Pseudomonas leptonychotis sp. nov., isolated from Weddell seals in Antarctica.

A taxonomic study was carried out on four Gram-stain-negative strains P5773T, P6169, P4708 and P6245, isolated from anus or mouth samples of Weddell seals at James Ross Island, Antarctica. The results of initial 16S rRNA gene sequence analysis showed that all four strains formed a group placed in the genus Pseudomonas and found Pseudomonas guineae and Pseudomonas peli to be their closest neighbours with 99.9 and 99.2 % sequence similarity, respectively. Sequence analysis of rpoD, rpoB and gyrB housekeeping genes confirmed the highest similarity of isolates to P. peli (rpoD) and to P. guineae (rpoB and gyrB). The average nucleotide identity value below 86 %, as calculated from the whole-genome sequence data, showed the low genomic relatedness of P5773T to its phylogenetic neighbours. The complete genome of strain P5773T was 4.4 Mb long and contained genes encoding proteins with biotechnological potential. The major fatty acids of the seal isolates were summed feature 8 (C18 : 1 ω7c), summed feature 3 (C16 : 1 ω 7 c/C16  : 1 ω6c) and C16:0. The major respiratory quinone was Q9. The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. Putrescine and spermidine are predominant in the polyamine pattern. Further characterization performed using repetitive sequence-based PCR fingerprinting and MALDI-TOF MS analysis showed that the studied isolates formed a coherent cluster separated from the remaining Pseudomonas species and confirmed that they represent a novel species within the genus Pseudomonas, for which the name Pseudomonas leptonychotis sp. nov. is suggested. The type strain is P5773T (=CCM 8849T=LMG 30618T).

Staphylococcus petrasii diagnostics and its pathogenic potential enhanced by mobile genetic elements.

Staphylococcus petrasii is recently described coagulase negative staphylococcal species and an opportunistic human pathogen, still often misidentified in clinical specimens. Four subspecies are distinguished in S. petrasii by polyphasic taxonomical analyses, however a comparative study has still not been done on the majority of isolates and their genome properties have not yet been thoroughly analysed. Here, we describe the phenotypic and genotypic characteristics of 65 isolates and the results of de novo sequencing, whole genome assembly and annotation of draft genomes of five strains. The strains were identified by MALDI-TOF mass spectrometry to the species level and the majority of the strains were identified to the subspecies level by fingerprinting methods, (GTG)5 repetitive PCR and ribotyping. Macrorestriction profiling by pulsed-field gel electrophoresis was confirmed to be a suitable strain typing method. Comparative genomics revealed the presence of new mobile genetic elements carrying antimicrobial resistance factors such as staphylococcal cassette chromosome (SCC) mec, transposones, phage-inducible genomic islands, and plasmids. Their mosaic structure and similarity across coagulase-negative staphylococci and Staphylococcus aureus suggest the possible exchange of these elements. Numerous putative virulence factors such as adhesins, autolysins, exoenzymes, capsule formation genes, immunomodulators, the phage-associated sasX gene, and SCC-associated spermidine N-acetyltransferase gene, pseudouridine and sorbitol utilization operons might explain clinical manifestations of S. petrasii isolates. The increasing recovery of S. petrasii isolates from human clinical material, the multi-drug resistance including methicillin resistance of S. petrasii subsp. jettensis strains, and virulence factors homologous to other pathogenic staphylococci demonstrate the importance of the species in human disease.

Staphylococcus edaphicus sp. nov., Isolated in Antarctica, Harbors the mecC Gene and Genomic Islands with a Suspected Role in Adaptation to Extreme Environments.

Two Gram-stain-positive, coagulase-negative staphylococcal strains were isolated from abiotic sources comprising stone fragments and sandy soil in James Ross Island, Antarctica. Here, we describe properties of a novel species of the genus Staphylococcus that has a 16S rRNA gene sequence nearly identical to that of Staphylococcus saprophyticus However, compared to S. saprophyticus and the next closest relatives, the new species demonstrates considerable phylogenetic distance at the whole-genome level, with an average nucleotide identity of <85% and inferred DNA-DNA hybridization of <30%. It forms a separate branch in the S. saprophyticus phylogenetic clade as confirmed by multilocus sequence analysis of six housekeeping genes, rpoB, hsp60, tuf, dnaJ, gap, and sod Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and key biochemical characteristics allowed these bacteria to be distinguished from their nearest phylogenetic neighbors. In contrast to S. saprophyticus subsp. saprophyticus, the novel strains are pyrrolidonyl arylamidase and ß-glucuronidase positive and ß-galactosidase negative, nitrate is reduced, and acid produced aerobically from d-mannose. Whole-genome sequencing of the 2.69-Mb large chromosome revealed the presence of a number of mobile genetic elements, including the 27-kb pseudo-staphylococcus cassette chromosome mec of strain P5085T (ψSCCmecP5085), harboring the mecC gene, two composite phage-inducible chromosomal islands probably essential to adaptation to extreme environments, and one complete and one defective prophage. Both strains are resistant to penicillin G, ampicillin, ceftazidime, methicillin, cefoxitin, and fosfomycin. We hypothesize that antibiotic resistance might represent an evolutionary advantage against beta-lactam producers, which are common in a polar environment. Based on these results, a novel species of the genus Staphylococcus is described and named Staphylococcus edaphicus sp. nov. The type strain is P5085T (= CCM 8730T = DSM 104441T).IMPORTANCE The description of Staphylococcus edaphicus sp. nov. enables the comparison of multidrug-resistant staphylococci from human and veterinary sources evolved in the globalized world to their geographically distant relative from the extreme Antarctic environment. Although this new species was not exposed to the pressure of antibiotic treatment in human or veterinary practice, mobile genetic elements carrying antimicrobial resistance genes were found in the genome. The genomic characteristics presented here elucidate the evolutionary relationships in the Staphylococcus genus with a special focus on antimicrobial resistance, pathogenicity, and survival traits. Genes encoded on mobile genetic elements were arranged in unique combinations but retained conserved locations for the integration of mobile genetic elements. These findings point to enormous plasticity of the staphylococcal pangenome, shaped by horizontal gene transfer. Thus, S. edaphicus can act not only as a reservoir of antibiotic resistance in a natural environment but also as a mediator for the spread and evolution of resistance genes.

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).

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).

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.

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.

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).

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).

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 alcedinis sp. nov., isolated from common kingfisher (Alcedo atthis).

Two Gram-positive, catalase-negative bacterial strains were isolated from the cloaca of common kingfishers (Alcedo atthis). Repetitive sequence-based PCR fingerprinting using the (GTG)5 primer grouped these isolates into a single cluster separated from all known enterococcal species. The two strains revealed identical 16S rRNA gene sequences placing them within the genus Enterococcus with Enterococcus aquimarinus LMG 16607(T) as the closest relative (97.14 % similarity). Further taxonomic investigation using sequencing of the genes for the superoxide dismutase (sodA), phenylalanyl-tRNA synthase alpha subunit (pheS) and the RNA polymerase alpha subunit (rpoA) as well as application of whole-cell protein fingerprinting, automated ribotyping and extensive phenotyping confirmed that both strains belong to the same species. Based on data from this polyphasic study, these strains represent a novel species of the genus Enterococcus, for which the name Enterococcus alcedinis sp. nov. is proposed. The type strain is L34(T) (= CCM 8433(T) = LMG 27164(T)).

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.

Pseudomonas prosekii sp. nov., a novel psychrotrophic bacterium from Antarctica.

During Czech expeditions at James Ross Island, Antarctica, in the years 2007-2009, the bacterial diversity of the genus Pseudomonas was studied. Twelve fluorescent Pseudomonas strains were isolated from various samples and were subjected to a detailed taxonomic study. A polyphasic approach included genotypic and phenotypic analyses. The genotypic analysis involved sequencing of rrs, rpoB and rpoD genes, DNA-DNA hybridization (DDH) studies as well as manual ribotyping using HindIII endonuclease. The phenotypic characterization included conventional tests as well as biotyping using the Biolog system, protein profiling by SDS-PAGE, and MALDI-TOF MS analysis. Our taxonomic study revealed that all isolates belonged to the same Pseudomonas species with psychrotrophic growth not exceeding 37 °C. The cultures showed a unique position among the phylogenetically related pseudomonads. DDH experiment between the proposed type strain of the antarctic isolates and the closest neighbour P. arsenicoxydans CCM 8423(T) showed only 40.9-50.1 % similarity, thus confirming that the characterized strains do not belong to the P. arsenicoxydans species. According to the results obtained we propose the name P. prosekii sp. nov. for this novel Pseudomonas taxon with type strain AN/28/1(T) (=CCM 7990(T) and LMG 26867(T)).

Pollutant interactions during the biodegradation of phenolic mixtures with either 2- or 3-mononitrophenol in a continuously operated packed bed reactor.

Pollutant interactions during the aerobic biodegradation of phenolic mixtures with either 2-nitrophenol (2-NP) or 3-nitrophenol (3-NP) by a NP-adapted microbial consortium in simulated wastewater were studied in a packed-bed bench scale bioreactor continuously operated in a flow mode. Phenol/2-NP and phenol/3-NP mixtures with varied phenol/nitrophenol ratios were shown to exhibit different biodegradability patterns. The presence of 2-NP led to a much lower overall elimination capacity and lower process stability in comparison to mixtures with 3-NP. In contrast to the expected greater degradation of a more biodegradable substrate in mixtures, phenol was degraded with a lower efficiency at higher phenol concentrations than NPs, although this difference became less pronounced with the gradual biofilm adaptation to phenol. This unusual substrate interaction, which appears to be common in the biotreatment of substituted phenol mixtures, was explained by prior biofilm adaptation to less degradable substrates, NPs. The biofilm composition was significantly altered during the long-term reactor operation. Although eukaryotes were not present in the inoculum, four fungal species were isolated from the biofilm after 1.5 years of operation. Of the initially present strains, only Chryseobacterium sp. and several Pseudomonas species persisted till the end of operation.

Staphylococcus aureus Prophage-Encoded Protein Causes Abortive Infection and Provides Population Immunity against Kayviruses.

Both temperate and obligately lytic phages have crucial roles in the biology of staphylococci. While superinfection exclusion among closely related temperate phages is a well-characterized phenomenon, the interactions between temperate and lytic phages in staphylococci are not understood. Here, we present a resistance mechanism toward lytic phages of the genus Kayvirus, mediated by the membrane-anchored protein designated PdpSau encoded by Staphylococcus aureus prophages, mostly of the Sa2 integrase type. The prophage accessory gene pdpSau is strongly linked to the lytic genes for holin and ami2-type amidase and typically replaces genes for the toxin Panton-Valentine leukocidin (PVL). The predicted PdpSau protein structure shows the presence of a membrane-binding α-helix in its N-terminal part and a cytoplasmic positively charged C terminus. We demonstrated that the mechanism of action of PdpSau does not prevent the infecting kayvirus from adsorbing onto the host cell and delivering its genome into the cell, but phage DNA replication is halted. Changes in the cell membrane polarity and permeability were observed from 10 min after the infection, which led to prophage-activated cell death. Furthermore, we describe a mechanism of overcoming this resistance in a host-range Kayvirus mutant, which was selected on an S. aureus strain harboring prophage 53 encoding PdpSau, and in which a chimeric gene product emerged via adaptive laboratory evolution. This first case of staphylococcal interfamily phage-phage competition is analogous to some other abortive infection defense systems and to systems based on membrane-destructive proteins. IMPORTANCE Prophages play an important role in virulence, pathogenesis, and host preference, as well as in horizontal gene transfer in staphylococci. In contrast, broad-host-range lytic staphylococcal kayviruses lyse most S. aureus strains, and scientists worldwide have come to believe that the use of such phages will be successful for treating and preventing bacterial diseases. The effectiveness of phage therapy is complicated by bacterial resistance, whose mechanisms related to therapeutic staphylococcal phages are not understood in detail. In this work, we describe a resistance mechanism targeting kayviruses that is encoded by a prophage. We conclude that the defense mechanism belongs to a broader group of abortive infections, which is characterized by suicidal behavior of infected cells that are unable to produce phage progeny, thus ensuring the survival of the host population. Since the majority of staphylococcal strains are lysogenic, our findings are relevant for the advancement of phage therapy.

Staphylococcus brunensis sp. nov. isolated from human clinical specimens with a staphylococcal cassette chromosome-related genomic island outside of the rlmH gene bearing the ccrDE recombinase gene complex.

Novel species of coagulase-negative staphylococci, which could serve as reservoirs of virulence and antimicrobial resistance factors for opportunistic pathogens from the genus Staphylococcus, are recognized in human and animal specimens due to advances in diagnostic techniques. Here, we used whole-genome sequencing, extensive biotyping, MALDI-TOF mass spectrometry, and chemotaxonomy to characterize five coagulase-negative strains from the Staphylococcus haemolyticus phylogenetic clade obtained from human ear swabs, wounds, and bile. Based on the results of polyphasic taxonomy, we propose the species Staphylococcus brunensis sp. nov. (type strain NRL/St 16/872T = CCM 9024T = LMG 31872T = DSM 111349T). The genomic analysis revealed numerous variable genomic elements, including staphylococcal cassette chromosome (SCC), prophages, plasmids, and a unique 18.8 kb-long genomic island SbCIccrDE integrated into the ribosomal protein L7 serine acetyltransferase gene rimL. SbCIccrDE has a cassette chromosome recombinase (ccr) gene complex with a typical structure found in SCCs. Based on nucleotide and amino acid identity to other known ccr genes and the distinct integration site that differs from the canonical methyltransferase gene rlmH exploited by SCCs, we classified the ccr genes as novel variants, ccrDE. The comparative genomic analysis of SbCIccrDE with related islands shows that they can accumulate virulence and antimicrobial resistance factors creating novel resistance elements, which reflects the evolution of SCC. The spread of these resistance islands into established pathogens such as Staphylococcus aureus would pose a great threat to the healthcare system. IMPORTANCE The coagulase-negative staphylococci are important opportunistic human pathogens, which cause bloodstream and foreign body infections, mainly in immunocompromised patients. The mobile elements, primarily the staphylococcal cassette chromosome mec, which confers resistance to methicillin, are the key to the successful dissemination of staphylococci into healthcare and community settings. Here, we present a novel species of the Staphylococcus genus isolated from human clinical material. The detailed analysis of its genome revealed a previously undescribed genomic island, which is closely related to the staphylococcal cassette chromosome and has the potential to accumulate and spread virulence and resistance determinants. The island harbors a set of conserved genes required for its mobilization, which we recognized as novel cassette chromosome recombinase genes ccrDE. Similar islands were revealed not only in the genomes of coagulase-negative staphylococci but also in S. aureus. The comparative genomic study contributes substantially to the understanding of the evolution and pathogenesis of staphylococci.

Characteristics and distribution of plasmids in a clonally diverse set of methicillin-resistant Staphylococcus aureus strains.

The aim of this study was to compare the plasmid contents of methicillin-resistant Staphylococcus aureus (MRSA) strains classified into different clonal clusters (CCs). The isolates were collected from 15 Czech hospitals in 2000-2008. Plasmid DNA was detected in 65 (89%) strains, and 33 of them harbored more than one plasmid type. Altogether 24 different types of plasmids were identified, ranging in size from 1.3 to 55 kb. Restriction endonuclease analysis, plasmid elimination, DNA hybridization, and sequencing were used for their further characterization. It has been found that the conjugative, erythromycin resistance and enterotoxin D encoding plasmids are harbored by strains from different CCs. On the other hand, chloramphenicol and tetracycline resistance plasmids, and most of the penicillinase and cryptic plasmids were only detected in certain CCs. Especially, the pUSA300-like plasmids were found exclusively in the USA300 clone strains. The high diversity in plasmid content detected in the study strains implies that plasmids play a major role in evolution of MRSA clonal lineages.

Enterococcus plantarum sp. nov., isolated from plants.

Eight Gram-positive, catalase-negative bacterial strains were isolated during screening of enterococcal populations on plants. rep-PCR fingerprinting using the (GTG)(5) primer showed that the isolates constituted a single cluster that was separate from all known enterococcal species. 16S rRNA gene sequence phylogenetic analysis of three representative strains showed that the isolates belonged to the genus Enterococcus and that they clustered with the Enterococcus faecalis species group. Sequencing of the genes for the phenylalanyl-tRNA synthase alpha subunit (pheS) and the RNA polymerase alpha subunit (rpoA) also revealed the isolates' separate taxonomic position. Application of whole-cell protein fingerprinting, automated ribotyping and extensive phenotyping demonstrated the genetic and phenotypic homogeneity of the isolates and confirmed their separate position within the E. faecalis species group. The isolates represent a novel species of the genus Enterococcus, for which the name Enterococcus plantarum sp. nov. is proposed; the type strain is CCM 7889(T) (=LMG 26214(T)=C27(T)).

Global Transcriptomic Analysis of Bacteriophage-Host Interactions between a Kayvirus Therapeutic Phage and Staphylococcus aureus.

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.

Staphylococcus ratti sp. nov. Isolated from a Lab Rat.

Staphylococci from the Staphylococcus intermedius-Staphylococcus hyicus species group include numerous animal pathogens and are an important reservoir of virulence and antimicrobial resistance determinants. Due to their pathogenic potential, they are possible causative agents of zoonoses in humans; therefore, it is important to address the properties of these strains. Here we used a polyphasic taxonomic approach to characterize the coagulase-negative staphylococcal strain NRL/St 03/464T, isolated from the nostrils of a healthy laboratory rat during a microbiological screening of laboratory animals. The 16S rRNA sequence, MALDI-TOF mass spectrometry and positive urea hydrolysis and beta-glucuronidase tests clearly distinguished it from closely related Staphylococcus spp. All analyses have consistently shown that the closest relative is Staphylococcus chromogenes; however, values of digital DNA-DNA hybridization <35.3% and an average nucleotide identity <81.4% confirmed that the analyzed strain is a distinct Staphylococcus species. Whole-genome sequencing and expert annotation of the genome revealed the presence of novel variable genetic elements, including two plasmids named pSR9025A and pSR9025B, prophages, genomic islands and a composite transposon that may confer selective advantages to other bacteria and enhance their survival. Based on phenotypic, phylogenetic and genomic data obtained in this study, the strain NRL/St 03/464T (= CCM 9025T = LMG 31873T = DSM 111348T) represents a novel species with the suggested name Staphylococcus ratti sp. nov.