a reported penicillin and tetracycline allergy. Molecular detection revealed treponemal DNA in the blood of the patient and sequencing of the 23S rDNA identified an A to G transition at the gene position corresponding to position 2059 in the Escherichia coli 23S rRNA gene. The occurrence of this novel 23S rDNA mutation was examined among 7 rabbit-propagated syphilitic strains of Treponema pallidum and among 22 syphilis patient isolates from the Czech Republic. The prevalence of A2058G and A2059G mutations among clinical specimens was 18.2 and 18.2 %, respectively.

Complete genome sequence of Treponema pallidum ssp. pallidum strain SS14 determined with oligonucleotide arrays.

BACKGROUND: Syphilis spirochete Treponema pallidum ssp. pallidum remains the enigmatic pathogen, since no virulence factors have been identified and the pathogenesis of the disease is poorly understood. Increasing rates of new syphilis cases per year have been observed recently. RESULTS: The genome of the SS14 strain was sequenced to high accuracy by an oligonucleotide array strategy requiring hybridization to only three arrays (Comparative Genome Sequencing, CGS). Gaps in the resulting sequence were filled with targeted dideoxy-terminators (DDT) sequencing and the sequence was confirmed by whole genome fingerprinting (WGF). When compared to the Nichols strain, 327 single nucleotide substitutions (224 transitions, 103 transversions), 14 deletions, and 18 insertions were found. On the proteome level, the highest frequency of amino acid-altering substitution polymorphisms was in novel genes, while the lowest was in housekeeping genes, as expected by their evolutionary conservation. Evidence was also found for hypervariable regions and multiple regions showing intrastrain heterogeneity in the T. pallidum chromosome. CONCLUSION: The observed genetic changes do not have influence on the ability of Treponema pallidum to cause syphilitic infection, since both SS14 and Nichols are virulent in rabbit. However, this is the first assessment of the degree of variation between the two syphilis pathogens and paves the way for phylogenetic studies of this fascinating organism.

A novel Treponema pallidum antigen, TP0136, is an outer membrane protein that binds human fibronectin.

The antigenicity, structural location, and function of the predicted lipoprotein TP0136 of Treponema pallidum subsp. pallidum were investigated based on previous screening studies indicating that anti-TP0136 antibodies are present in the sera of syphilis patients and experimentally infected rabbits. Recombinant TP0136 (rTP0136) protein was purified and shown to be strongly antigenic during human and experimental rabbit infection. The TP0136 protein was exposed on the surface of the bacterial outer membrane and bound to the host extracellular matrix glycoproteins fibronectin and laminin. In addition, the TP0136 open reading frame was shown to be highly polymorphic among T. pallidum subspecies and strains at the nucleotide and amino acid levels. Finally, the ability of rTP0136 protein to act as a protective antigen to subsequent challenge with infectious T. pallidum in the rabbit model of infection was assessed. Immunization with rTP0136 delayed ulceration but did not prevent infection or the formation of lesions. These results demonstrate that TP0136 is expressed on the outer membrane of the treponeme during infection and may be involved in attachment to host extracellular matrix components.

Complete sequence of low-copy-number plasmid MccC7-H22 of probiotic Escherichia coli H22 and the prevalence of mcc genes among human E. coli.

The complete sequence of the plasmid MccC7-H22 encoding microcin C7, isolated from probiotic E. coli H22, was determined and analyzed. DNA of pMccC7-H22 comprises 32,014 bp and contains 39 predicted ORFs. Two main gene clusters, i.e., genes involved in plasmid replication and maintenance and genes encoding microcin C7 synthesis, are separated by several ORFs homologous to ORFs present in IS (insertion sequence) elements and transposons. Additional 14 ORFs code for proteins with similarities to known proteins (4 ORFs) or for hypothetical proteins with unknown function (10 ORFs). The differences in G+C content of individual ORFs and gene clusters of pMccC7-H22 indicate a mosaic structure for the plasmid, resulting from recombination events. Real-time PCR quantification was applied to measure the copy number of pMccC7-H22. Escherichia coli H22 carries approximately 5 copies of pMccC7-H22 per chromosome and thus pMccC7-H22 belongs to the group of relatively low-copy-number plasmids. Following 360 generations, all bacterial colonies (out of 100 tested) synthesized microcin C7 indicating that pMccC7-H22 is stably maintained in E. coli H22. Screening of 105 E. coli strains isolated from human fecal samples revealed 2 (1.9%) strains that produced microcin C7.

Genome differences between Treponema pallidum subsp. pallidum strain Nichols and T. paraluiscuniculi strain Cuniculi A.

The genome of Treponema paraluiscuniculi strain Cuniculi A was compared to the genome of the syphilis spirochete Treponema pallidum subsp. pallidum strain Nichols using DNA microarray hybridization, whole-genome fingerprinting, and DNA sequencing. A DNA microarray of T. pallidum subsp. pallidum Nichols containing all 1,039 predicted open reading frame PCR products was used to identify deletions and major sequence changes in the Cuniculi A genome. Using these approaches, deletions, insertions, and prominent sequence changes were found in 38 gene homologs and six intergenic regions of the Cuniculi A genome when it was compared to the genome of T. pallidum subsp. pallidum Nichols. Most of the observed differences were localized in tpr loci and the vicinity of these loci. In addition, 14 other genes were found to contain frameshift mutations resulting in major changes in protein sequences. Analysis of restriction target sites representing 0.34% of the total genome length and DNA sequencing of three PCR products (0.46% of the total genome length) amplified from Cuniculi A chromosomal regions and comparison to the Nichols genome revealed a sequence similarity of 98.6 to 99.3%. These results are consistent with a close genetic relationship among the T. pallidum strains and subspecies and a strong, but relatively divergent connection between the human and rabbit pathogens.

Detection of Treponema pallidum subsp. pallidum from skin lesions, serum, and cerebrospinal fluid in an infant with congenital syphilis after clindamycin treatment of the mother during pregnancy.

We report here a case of congenital syphilis in a newborn after clindamycin treatment in pregnancy. Using PCR detection of tmpC (TP0319) and DNA sequencing of the genes TP0136 and TP0548, DNA sequences identical to Treponema pallidum subsp. pallidum strain SS14 were detected in the infant's skin lesions, serum, and cerebrospinal fluid.

Recognition of pore-forming colicin Y by its cognate immunity protein.

Construction of hybrid immunity genes between colicin U (cui) and Y (cyi) immunity genes and site-directed mutagenesis of cyi were used to identify amino-acid residues of the colicin Y immunity protein (Cyi) involved in recognition of colicin Y. These amino-acid residues were localized close to the cytoplasmic site of the Cyi transmembrane helices T3 (S104, S107, F110, A112) and T4 (A159). Mutations in cui, which converted Cui sequence to Cyi sequence in positions 104, 107, 110, 112 and 159, resulted in an immunity gene that also conferred (besides immunity to colicin U) a high degree of immunity to colicin Y.