AIM: Staphylococcus aureus strains are the cause of frightening hospital and community infections, especially when they are resistant to antimicrobials, have important pathogenicity factors, or have biofilm production ability. Looking for novel therapeutic options which would be effective against such strains is one of the highest priorities of medicine and medical research. The study aim was to describe the occurrence of S. aureus strains and proportion of methicillin resistant strains (MRSA) detected in laboratories of the Microbiological Institute, Faculty of Medicine, Masaryk University (FM MU) and St. Anne's University Hospital, Brno in 2011-2018. Selected strains of S. aureus were tested for biofilm production ability and susceptibility to antimicrobials and Stafal®, a phage therapeutic agent. A prerequisite was to develop a simple routine method suitable for phage susceptibility testing of bacteria. MATERIAL AND METHODS: Altogether 867 clinical isolates of S. aureus and 132 strains of other species of the genus Staphylococcus (isolated in 2011-2017) were tested for susceptibility to the phage therapy preparation Stafal® using the double-layer agar method. All strains of S. aureus were tested for biofilm production ability by the modified Christensen method with the use of titration microplates and for susceptibility to antistaphylococcal antibiotics by the disk diffusion test. For 95 S. aureus strains, the outcome of the double-layer agar method (DAM) was compared with that of our newly designed method (ODM) based on optical density decrease of the bacterial suspension. RESULTS: During the study period, the laboratories of the Faculty of Medicine, Masaryk University (FM MU) and St. Anne's University Hospital, Brno detected 2900 strains of S. aureus per year on average. The proportion of MRSA among S. aureus isolates from blood culture and venous catheters ranged between 8.8-15.2 %. S. aureus strains recovered from venous catheters and blood culture were confirmed as stronger biofilm producers than those from other clinical specimens. MRSA strains showed higher biofilm production than methicillin susceptible strains (MSSA). As many as 90.4 % of S. aureus strains tested susceptible to the Stafal® preparation. Even a higher proportion, i.e. 99.0 %, of MRSA strains were Stafal® susceptible. No relationship was found between Stafal® susceptibility and biofilm production ability. Although Stafal® targets primarily S. aureus, some susceptibility (26.5 %) was also found for other staphylococcal species. A novel simple method designed for routine testing of susceptibility to phage therapy preparations based on optical density decrease was comparably sensitive and reliable as the commonly used double-layer agar method (DAM) and, in addition to being easy and rapid to perform, after prolonged suspension culture and at higher measurement frequency, it has an extra advantage of providing the possibility for monitoring also phage action dynamics. CONCLUSIONS: The proportion of MRSA strains detected in this study is comparable to that reported for the whole Czech Republic, and the biofilm production data are consistent with scientific evidence. The host range of the Stafal® preparation is relatively wide and covers most strains of S. aureus and some coagulase negative staphylococci. The highest efficiency of Stafal® (99.4 %) was observed against MRSA strains with multiple types of antibiotic resistance. In vitro testing of 867 strains of S. aureus and 132 other staphylococcal species has shown the phage therapy preparation Stafal® to be a suitable candidate therapeutic option for the treatment of staphylococcal infections, especially in case of failure of conventional antibiotic therapy. Moreover, a simple method for routine phage susceptibility testing of clinical bacterial isolates has been designed, which is an essential tool to be used in phage therapy.
- Klíčová slova
- MRSA, Phage therapy, Stafal®, Staphylococcus, biofilm,
- MeSH
- antibakteriální látky terapeutické užití MeSH
- bakteriofágy * fyziologie MeSH
- lidé MeSH
- methicilin rezistentní Staphylococcus aureus virologie MeSH
- stafylokokové infekce * terapie virologie MeSH
- Staphylococcus * virologie MeSH
- techniky in vitro MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Česká republika MeSH
- Názvy látek
- antibakteriální látky MeSH
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.
- Klíčová slova
- Siphoviridae, Staphylococcus pseudintermedius, bacteriophages, comparative genomics, viral taxonomy,
- 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
The spontaneous host-range mutants 812F1 and K1/420 are derived from polyvalent phage 812 that is almost identical to phage K, belonging to family Myoviridae and genus Kayvirus. Phage K1/420 is used for the phage therapy of staphylococcal infections. Endolysin of these mutants designated LysF1, consisting of an N-terminal cysteine-histidine-dependent aminohydrolase/peptidase (CHAP) domain and C-terminal SH3b cell wall-binding domain, has deleted middle amidase domain compared to wild-type endolysin. In this work, LysF1 and both its domains were prepared as recombinant proteins and their function was analyzed. LysF1 had an antimicrobial effect on 31 Staphylococcus species of the 43 tested. SH3b domain influenced antimicrobial activity of LysF1, since the lytic activity of the truncated variant containing the CHAP domain alone was decreased. The results of a co-sedimentation assay of SH3b domain showed that it was able to bind to three types of purified staphylococcal peptidoglycan 11.2, 11.3, and 11.8 that differ in their peptide bridge, but also to the peptidoglycan type 11.5 of Streptococcus uberis, and this capability was verified in vivo using the fusion protein with GFP and fluorescence microscopy. Using several different approaches, including NMR, we have not confirmed the previously proposed interaction of the SH3b domain with the pentaglycine bridge in the bacterial cell wall. The new naturally raised deletion mutant endolysin LysF1 is smaller than LysK, has a broad lytic spectrum, and therefore is an appropriate enzyme for practical use. The binding spectrum of SH3b domain covering all known staphylococcal peptidoglycan types is a promising feature for creating new chimeolysins by combining it with more effective catalytic domains.
- Klíčová slova
- Endolysin, Endopeptidases, Enzybiotics, Src homology domains, Staphylococcal infections, Staphylococcus bacteriophage,
- MeSH
- endopeptidasy genetika izolace a purifikace metabolismus MeSH
- hostitelská specificita * MeSH
- mutantní proteiny genetika izolace a purifikace metabolismus MeSH
- Myoviridae enzymologie genetika fyziologie MeSH
- peptidoglykan metabolismus MeSH
- proteinové domény MeSH
- sekvenční delece * MeSH
- Staphylococcus virologie MeSH
- vazba proteinů MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- endolysin MeSH Prohlížeč
- endopeptidasy MeSH
- mutantní proteiny MeSH
- peptidoglykan MeSH
Staphylococcus sciuri is a bacterial pathogen associated with infections in animals and humans, and represents a reservoir for the mecA gene encoding methicillin-resistance in staphylococci. No S. sciuri siphophages were known. Here the identification and characterization of two temperate S. sciuri phages from the Siphoviridae family designated ϕ575 and ϕ879 are presented. The phages have icosahedral heads and flexible noncontractile tails that end with a tail spike. The genomes of the phages are 42,160 and 41,448 bp long and encode 58 and 55 ORFs, respectively, arranged in functional modules. Their head-tail morphogenesis modules are similar to those of Staphylococcus aureus ϕ13-like serogroup F phages, suggesting their common evolutionary origin. The genome of phage ϕ575 harbours genes for staphylokinase and phospholipase that might enhance the virulence of the bacterial hosts. In addition both of the phages package a homologue of the mecA gene, which is a requirement for its lateral transfer. Phage ϕ879 transduces tetracycline and aminoglycoside pSTS7-like resistance plasmids from its host to other S. sciuri strains and to S. aureus. Furthermore, both of the phages efficiently adsorb to numerous staphylococcal species, indicating that they may contribute to interspecies horizontal gene transfer.
- MeSH
- bakteriální geny * MeSH
- fosfolipasy metabolismus MeSH
- genom virový MeSH
- genomika metody MeSH
- hostitelská specificita MeSH
- metaloendopeptidasy metabolismus MeSH
- plazmidy genetika MeSH
- přenos genů horizontální MeSH
- přichycení viru MeSH
- stafylokokové bakteriofágy fyziologie ultrastruktura MeSH
- Staphylococcus virologie MeSH
- transdukce genetická * MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- auR protein, Staphylococcus aureus MeSH Prohlížeč
- fosfolipasy MeSH
- metaloendopeptidasy MeSH
Temperate bacteriophages were induced in and released from isolates of Staphylococcus aureus and Streptococcus agalactiae using mitomycin C. Various specific indicator cultures were tested for providing clear plaques after phage infection. Specific lytic mixture of bacteriophages was prepared using the induced, modified and laboratory variants of phages. Under laboratory conditions, the mixture eliminated all isolates from the tested collection of microorganisms. The restriction barrier of some bacterial isolates to bacteriophage infection was overcome either by UV irradiation or in vitro modification of bacteriophage DNA with specific methyltransferases. Conjugative R plasmids, capable of replication in G+ and G- bacteria, were detected and isolated from S. aureus and S. agalactiae antibiotic-resistant strains.
- MeSH
- aktivace viru MeSH
- DNA virů genetika izolace a purifikace MeSH
- lidé MeSH
- mnohočetná bakteriální léková rezistence genetika MeSH
- R-plasmidy genetika MeSH
- stafylokokové bakteriofágy fyziologie MeSH
- Staphylococcus aureus účinky léků genetika izolace a purifikace virologie MeSH
- Staphylococcus účinky léků genetika izolace a purifikace virologie MeSH
- techniky in vitro MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- DNA virů MeSH
