Bacteriophages of the significant veterinary pathogen Staphylococcus pseudintermedius are rarely described morphologically and genomically in detail, and mostly include phages of the Siphoviridae family. There is currently no taxonomical classification for phages of this bacterial species. Here we describe a new phage designated vB_SpsS_QT1, which is related to phage 2638A originally described as a Staphylococcus aureus phage. Propagating strain S. aureus 2854 of the latter was reclassified by rpoB gene sequencing as S. pseudintermedius 2854 in this work. Both phages have a narrow but different host range determined on 54 strains. Morphologically, both of them belong to the family Siphoviridae, share the B1 morphotype, and differ from other staphylococcal phage genera by a single long fibre at the terminus of the tail. The complete genome of phage vB_SpsS_QT1 was sequenced with the IonTorrent platform and expertly annotated. Its linear genome with cohesive ends is 43,029 bp long and encodes 60 predicted genes with the typical modular structure of staphylococcal siphophages. A global alignment found the genomes of vB_SpsS_QT1 and 2638A to share 84% nucleotide identity, but they have no significant similarity of nucleotide sequences with other phage genomes available in public databases. Based on the morphological, phylogenetic, and genomic analyses, a novel genus Fibralongavirus in the family Siphoviridae is described with phage species vB_SpsS_QT1 and 2638A.

• MeSH
• fylogeneze MeSH
• genom virový MeSH
• genomika metody   MeSH
• hostitelská specificita MeSH
• replikace viru MeSH
• Siphoviridae klasifikace   ultrastruktura   MeSH
• Staphylococcus virologie   MeSH
• virion ultrastruktura   MeSH
• virové geny MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Lytic bacteriophages are valuable therapeutic agents against bacterial infections. There is continual effort to obtain new phages to increase the effectivity of phage preparations against emerging phage-resistant strains. Here we described the genomic diversity of spontaneous host-range mutants of kayvirus 812. Five mutant phages were isolated as rare plaques on phage-resistant Staphylococcus aureus strains. The host range of phage 812-derived mutants was 42% higher than the wild type, determined on a set of 186 methicillin-resistant S. aureus strains representing the globally circulating human and livestock-associated clones. Comparative genomics revealed that single-nucleotide polymorphisms from the parental phage 812 population were fixed in next-step mutants, mostly in genes for tail and baseplate components, and the acquired point mutations led to diverse receptor binding proteins in the phage mutants. Numerous genome changes associated with rearrangements between direct repeat motifs or intron loss were found. Alterations occurred in host-takeover and terminal genomic regions or the endolysin gene of mutants that exhibited the highest lytic activity, which implied various mechanisms of overcoming bacterial resistance. The genomic data revealed that Kayvirus spontaneous mutants are free from undesirable genes and their lytic properties proved their suitability for rapidly updating phage therapeutics.

• MeSH
• bakteriální léková rezistence MeSH
• bakteriofágy genetika   MeSH
• délka genomu MeSH
• genom virový MeSH
• genomika MeSH
• jednonukleotidový polymorfismus MeSH
• methicilin farmakologie   MeSH
• mutace * MeSH
• Staphylococcus aureus růst a vývoj   virologie   MeSH
• zastoupení bazí MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The genus Macrococcus is a close relative of the genus Staphylococcus. Whilst staphylococci are widespread as human pathogens, macrococci have not yet been reported from human clinical specimens. Here we investigated Gram-positive and catalase-positive cocci recovered from human clinical material and identified as Macrococcus sp. by a polyphasic taxonomic approach and by comparative genomics. Relevant phenotypic, genotypic and chemotaxonomic methods divided the analyzed strains into two separate clusters within the genus Macrococcus. Comparative genomics of four representative strains revealed enormous genome structural plasticity among the studied isolates. We hypothesize that high genomic variability is due to the presence of a com operon, which plays a key role in the natural transformation of bacilli and streptococci. The possible uptake of exogenous DNA by macrococci can contribute to a different mechanism of evolution from staphylococci, where phage-mediated horizontal gene transfer predominates. The described macrococcal genomes harbor novel plasmids, genomic islands and islets, as well as prophages. Capsule gene clusters, intracellular protease, and a fibronectin-binding protein enabling opportunistic pathogenesis were found in all four strains. Furthermore, the presence of a CRISPR-Cas system with 90 spacers in one of the sequenced genomes corresponds with the need to limit the burden of foreign DNA. The highly dynamic genomes could serve as a platform for the exchange of virulence and resistance factors, as was described for the methicillin resistance gene, which was found on the novel composite SCCmec-like element containing a unique mec gene complex that is considered to be one of the missing links in SCC evolution. The phenotypic, genotypic, chemotaxonomic and genomic results demonstrated that the analyzed strains represent one novel subspecies and three novel species of the genus Macrococcus, for which the names Macrococcus caseolyticus subsp. hominis subsp. nov. (type strain CCM 7927T = DSM 103682T), Macrococcus goetzii sp. nov. (type strain CCM 4927T = DSM 103683T), Macrococcus epidermidis sp. nov. (type strain CCM 7099T = DSM 103681T), and Macrococcus bohemicus sp. nov. (type strain CCM 7100T = DSM 103680T) are proposed. Moreover, a formal description of Macrococcus caseolyticus subsp. caseolyticus subsp. nov. and an emended description of the genus Macrococcus are provided.

• Publikační typ
• časopisecké články MeSH

Staphylococcus aureus je významný oportunní patogen a častý původce bakteriálních nozokomiálních nákaz.Patogenní vlastnosti této bakterie jsou často kódovány mobilními genetickými elementy, jako jsou např. plazmi-dy. Na plazmidu se nachází i gen etb pro exfoliativní toxin B (ETB), který je příčinou puchýřnatého onemocnění(bulózní impetigo), jehož generalizovaná a život ohrožující forma se označuje jako stafylokokový syndrom opařené kůže. U pěti etb-pozitivních plazmidů, izolovaných během let 1999–2015 z kmenů S. aureus pocházejícíchz českých nemocnic, byla provedena srovnávací sekvenční analýza. Bylo zjištěno, že velká většina z nich sdílírozsáhlou oblast sekvence DNA, která zahrnuje typické geny virulence, avšak obsahují i variabilní úseky umož-ňující jejich diferenciaci. Přestože se dosud předpokládalo, že jsou ETB plazmidy relativně uniformní, byl izolo-ván a popsán nový typ, který s ostatními ETB plazmidy vykazuje jen minimální sekvenční shodu. Nese navíc geny pro horizontální přenos konjugací a nové varianty genů pro faktory virulence včetně etb, čímž reprezentuje zcela novou linii plazmidů kódujících exfoliatin B.

Staphylococcus aureus is an important opportunistic pathogen and a common cause of bacterial nosocomialinfections. Pathogenic properties of this bacterium are often encoded by mobile genetic elements, such as plas-mids. Plasmids also carry the etb gene encoding exfoliative toxin B(ETB), which is the cause of ablistering disease(bullous impetigo)whose generalized and life-threatening form is known as staphylococcal scalded skin syn-drome. Comparative sequence analysis was conducted to study five etb-positive plasmids, isolated from S. aureusstrains from Czech hospitals between 1999 and 2015. They were found to share a large part of the DNA sequencecomprising typical virulence genes. However, they also contain variable fragments, which help us to differentiatebetween them. Although ETB plasmids were previously assumed to be relatively uniform, a new type showingminimal sequence similarity to other ETB plasmids was isolated and described. Moreover, it carries genes forhorizontal transfer by conjugation as well as new variants of genes for virulence factors, including etb, and assuch it represents a completely new lineage of exfoliative toxin B-encoding plasmids.

• MeSH
• exfoliatiny genetika   MeSH
• faktory virulence genetika   MeSH
• genotypizační techniky MeSH
• plazmidy genetika   MeSH
• polymerázová řetězová reakce metody   MeSH
• Staphylococcus aureus genetika   izolace a purifikace   patogenita   MeSH

Exfoliative toxin B (ETB) encoded by some large plasmids plays a crucial role in epidermolytic diseases caused by Staphylococcus aureus. We have found as yet unknown types of etb gene-positive plasmids isolated from a set of impetigo strains implicated in outbreaks of pemphigus neonatorum in Czech maternity hospitals. Plasmids from the strains of clonal complex CC121 were related to archetypal plasmid pETBTY4. Sharing a 33-kb core sequence including virulence genes for ETB, EDIN C, and lantibiotics, they were assigned to a stand-alone lineage, named pETBTY4-based plasmids. Differing from each other in the content of variable DNA regions, they formed four sequence types. In addition to them, a novel unique plasmid pETB608 isolated from a strain of ST130 was described. Carrying conjugative cluster genes, as well as new variants of etb and edinA genes, pETB608 could be regarded as a source of a new lineage of ETB plasmids. We have designed a helpful detection assay, which facilitates the precise identification of the all described types of ETB plasmids.

• MeSH
• bakteriální proteiny genetika   MeSH
• bakteriociny genetika   MeSH
• dermotoxiny genetika   MeSH
• DNA bakterií genetika   MeSH
• exfoliatiny genetika   MeSH
• fylogeneze MeSH
• impetigo epidemiologie   mikrobiologie   MeSH
• lidé MeSH
• pemfigus epidemiologie   mikrobiologie   MeSH
• plazmidy genetika   izolace a purifikace   MeSH
• sekvenování celého genomu MeSH
• Staphylococcus aureus klasifikace   genetika   MeSH
• virulence genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Česká republika epidemiologie   MeSH

The newly developed molecular methods for direct detection of S. aureus, MRSA, small colony variants (SCV) of S. aureus and SCV-MRSA and special culture methods will be used for repeat analyses of clinical specimens from CF patients (about 300 strains per year). The molecular diagnostic and typing methods will be helpful not only in direct detection and clonal characterization of the pathogen but also in the analysis of resistance (resistance determinants, hypermutator phenotype) and virulence (superantigens, the pvl gene). The obtained results and their interpretation will be vital for tailored treatment of CF patients. The analysis of intracellular persistence of strains and their in vitro susceptibility to bacteriophages, with emphasis on SCV and SCV-MRSA strains, will be a point of departure for the development of candidate phage therapy in CF patients.

Nově zavedenými molekulárními metodami na přímý průkaz infekcí S. aureus, MRSA, small colny variants (SCV) S. aureus a SCV-MRSA a speciálními kultivačními metodami opakovaně vyšetříme klinický materiál pacientů s CF (ročně zhruba 300 kmenů). Nově zavedené molekulárně diagnostické a typizační metody umožní přímý průkaz a klonální charakterizaci patogena, ale i analýzu rezistence (determinanty rezistence, hypermutatorový typ rezistence) a virulence (superantigeny, gen pvl). Aplikace těchto progresivních metod a interpretace jejich výsledků poslouží k přímé racionalizaci léčby konkrétních pacientů s CF. Analýza intracelulární perzistence a citlivosti kmenů k bakteriofágům, s důrazem na SCV a SCV-MRSA, in vitro vytvoří předpoklady pro další studium specifické fagoterapie u pacientů s CF.

• MeSH
• cystická fibróza komplikace   mikrobiologie   MeSH
• diagnostické techniky molekulární MeSH
• methicilin rezistentní Staphylococcus aureus MeSH
• molekulární biologie MeSH
• polymerázová řetězová reakce MeSH
• stafylokokové infekce MeSH
• Staphylococcus aureus patogenita   MeSH

• Konspekt
• Mikrobiologie
• NLK Obory
• bakteriologie
• genetika, lékařská genetika
• NLK Publikační typ
• závěrečné zprávy o řešení grantu IGA MZ ČR

In Staphylococcus aureus, generalized transduction mediated by temperate bacteriophages represents a highly efficient way of transferring antibiotic resistance genes between strains. In the present study, we identified and characterized in detail a new efficiently transducing bacteriophage of the family Siphoviridae, designated ϕJB, which resides as a prophage in the meticillin-resistant S. aureus (MRSA) strain Jevons B. Whole-genome sequencing followed by detailed in silico analysis uncovered a linear dsDNA genome consisting of 43 ,12 bp and comprising 70 ORFs, of which ∼40 encoded proteins with unknown function. A global genome alignment of ϕJB and other efficiently transducing phages ϕ11, ϕ53, ϕ80, ϕ80α and ϕNM4 showed a high degree of homology with ϕNM4 and substantial differences with regard to other phages. Using a model transduction system with a well-defined donor and recipient, ϕJB transferred the tetracycline resistance plasmid pT181 and a penicillinase plasmid with outstanding frequencies, beating most of the above-mentioned phages by an order of magnitude. Moreover, ϕJB demonstrated high frequencies of transferring antibiotic resistance plasmids even upon induction from a lysogenic donor strain. Considering such transducing potential, ϕJB and related bacteriophages may serve as a suitable tool for elucidating the nature of transduction and its contribution to the spread of antibiotic resistance genes in naturally occurring MRSA populations.

• MeSH
• aktivace viru MeSH
• bakteriální léková rezistence MeSH
• DNA virů chemie   genetika   MeSH
• fylogeneze MeSH
• genom virový MeSH
• lyzogenie MeSH
• methicilin rezistentní Staphylococcus aureus virologie   MeSH
• molekulární sekvence - údaje MeSH
• otevřené čtecí rámce MeSH
• plazmidy MeSH
• pořadí genů MeSH
• přenos genů horizontální MeSH
• profágy genetika   izolace a purifikace   ultrastruktura   MeSH
• sekvenční analýza DNA MeSH
• sekvenční homologie MeSH
• Siphoviridae genetika   izolace a purifikace   ultrastruktura   MeSH
• syntenie MeSH
• transdukce genetická * MeSH
• transmisní elektronová mikroskopie MeSH
• výpočetní biologie MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Exfoliative toxin A (ETA)-coding temperate bacteriophages are leading contributors to the toxic phenotype of impetigo strains of Staphylococcus aureus. Two distinct eta gene-positive bacteriophages isolated from S. aureus strains which recently caused massive outbreaks of pemphigus neonatorum in Czech maternity hospitals were characterized. The phages, designated φB166 and φB236, were able to transfer the eta gene into a prophageless S. aureus strain which afterwards converted into an ETA producer. Complete phage genome sequences were determined, and a comparative analysis of five designed genomic regions revealed major variances between them. They differed in the genome size, number of open reading frames, genome architecture, and virion protein patterns. Their high mutual sequence similarity was detected only in the terminal regions of the genome. When compared with the so far described eta phage genomes, noticeable differences were found. Thus, both phages represent two new lineages of as yet not characterized bacteriophages of the Siphoviridae family having impact on pathogenicity of impetigo strains of S. aureus.

• MeSH
• DNA virů chemie   genetika   MeSH
• DNA viry genetika   izolace a purifikace   MeSH
• epidemický výskyt choroby MeSH
• exfoliatiny genetika   MeSH
• fylogeneze MeSH
• genom virový * MeSH
• impetigo epidemiologie   mikrobiologie   MeSH
• infekce spojené se zdravotní péčí epidemiologie   MeSH
• lidé MeSH
• molekulární sekvence - údaje MeSH
• novorozenec MeSH
• otevřené čtecí rámce MeSH
• polymorfismus délky restrikčních fragmentů MeSH
• pořadí genů MeSH
• porodnice MeSH
• přenos genů horizontální MeSH
• profágy klasifikace   genetika   izolace a purifikace   MeSH
• sekvenční analýza DNA MeSH
• sekvenční homologie MeSH
• shluková analýza MeSH
• stafylokokové bakteriofágy klasifikace   genetika   izolace a purifikace   MeSH
• stafylokokové infekce epidemiologie   mikrobiologie   MeSH
• Staphylococcus aureus izolace a purifikace   virologie   MeSH
• syntenie MeSH
• transdukce genetická MeSH
• Check Tag
• lidé MeSH
• novorozenec MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Česká republika MeSH

Staphylococcus aureus is one of the most frequent pathogens infecting the respiratory tract of patients with cystic fibrosis (CF). This study was the first to examine S. aureus isolates from CF patients in the Czech Republic. Among 100 S. aureus isolates from 92 of 107 observed patients, we found a high prevalence of resistance to macrolide-lincosamide-streptogramin B (MLS(B)) antibiotics (56%). More than half of the resistant strains (29 of 56) carried a mutation in the MLS(B) target site. The emergence of MLS(B) resistance and mutations conferring resistance to MLS(B) antibiotics was associated with azithromycin treatment (p=0.000000184 and p=0.000681, respectively). Methicillin resistance was only detected in 3% of isolates and the rate of resistance to other antibiotics did not exceed 12%. The prevalence of small-colony variant (SCV) strains was relatively low (9%) and eight of nine isolates with the SCV phenotype were thymidine dependent. The study population of S. aureus was heterogeneous in structure and both the most prevalent community-associated and hospital-acquired clonal lineages were represented. Of the virulence genes, enterotoxin genes seg (n=52), sei (n=49), and sec (n=16) were the most frequently detected among the isolates. The PVL genes (lukS-PV and lukF-PV) have not been revealed in any of the isolates.

• MeSH
• antibakteriální látky farmakologie   terapeutické užití   MeSH
• azithromycin terapeutické užití   MeSH
• bakteriální léková rezistence genetika   MeSH
• cystická fibróza mikrobiologie   MeSH
• dlouhodobá péče MeSH
• infekce spojené se zdravotní péčí mikrobiologie   MeSH
• lidé MeSH
• methicilin rezistentní Staphylococcus aureus účinky léků   MeSH
• mikrobiální testy citlivosti MeSH
• mutace genetika   MeSH
• ribozomy genetika   MeSH
• stafylokokové infekce farmakoterapie   mikrobiologie   MeSH
• Staphylococcus aureus účinky léků   genetika   MeSH
• thymidin genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Česká republika MeSH

• Publikační typ
• abstrakt z konference MeSH