Kayviruses are polyvalent broad host range staphylococcal phages with a potential to combat staphylococcal infections. However, the implementation of rational phage therapy in medicine requires a thorough understanding of the interactions between bacteriophages and pathogens at omics level. To evaluate the effect of a phage used in therapy on its host bacterium, we performed differential transcriptomic analysis by RNA-Seq from bacteriophage K of genus Kayvirus infecting two Staphylococcus aureus strains, prophage-less strain SH1000 and quadruple lysogenic strain Newman. The temporal transcriptional profile of phage K was comparable in both strains except for a few loci encoding hypothetical proteins. Stranded sequencing revealed transcription of phage noncoding RNAs that may play a role in the regulation of phage and host gene expression. The transcriptional response of S. aureus to phage K infection resembles a general stress response with differential expression of genes involved in a DNA damage response. The host transcriptional changes involved upregulation of nucleotide, amino acid and energy synthesis and transporter genes and downregulation of host transcription factors. The interaction of phage K with variable genetic elements of the host showed slight upregulation of gene expression of prophage integrases and antirepressors. The virulence genes involved in adhesion and immune evasion were only marginally affected, making phage K suitable for therapy. IMPORTANCE Bacterium Staphylococcus aureus is a common human and veterinary pathogen that causes mild to life-threatening infections. As strains of S. aureus are becoming increasingly resistant to multiple antibiotics, the need to search for new therapeutics is urgent. A promising alternative to antibiotic treatment of staphylococcal infections is a phage therapy using lytic phages from the genus Kayvirus. Here, we present a comprehensive view on the phage-bacterium interactions on transcriptomic level that improves the knowledge of molecular mechanisms underlying the Kayvirus lytic action. The results will ensure safer usage of the phage therapeutics and may also serve as a basis for the development of new antibacterial strategies.
In order to characterize the most commonly detected Salmonella serotypes, we tested 124 isolates of S. Typhimurium and 89 isolates of the monophasic variant of S. Typhimurium (S. 1,4, [5],12:i:-) for their antimicrobial susceptibility by means of the Kirby-Bauer disk-diffusion method, and for the detection of 19 genes (four Phage Markers (g13, Sieb, eat, g8), ten prophage-related virulence genes (gipA, gtgB, nanH, gogB, grvA, sopE, sspH1, sspH2, sodC1, gtgE), and five plasmid-borne virulence genes (spvC, pefA, mig5, rcK, srgA)) by means of PCR-based assays. A total of 213 strains were analyzed from, humans (n = 122), animals (n = 25), food (n = 46), and irrigation water (n = 20). S. Typhimurium isolates showed higher variability, in both their resistance profiles and molecular typing, than S. 1,4, [5],12:i:-. Strains from irrigation water displayed significantly higher susceptibility to antibiotics than those from the other sources. Resistance to ampicillin, streptomycin, sulfonamide, and tetracycline was the most commonly detected resistance profile (R-type), being in serovar S. 1,4, [5],12:i:-, frequently associated to resistance to other antimicrobials. Significant differences in genetic profiles in the two abovementioned Salmonella serotypes were found. None of the plasmid-borne virulence genes investigated were detected in S. 1,4, [5],12:i:- isolates, while those genes, characterized 37.9% of the S. Typhimurium strains. Differences in the prevalence of some molecular targets between the two Salmonella serotypes deserve further study. Importantly, the grvA gene was found exclusively in S. Typhimurium strains, whereas sopE, sodC, gtgB, and gipA were mainly detected, with a statistically significant difference, in S. 1,4, [5],12:i:- isolates.
- MeSH
- antibakteriální látky farmakologie MeSH
- bakteriální geny genetika MeSH
- genetická variace MeSH
- lidé MeSH
- mikrobiální testy citlivosti MeSH
- mikrobiologie vody * MeSH
- mnohočetná bakteriální léková rezistence MeSH
- molekulární typizace MeSH
- plazmidy genetika MeSH
- potravinářská mikrobiologie * MeSH
- profágy genetika MeSH
- Salmonella typhimurium klasifikace účinky léků genetika izolace a purifikace MeSH
- salmonelóza epidemiologie mikrobiologie MeSH
- séroskupina MeSH
- virulence genetika MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
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
The transduction mediated by bacteriophages is considered to be one of the primary driving forces in horizontal gene transfer in staphylococci, which is crucial to their adaptation and successful evolution. For a transduction to be effective, it is generally accepted that the recipient strain should be susceptible to the transducing phage. In this study, we demonstrate that the plasmid DNAs are effectively transduced into the recipient Staphylococcus aureus strains in spite of their insensitivity to the lytic action of the transducing phage, provided that these phages adsorb effectively to the bacterial cells. The tetracycline and penicillinase plasmids were transduced to insensitive laboratory and clinical strains by bacteriophages ϕ29, ϕ52A and ϕ80α as well as by prophage ϕ53 and naturally occurring prophages induced from donor lysogenic strains. Comparable frequencies of transduction were achieved in both phage-sensitive and phage-insensitive recipient strains. We have demonstrated that such mechanisms as the restriction of DNA and lysogenic immunity which are responsible for insensitivity of cells to phages may not be a barrier to the transfer, maintenance and effective spread of plasmids to a wider range of potential recipients in the staphylococcal population.
- MeSH
- antibakteriální látky farmakologie MeSH
- lyzogenie MeSH
- mnohočetná bakteriální léková rezistence MeSH
- penicilinasa genetika MeSH
- plazmidy * MeSH
- profágy genetika fyziologie MeSH
- stafylokokové bakteriofágy genetika fyziologie MeSH
- stafylokokové infekce mikrobiologie MeSH
- Staphylococcus aureus genetika virologie MeSH
- tetracyklin farmakologie MeSH
- transdukce genetická * MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
UNLABELLED: The deep sea is a massive, largely oligotrophic ecosystem, stretched over nearly 65% of the planet's surface. Deep-sea planktonic communities are almost completely dependent upon organic carbon sinking from the productive surface, forming a vital component of global biogeochemical cycles. However, despite their importance, viruses from the deep ocean remain largely unknown. Here, we describe the first complete genomes of deep-sea viruses assembled from metagenomic fosmid libraries. "Candidatus Pelagibacter" (SAR11) phage HTVC010P and Puniceispirillum phage HMO-2011 are considered the most abundant cultured marine viruses known to date. Remarkably, some of the viruses described here recruited as many reads from deep waters as these viruses do in the photic zone, and, considering the gigantic scale of the bathypelagic habitat, these genomes provide information about what could be some of the most abundant viruses in the world at large. Their role in the viral shunt in the global ocean could be very significant. Despite the challenges encountered in inferring the identity of their hosts, we identified one virus predicted to infect members of the globally distributed SAR11 cluster. We also identified a number of putative proviruses from diverse taxa, including deltaproteobacteria, bacteroidetes, SAR11, and gammaproteobacteria. Moreover, our findings also indicate that lysogeny is the preferred mode of existence for deep-sea viruses inhabiting an energy-limited environment, in sharp contrast to the predominantly lytic lifestyle of their photic-zone counterparts. Some of the viruses show a widespread distribution, supporting the tenet "everything is everywhere" for the deep-ocean virome. IMPORTANCE: The deep sea is among the largest known habitats and a critical cog in biogeochemical cycling but remains underexplored in its microbiology. Even more than is the case for its prokaryotic community, our knowledge of its viral component has remained limited by the paucity of information provided by studies dependent upon short sequence fragments. In this work, we attempt to fill this existing gap by using a combination of classical fosmid libraries with next-generation sequencing and assembly to recover long viral genomic fragments. We have sequenced ca. 6,000 fosmids from two metagenomics libraries made from prokaryotic biomass from the deep Mediterranean Sea and recovered twenty-eight complete viral genomes, all of them novel and quite distinct from all previously described viral genomes. They are preferentially found in deeper waters and are widely distributed all over the oceans. To our knowledge, this is the first report on complete and cosmopolitan viral genomes from the bathypelagic habitat.
- MeSH
- bakteriofágy klasifikace genetika izolace a purifikace MeSH
- fylogeneze MeSH
- genom virový * MeSH
- metagenomika MeSH
- mořská voda mikrobiologie MeSH
- oceány a moře * MeSH
- profágy klasifikace genetika izolace a purifikace MeSH
- sekvenční homologie MeSH
- syntenie MeSH
- virové proteiny genetika MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- oceány a moře * 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
The Staphylococcal Cassette Chromosome mec (SCCmec) confers methicillin resistance to Staphylococcus aureus. While SCCmec is generally regarded as a mobile genetic element, the precise mechanisms by which large SCCmec elements are exchanged between staphylococci have remained enigmatic. In the present studies, we observed that the clinical methicillin-resistant S. aureus (MRSA) isolate UMCG-M4 with the sequence type 398 contains four prophages belonging to the serological groups A, B and Fa. Previous studies have shown that certain serological group B bacteriophages of S. aureus are capable of generalized transduction. We therefore assessed the transducing capabilities of the phages from strain UMCG-M4. The results show that some of these phages can indeed transduce plasmid pT181 to the recipient S. aureus strain RN4220. Therefore, we also investigated the possible involvement of these transducing phages in the transmission of the large SCCmec type V (5C2&5) element of S. aureus UMCG-M4. While no transduction of the complete SCCmec element was observed, we were able to demonstrate that purified phage particles did contain large parts of the SCCmec element of the donor strain, including the methicillin resistance gene mecA. This shows that staphylococcal phages can encapsulate the resistance determinant mecA of a large SCCmec type V (5C2&5) element, which may lead to its transfer to other staphylococci.
- MeSH
- bakteriální geny * MeSH
- methicilin rezistentní Staphylococcus aureus genetika virologie MeSH
- plazmidy MeSH
- profágy genetika fyziologie MeSH
- rezistence na methicilin MeSH
- sestavení viru * MeSH
- stafylokokové bakteriofágy klasifikace genetika MeSH
- transdukce genetická * MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The epidemic community-associated methicillin-resistant clone Staphylococcus aureus USA300 is a major source of skin and soft tissue infections and involves strains with a diverse set of resistance genes. In this study, we report efficient transduction of penicillinase and tetracycline resistance plasmids by bacteriophages φ80α and φJB between clinical isolates belonging to the USA300 clone. High transduction frequencies (10(-5) - 10(-6) CFU/PFU) were observed using phages propagated on donor strains as well as prophages induced from donors by ultraviolet light. Quantitative real-time PCR was employed to detect penicillinase plasmids in transducing phage particles and determine the ratio of transducing particles in phage lysates to infectious phage particles (determined as approximately 1 : 1700). Successful transfer of plasmids between strains in USA300 clone proves transduction is an effective mechanism for spreading plasmids within the clone. Such events contribute to its evolution and to emergence of new multiple drug-resistant strains of this successful clone.
- MeSH
- bakteriální léková rezistence MeSH
- bakteriofágy genetika MeSH
- lidé MeSH
- methicilin rezistentní Staphylococcus aureus genetika izolace a purifikace virologie MeSH
- plazmidy MeSH
- přenos genů horizontální MeSH
- profágy genetika MeSH
- stafylokokové infekce mikrobiologie MeSH
- transdukce genetická MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- dopisy MeSH
- práce podpořená grantem MeSH
The exfoliative toxin A (ETA) is encoded by the gene located on Staphylococcus aureus prophages. We have developed a single-reaction multiplex polymerase chain reaction (PCR) assay for rapid and specific detection of various phiETA prophages of serogroup B responsible for dissemination of eta gene and ETA production in clinical strains. This PCR strategy enabled to classify the ETA-positive strains into 6 groups designated ETA-B1, ETA-B2, ETA-B3, ETA-B4, ETA-B5, and ETA-B6. The method was tested on a diverse set of 101 ETA and/or ETB-positive S. aureus strains isolated in 22 Czech maternity hospitals and 1 Slovak maternity hospital between 1998 and 2009. This novel PCR strategy is reliable in the rapid identification of yet undescribed ETA-converting B prophages and differentiation of the closely related ETA-positive strains, and it is a convenient tool for hospital epidermolytic infection control.
- MeSH
- exfoliatiny genetika MeSH
- kůže mikrobiologie MeSH
- lidé MeSH
- novorozenec MeSH
- pemfigus mikrobiologie MeSH
- polymerázová řetězová reakce metody MeSH
- porodnice MeSH
- profágy genetika MeSH
- stafylokokové infekce mikrobiologie MeSH
- Staphylococcus aureus klasifikace genetika MeSH
- techniky typizace bakterií metody MeSH
- těhotenství MeSH
- Check Tag
- lidé MeSH
- novorozenec MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Československo MeSH
Given the great biological importance and high diversity of temperate Staphylococcus aureus bacteriophages, a method is needed for the description of their genomic structure. Here we have updated a multiplex PCR strategy for the complex characterization of S. aureus phages of the family Siphoviridae. Based on the comparative genomic analysis of the available phage sequences, a multilocus PCR strategy for typing the major modules of the phage genome was designed. The genomic modules were classified on the basis of the genes for integrase (10 types), anti-repressor (five types), replication proteins polA, dnaC and dnaD (four types), dUTPase (four types), portal protein (eight types), tail appendices (four types) and endolysin (four types) corresponding to the integrase locus, lysogeny control region, and modules for DNA replication, transcription regulation, packaging, tail appendices and lysis respectively. The nine PCR assays designed for the above sequences were shown to be capable to identify the bacteriophage gene pool present both in the phage and bacterial genomes and their extensive mosaic structure. The established multiplex PCR-based multilocus diagnostic scheme is convenient for rapid and reliable phage and prophage classification and for the study of bacteriophage evolution.
- MeSH
- DNA virů genetika MeSH
- genom virový MeSH
- multilokusová sekvenční typizace MeSH
- polymerázová řetězová reakce metody MeSH
- profágy klasifikace genetika MeSH
- Siphoviridae klasifikace genetika MeSH
- srovnávací genomová hybridizace MeSH
- Staphylococcus aureus genetika virologie MeSH
- virové proteiny genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
