Pavla Holochová

Multilocus PCR typing strategy for differentiation of Staphylococcus aureus siphoviruses reflecting their modular genome structure

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.

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.

Rapid detection and differentiation of the exfoliative toxin A-producing Staphylococcus aureus strains based on ϕETA prophage polymorphisms

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.

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).

Genomic diversity of two lineages of exfoliative toxin A-converting phages predominating in Staphylococcus aureus strains in the Czech Republic

We have isolated and characterized two distinct types of exfoliative toxin A (ETA)-converting bacteriophages originating from Staphylococcus aureus strains responsible for massive outbreaks of pemphigus neonatorum in the Czech Republic. Three induced phages designated as ph iB531, phi B557 and phi B122 were found to be capable of transferring the eta gene into the prophageless non-toxigenic S. aureus strain and converting it into an ETA producer. Comparisons of the phage sequences derived from 12 selected genes and 2 genomic segments (polymorphic P2 and conserved C4) revealed that phi B531 and phi B557 were identical each other, but phi B122 differed from them in 5 gene sequences, the xis gene content and the virion protein profile. Thus, phi B122 represents a new type of still undescribed ETA-converting phage. This study highlights not only the conclusive genomic diversity of eta gene-positive phages, but also their virulence implications in impetigo S. aureus strains.

Mucilaginibacter terrae sp. nov., isolated from Antarctic soil

A bacterial strain designated CCM 8645T was isolated from a soil sample collected nearby a mummified seal carcass in the northern part of James Ross Island, Antarctica. The cells were short rods, Gram-stain-negative, non-motile, catalase and oxidase positive, and produced a red-pink pigment on R2A agar. A polyphasic taxonomic approach based on 16S rRNA gene sequencing, extensive biotyping using conventional tests and commercial identification kits and chemotaxonomic analyses were applied to clarify its taxonomic position. Phylogenetic analysis based on the 16S rRNA gene placed strain CCM 8645T in the genus Mucilaginibacter with the closest relative being Mucilaginibacter daejeonensis Jip 10T, exhibiting 96.5 % 16S rRNA pairwise similarity which was clearly below the 97 % threshold value recommended for species demarcation. The major components in fatty acid profiles were Summed feature 3 (C16 : 1ω7c/C16 : 1ω6c), C15 : 0 iso and C17 : 0 iso 3OH. The cellular quinone content was exclusively menaquinone MK-7. The major polyamine was sym-homospermidine and predominant polar lipids were phosphatidylethanolamine and phosphatidylserine. Based on presented results, we propose a novel species for which the name Mucilaginibacter terrae sp. nov. is suggested, with the type strain CCM 8645T (=LMG 29437T).

Pedobacter jamesrossensis sp. nov., Pedobacter lithocola sp. nov., Pedobacter mendelii sp. nov. and Pedobacter petrophilus sp. nov., isolated from the Antarctic environment

A taxonomic study performed on 17 Gram-stain-negative rod-shaped bacterial strains originating from the Antarctic environment is described. Initial phylogenetic analysis using 16S rRNA gene sequencing differentiated the strains into four groups belonging to the genus Pedobacter but they were separated from all hitherto described Pedobacter species. Group I (n=8) was closest to Pedobacter aquatilis (97.8 % 16S rRNA gene sequence similarity). Group II (n=2) and group III (n=4) were closely related (98.8 % 16S rRNA gene sequence similarity) and had Pedobacter jejuensis as their common nearest neighbour. Group IV (n=3) was distantly delineated from the remaining Pedobacter species. Differentiation of the analysed strains into four clusters was further confirmed by repetitive sequence-based PCR fingerprinting, ribotyping, DNA-DNA hybridization and phenotypic traits. Common to representative strains for the four groups were the presence of major menaquinone MK-7, sym-homospermidine as the major polyamine, phosphatidylethanolamine, two unidentified lipids (L2, L5) and an unidentified aminolipid (AL2) as the major polar lipids, presence of an alkali-stable lipid, and C16:1ω7c/C16:1ω6c (summed feature 3), iso-C15:0 and iso-C 17:0 3-OH as the major fatty acids, which corresponded to characteristics of the genus Pedobacter. The obtained results showed that the strains analysed represent four novel species of the genus Pedobacter, for which the names Pedobacter jamesrossensis sp. nov. (type strain CCM 8689T=LMG 29684T), Pedobacter lithocola sp. nov. (CCM 8691T=LMG 29685T), Pedobacter mendelii sp. nov. (CCM 8685T=LMG 29688T) and Pedobacter petrophilus sp. nov. (CCM 8687T=LMG 29686T) are proposed.

Pedobacter psychrophilus sp. nov., isolated from fragmentary rock

Strain P4487AT was isolated during investigation of cultivable bacterial populations of environmental materials sampled at James Ross Island, Antarctica. It revealed Gram-stain-negative short rod-shaped cells producing a pink pigment. Phylogenetic analysis based on 16S rRNA gene sequences allocated strain P4487AT to the genus Pedobacter but showed that the strain represents a distinct intrageneric phylogenetic lineage clearly separated from remaining Pedobacter species. Phylogenetically, strain P4487AT formed a common branch with the Pedobacter arcticus and Pedobacter lignilitoris cluster while the highest value of 94.4 % 16S rRNA gene sequence similarity suggested that Pedobacter lentus is the most closely related species. Biochemical and physiological test results enabled the differentiation of strain P4487AT from all phylogenetically closely related species. Chemotaxonomic analyses of strain P4487AT showed MK-7 as the respiratory menaquinone, sym-homospermidine as the major polyamine, phosphatidylethanolamine and two unidentified lipids as the major polar lipids, presence of sphingolipids, and C16 : 1ω7c/C16 : 1ω6c (summed feature 3), iso-C15 : 0 and iso-C17 : 0 3-OH as the major fatty acids, all of which corresponded with characteristics of the genus Pedobacter. The results showed that strain P4487AT represents a novel species within the genus Pedobacter, for which the name Pedobacter psychrophilus sp. nov. is proposed. The type strain is P4487AT (=CCM 8644T=LMG 29436T).

Rufibacter ruber sp. nov., isolated from fragmentary rock

A red-pigmented, Gram-stain-negative, rod-shaped, aerobic bacterium, designated strain CCM 8646T, was isolated from stone fragments in James Ross Island, Antarctica. Strain CCM 8646T was able to grow from 10 to 40 °C, in the presence of up to 1 % (w/v) NaCl and at pH 7.0-11.0. Analysis of the 16S rRNA gene sequence placed strain CCM 8646T in the genus Rufibacter with the closest relative being Rufibacter roseus H359T (97.07 % 16S rRNA gene sequence similarity). The digital DNA-DNA hybridization values between strain CCM 8646T and R. roseus H359T were low (21.30±2.34 %). The major quinone was menaquinone MK-7. The polar lipids comprised phosphatidylethanolamine, an unknown aminoglycolipid and six unknown polar lipids. The G+C content of strain CCM 8646T was 51.54 mol%. On the basis of phenotypic, chemotaxonomic and genotyping results, strain CCM 8646T is considered to represent a novel species within the genus Rufibacter, for which the name Rufibacter ruber sp. nov. is proposed. The type strain is CCM 8646T (=LMG 29438T).