C-terminal intrinsically disordered region-dependent organization of the mycobacterial genome by a histone-like protein.

The architecture of the genome influences the functions of DNA from bacteria to eukaryotes. Intrinsically disordered regions (IDR) of eukaryotic histones have pivotal roles in various processes of gene expression. IDR is rare in bacteria, but interestingly, mycobacteria produce a unique histone-like protein, MDP1 that contains a long C-terminal IDR. Here we analyzed the role of IDR in MDP1 function. By employing Mycobacterium smegmatis that inducibly expresses MDP1 or its IDR-deficient mutant, we observed that MDP1 induces IDR-dependent DNA compaction. MDP1-IDR is also responsible for the induction of growth arrest and tolerance to isoniazid, a front line tuberculosis drug that kills growing but not growth-retardated mycobacteria. We demonstrated that MDP1-deficiency and conditional knock out of MDP1 cause spreading of the M. smegmatis genome in the stationary phase. This study thus demonstrates for the first time a C-terminal region-dependent organization of the genome architecture by MDP1, implying the significance of IDR in the function of bacterial histone-like protein.

Genomic comparison between Staphylococcus aureus GN strains clinically isolated from a familial infection case: IS1272 transposition through a novel inverted repeat-replacing mechanism.

A bacterial insertion sequence (IS) is a mobile DNA sequence carrying only the transposase gene (tnp) that acts as a mutator to disrupt genes, alter gene expressions, and cause genomic rearrangements. "Canonical" ISs have historically been characterized by their terminal inverted repeats (IRs), which may form a stem-loop structure, and duplications of a short (non-IR) target sequence at both ends, called target site duplications (TSDs). The IS distributions and virulence potentials of Staphylococcus aureus genomes in familial infection cases are unclear. Here, we determined the complete circular genome sequences of familial strains from a Panton-Valentine leukocidin (PVL)-positive ST50/agr4 S. aureus (GN) infection of a 4-year old boy with skin abscesses. The genomes of the patient strain (GN1) and parent strain (GN3) were rich for "canonical" IS1272 with terminal IRs, both having 13 commonly-existing copies (ce-IS1272). Moreover, GN1 had a newly-inserted IS1272 (ni-IS1272) on the PVL-converting prophage, while GN3 had two copies of ni-IS1272 within the DNA helicase gene and near rot. The GN3 genome also had a small deletion. The targets of ni-IS1272 transposition were IR structures, in contrast with previous "canonical" ISs. There were no TSDs. Based on a database search, the targets for ce-IS1272 were IRs or "non-IRs". IS1272 included a larger structure with tandem duplications of the left (IRL) side sequence; tnp included minor cases of a long fusion form and truncated form. One ce-IS1272 was associated with the segments responsible for immune evasion and drug resistance. Regarding virulence, GN1 expressed cytolytic peptides (phenol-soluble modulin α and δ-hemolysin) and PVL more strongly than some other familial strains. These results suggest that IS1272 transposes through an IR-replacing mechanism, with an irreversible process unlike that of "canonical" transpositions, resulting in genomic variations, and that, among the familial strains, the patient strain has strong virulence potential based on community-associated virulence factors.

Molecular characterization of Streptococcus pneumoniae, particularly serotype19A/ST320, which emerged in Krasnoyarsk, Russia.

Streptococcus pneumoniae, a common human pathogen, colonizes the nasopharynx and causes diseases including acute otitis media (AOM). Herein, pneumococcal serotype distributions in children before and after PCV7 vaccination and in patients with pneumococcal disease in Siberian Russia (Krasnoyarsk) are reported. Analyses included antimicrobial susceptibility testing, sequence typing (ST), pulsed field gel electrophoresis, virulence-related surface protein gene (VSG) typing with novel primers and structural analysis by scanning electron microscopy. In healthy children (HC) prior to administration of PCV7, drug-susceptible serotype23F/ST1500 was a major pneumococcal genotype. In the PCV7 trial, multidrug-resistant serotype19A/ST320 emerged in vaccinees after PCV7, exhibiting a PCV7-induced serotype replacement. Multidrug-resistant serotype19A/ST320 was evident in patients with AOM. Community-acquired pneumonia (CAP) isolates showed genetic similarities to the AOM (ST320) genotype, constituting a common non-invasive AOM-CAP group. In contrast, meningitis isolates were more divergent. Overall, 25 ST types were identified; five (20%) of which were Krasnoyarsk-native. Regarding VSGs, PI-1 (rlrA/rrgB), PI-2 (pitA/B), psrP and cbpA were present at 54.3%, 38.6%, 48.6%, and 95.7%, respectively, with two major VSG content types, PI-1- /PI-2- /psrP+ /cbpA+ and PI-1+ /PI-2+ /psrP- /cbpA+ , being found for HC and non-invasive diseases, respectively. A major clone of serotype19A/ST320 (PI-1+ /PI-2+ ) produced the longest pneumococcal wire (pilus) structures in colonies. ST1016 (PI-1- /PI-2- ) in HC had HEp-2 cell-adherent pili. These results suggest that serotype19A/ST320 and related genotypes, with the VSG content type PI-1+ /PI-2+ /psrP- /cbpA+ , emerged in vaccinees after PCV7 in Siberia, accompanying diseases in non-vaccinated children, and that some genotypes (serotypes19A/ST320 and 18/ST1016) produced novel pneumococcal structures, predicting their roles in colony formation and adherence.

Complete Circular Genome Sequence of Successful ST8/SCCmecIV Community-Associated Methicillin-Resistant Staphylococcus aureus (OC8) in Russia: One-Megabase Genomic Inversion, IS256's Spread, and Evolution of Russia ST8-IV.

ST8/SCCmecIV community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) has been a common threat, with large USA300 epidemics in the United States. The global geographical structure of ST8/SCCmecIV has not yet been fully elucidated. We herein determined the complete circular genome sequence of ST8/SCCmecIVc strain OC8 from Siberian Russia. We found that 36.0% of the genome was inverted relative to USA300. Two IS256, oppositely oriented, at IS256-enriched hot spots were implicated with the one-megabase genomic inversion (MbIN) and vSaß split. The behavior of IS256 was flexible: its insertion site (att) sequences on the genome and junction sequences of extrachromosomal circular DNA were all divergent, albeit with fixed sizes. A similar multi-IS256 system was detected, even in prevalent ST239 healthcare-associated MRSA in Russia, suggesting IS256's strong transmission potential and advantage in evolution. Regarding epidemiology, all ST8/SCCmecIVc strains from European, Siberian, and Far Eastern Russia, examined had MbIN, and geographical expansion accompanied divergent spa types and resistance to fluoroquinolones, chloramphenicol, and often rifampicin. Russia ST8/SCCmecIVc has been associated with life-threatening infections such as pneumonia and sepsis in both community and hospital settings. Regarding virulence, the OC8 genome carried a series of toxin and immune evasion genes, a truncated giant surface protein gene, and IS256 insertion adjacent to a pan-regulatory gene. These results suggest that unique single ST8/spa1(t008)/SCCmecIVc CA-MRSA (clade, Russia ST8-IVc) emerged in Russia, and this was followed by large geographical expansion, with MbIN as an epidemiological marker, and fluoroquinolone resistance, multiple virulence factors, and possibly a multi-IS256 system as selective advantages.

Healthcare- and Community-Associated Methicillin-Resistant Staphylococcus aureus (MRSA) and Fatal Pneumonia with Pediatric Deaths in Krasnoyarsk, Siberian Russia: Unique MRSA's Multiple Virulence Factors, Genome, and Stepwise Evolution.

Methicillin-resistant Staphylococcus aureus (MRSA) is a common multidrug-resistant (MDR) pathogen. We herein discussed MRSA and its infections in Krasnoyarsk, Siberian Russia between 2007 and 2011. The incidence of MRSA in 3,662 subjects was 22.0% and 2.9% for healthcare- and community-associated MRSA (HA- and CA-MRSA), respectively. The 15-day mortality rates for MRSA hospital- and community-acquired pneumonia (HAP and CAP) were 6.5% and 50%, respectively. MRSA CAP cases included pediatric deaths; of the MRSA pneumonia episodes available, ≥27.3% were associated with bacteremia. Most cases of HA-MRSA examined exhibited ST239/spa3(t037)/SCCmecIII.1.1.2 (designated as ST239Kras), while all CA-MRSA cases examined were ST8/spa1(t008)/SCCmecIV.3.1.1(IVc) (designated as ST8Kras). ST239Kras and ST8Kras strongly expressed cytolytic peptide (phenol-soluble modulin α, PSMα; and δ-hemolysin, Hld) genes, similar to CA-MRSA. ST239Kras pneumonia may have been attributed to a unique set of multiple virulence factors (MVFs): toxic shock syndrome toxin-1 (TSST-1), elevated PSMα/Hld expression, α-hemolysin, the staphylococcal enterotoxin SEK/SEQ, the immune evasion factor SCIN/SAK, and collagen adhesin. Regarding ST8Kras, SEA was included in MVFs, some of which were common to ST239Kras. The ST239Kras (strain OC3) genome contained: a completely unique phage, φSa7-like (W), with no att repetition; S. aureus pathogenicity island SaPI2R, the first TSST-1 gene-positive (tst+) SaPI in the ST239 lineage; and a super copy of IS256 (≥22 copies/genome). ST239Kras carried the Brazilian SCCmecIII.1.1.2 and United Kingdom-type tst. ST239Kras and ST8Kras were MDR, with the same levofloxacin resistance mutations; small, but transmissible chloramphenicol resistance plasmids spread widely enough to not be ignored. These results suggest that novel MDR and MVF+ HA- and CA-MRSA (ST239Kras and ST8Kras) emerged in Siberian Russia (Krasnoyarsk) associated with fatal pneumonia, and also with ST239Kras, a new (Siberian Russian) clade of the ST239 lineage, which was created through stepwise evolution during its potential transmission route of Brazil-Europe-Russia/Krasnoyarsk, thereby selective advantages from unique MVFs and the MDR.

An in vivo study of osteoplastic properties of resorbable poly-3-hydroxybutyrate in models of segmental osteotomy and chronic osteomyelitis.

A series of 3D implants and filling materials prepared from powdered biodegradable polymers, polyhydroxyalkanoates (PHAs), have been designed for the purposes of reparative osteogenesis. The 3D implants are made of resorbable polymer of hydroxybutyric acid (poly-3-hydroxybutyrate, P3HB) and a composite of this polymer with hydroxyapatite (HA) (P3HB/HA). The properties of the implants were studied in vivo in a model of segmental osteotomy and compared with commercial material Bio-Oss(®). All implants containing P3HB as the main component facilitate reconstructive osteogenesis. P3HB and P3HB/HA show pronounced osteoplastic properties; their in vivo degradation is slow and corresponds to the growth of a new bone tissue, facilitating normal reparative osteogenesis. Also, powdered P3HB and P3HB/tienam can be used as filling materials for osteoplasty of bone cavities infected by Staphylococcus aureus. Biodegradable 3D implants and P3HB-based filling materials show pronounced osteoplastic properties and degrade in vivo at a slow rate, enabling normal reparative osteogenesis.