Ivana Mašlaňová

Bacteriophages of Staphylococcus aureus efficiently package various bacterial genes and mobile genetic elements including SCCmec with different frequencies

Staphylococcus aureus is a serious human and veterinary pathogen in which new strains with increasing virulence and antimicrobial resistance occur due to acquiring new genes by horizontal transfer. It is generally accepted that temperate bacteriophages play a major role in gene transfer. In this study, we proved the presence of various bacterial genes of the S. aureus COL strain directly within the phage particles via qPCR and quantified their packaging frequency. Non-parametric statistical analysis showed that transducing bacteriophages φ11, φ80 and φ80α of serogroup B, in contrast to serogroup A bacteriophage φ81, efficiently package selected chromosomal genes localized in 4 various loci of the chromosome and 8 genes carried on variable elements, such as staphylococcal cassette chromosome SCCmec, staphylococcal pathogenicity island SaPI1, genomic islands vSaα and vSaβ, and plasmids with various frequency. Bacterial gene copy number per ng of DNA isolated from phage particles ranged between 1.05 × 10(2) for the tetK plasmid gene and 3.86 × 10(5) for the SaPI1 integrase gene. The new and crucial finding that serogroup B bacteriophages can package concurrently ccrA1 (1.16 × 10(4)) and mecA (1.26 × 10(4)) located at SCCmec type I into their capsids indicates that generalized transduction plays an important role in the evolution and emergence of new methicillin-resistant clones.

Efficient transfer of antibiotic resistance plasmids by transduction within methicillin-resistant Staphylococcus aureus USA300 clone.

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.

Staphylococcus petrasii sp. nov. including S. petrasii subsp. petrasii subsp. nov. and S. petrasii subsp. croceilyticus subsp. nov., isolated from human clinical specimens and human ear infections

Thirteen coagulase-negative, oxidase-negative, and novobiocin-susceptible staphylococci were isolated from human clinical specimens. The isolates were differentiated from known staphylococcal species on the basis of 16S rRNA, hsp60, rpoB, dnaJ, tuf, and gap gene sequencing, automated ribotyping, (GTG)5-PCR fingerprinting, and MALDI-TOF MS analysis. Phylogenetic analysis based on the 16S rRNA gene sequence indicated phylogenetic relatedness of the analyzed strains to Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus devriesei, and Staphylococcus lugdunensis. DNA-DNA hybridization experiments between representative strains CCM 8418(T), CCM 8421(T), and the closest phylogenetic neighbors confirmed that the isolates represent novel Staphylococcus species, for which the name Staphylococcus petrasii sp. nov. is proposed. Genotypic and phenotypic analyses unambiguously split the strains into two closely related subclusters. Based on the results, two novel subspecies S. petrasii subsp. petrasii subsp. nov. and S. petrasii subsp. croceilyticus subsp. nov. are proposed, with type strains CCM 8418(T) (=CCUG 62727(T)) and CCM 8421(T) (=CCUG 62728(T)), respectively.

The Staphylococcal Cassette Chromosome mec type V from Staphylococcus aureus ST398 is packaged into bacteriophage capsids

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.

Enterococcus ureilyticus sp. nov. and Enterococcus rotai sp. nov., two urease-producing enterococci from the environment

A set of 25 urease-producing, yellow-pigmented enterococci was isolated from environmental sources. Phenotypic classification divided the isolates into two phena. Both phena were characterized using 16S rRNA gene sequence analysis, DNA base composition, rep-PCR fingerprinting and automated ribotyping. The obtained data distinguished the isolates from all members of the genus Enterococcus with validly published names and placed them in the Enterococcus faecalis species group. DNA-DNA hybridization experiments, pheS and rpoA sequencing and whole-cell protein electrophoresis provided conclusive evidence for the classification of each phenon as a novel species of the genus Enterococcus, for which the names Enterococcus ureilyticus sp. nov. (type strain CCM 4629(T)  = LMG 26676(T)  = CCUG 48799(T)), inhabiting water and plants, and Enterococcus rotai sp. nov. (type strain CCM 4630(T)  = LMG 26678(T)  = CCUG 61593(T)), inhabiting water, insects (mosquitoes) and plants, are proposed.

Red-pink pigmented Hymenobacter coccineus sp. nov., Hymenobacter lapidarius sp. nov. and Hymenobacter glacialis sp. nov., isolated from rocks in Antarctica

Four rod-shaped and Gram-stain-negative bacterial strains, CCM 8647, CCM 8649T, CCM 8643T and CCM 8648T, were isolated from rock samples collected on James Ross Island, Antarctica. Extensive biotyping, fatty acid profiling, chemotaxonomy, 16S rRNA gene sequencing and whole-genome sequencing was applied to isolates to clarify their taxonomic position. Phylogenetic analysis based on 16S rRNA gene sequencing indicated that all four isolates belonged to the genus Hymenobacter. Strains CCM 8649T and CCM 8647 were most closely related to Hymenobacter arizonensis OR362-8T (94.4 % 16S rRNA gene sequence similarity), strain CCM 8643T to Hymenobacter terrae DG7AT (96.3 %) and strain CCM 8648T to Hymenobacter glaciei VUG-A130T (96.3 %). The predominant fatty acids of CCM 8649T and CCM 8647 were summed feature 3 (C16 : 1ω7c/C16 : 1ω6c), C16 : 1ω5c and iso-C15 : 0, whereas those of CCM 8643T and CCM 8648T were summed feature 3 (C16 : 1ω7c/C16 : 1ω6c) and C16 : 1ω5c. The quinone systems contained exclusively menaquinone MK-7. The major polyamine was sym-homospermidine. All four strains contained the major polar lipid phosphatidylethanolamine. The G+C content of genomic DNA ranged from 60-63 mol%. Whole-genome sequencing data supported the finding that isolates represented distinct species of the genus Hymenobacter. On the basis of the results obtained, three novel species are proposed for which the names Hymenobacter coccineus sp. nov., Hymenobacter lapidarius sp. nov. and Hymenobacter glacialis sp. nov. are suggested, with the type strains CCM 8649T (=LMG 29441T=P5239T), CCM 8643T (=LMG 29435T=P3150T) and CCM 8648T (=LMG 29440T=P5086T), respectively.

Aquitalea pelogenes sp. nov., isolated from mineral peloid

Strain P1297T was isolated in the frame of a project aimed on the psychrotolerant microbiota occurring in water sources. The strain initially identified as a tentative species of the genus Aeromonas was rod-shaped, Gram-stain-negative, facultatively anaerobic and oxidase-positive. Subsequently, 16S rRNA gene sequence analysis placed strain P1297T within the class Betaproteobacteria and showed Aquitalea magnusonii TRO-001DR8T as the closest phylogenetic relative with 99.28 % 16S rRNA gene sequence similarity. Digital DDH and average nucleotide identity (ANI) were determined to evaluate the genomic relationship between strain P1297T and Aquitalea magnusonii CCM 7607T. Digital DDH estimation (31.3 ± 2.46 %) as well as ANI (85.6001 %; reciprocal value 85.3277 %) proved the dissimilarity of strain P1297T. Further investigation using phenotyping, automated ribotyping, whole-cell protein profiling and PCR-fingerprinting methods showed a distinct taxonomic position of strain P1297T among hitherto described species of the genus Aquitalea. DNA-DNA hybridization experiments revealed low binding values between strain P1297T and Aquitalea magnusonii CCM 7607T (57 ± 3 %) and Aquitalea denitrificans CCM 7935T (41 ± 5 %). The DNA G+C content of strain P1297T was 60.3 mol%. The predominant fatty acids were C16 : 1ω7c/ iso-C15 : 0 2-OH (47.0 %), C16 : 0 (24.5 %) and C18 : 1ω7c (10.6 %), and the quinone system contained predominantly ubiquinone Q-8. The polar lipids detected were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, two unidentified phospholipids and one unidentified aminophospholipid. Obtained results of genotypic and chemotaxonomic methods clearly proved that strain P1297T represents a novel species of the genus Aquitalea, for which the name Aquitalea pelogenes sp. nov. is proposed. The type strain is P1297T ( = CCM 7557T = LMG 28989T = CCUG 67440T).

Staphylococcus sciuri bacteriophages double-convert for staphylokinase and phospholipase, mediate interspecies plasmid transduction, and package mecA gene

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.

Classification of strain CCM 4446T as Rhodococcus degradans sp. nov.

Strain CCM 4446T, with notable biodegradation capabilities, was investigated in this study in order to elucidate its taxonomic position. Chemotaxonomic analyses of quinones, polar lipids, mycolic acids, polyamines and the diamino acid of the cell-wall peptidoglycan corresponded with characteristics of the genus Rhodococcus. Phylogenetic analysis, based on the 16S rRNA gene sequence, assigned strain CCM 4446T to the genus Rhodococcus and placed it in the Rhodococcus erythropolis 16S rRNA gene clade. Further analysis of catA and gyrB gene sequences, automated ribotyping with EcoRI restriction endonuclease, whole-cell protein profiling, DNA-DNA hybridization and extensive biotyping enabled differentiation of strain CCM 4446T from all phylogenetically closely related species, i.e., Rhodococcus baikonurensis, Rhodococcus qingshengii, Rhodococcus erythropolis and Rhodococcus globerulus. The results obtained show that the strain investigated represents a novel species within the genus Rhodococcus, for which the name Rhodococcus degradans sp. nov., is proposed. The type strain is CCM 4446T ( = LMG 28633T).

Expert opinion on three phage therapy related topics: bacterial phage resistance, phage training and prophages in bacterial production strains

Phage therapy is increasingly put forward as a “new” potential tool in the fight against antibiotic resistant infections. During the “Centennial Celebration of Bacteriophage Research” conference in Tbilisi, Georgia on 26–29 June 2017, an international group of phage researchers committed to elaborate an expert opinion on three contentious phage therapy related issues that are hampering clinical progress in the field of phage therapy. This paper explores and discusses bacterial phage resistance, phage training and the presence of prophages in bacterial production strains while reviewing relevant research findings and experiences. Our purpose is to inform phage therapy stakeholders such as policy makers, officials of the competent authorities for medicines, phage researchers and phage producers, and members of the pharmaceutical industry. This brief also points out potential avenues for future phage therapy research and development as it specifically addresses those overarching questions that currently call for attention whenever phages go into purification processes for application.