Marcel Kosina

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.

Enterococcus rivorum sp. nov., from water of pristine brooks

A significant number of Enterococcus strains from pristine waters of two brooks in Finland formed a distinct cluster on the basis of whole-cell protein fingerprinting by one-dimensional SDS-PAGE. The strains shared the following characteristics. Cells were ovoid, Gram-positive-staining and non-spore-forming, appearing singly or in pairs or chains. They were facultatively anaerobic and catalase-negative. Growth in broth containing 6.5 % NaCl or at 45 °C was weak or absent. Production of D antigen was variable. The strains tolerated 60 °C for 30 min, 40 % bile and tellurite, hydrolysed aesculin strongly and gelatin weakly, produced no acid from hippurate and did not reduce it, grew weakly at 10 °C, showed a strong reaction for the Voges-Proskauer test and produced acid from methyl α-d-glucoside, mannitol, sorbitol and sucrose, with weak or no production of acid from methyl α-d-mannoside, l-arabinose, gluconate and l-xylose. Several of the strains were selected for identification on the basis of sequencing of almost the whole 16S rRNA gene and partial atpA and pheS genes and of (GTG)(5)-PCR fingerprints. Partial atpA and pheS gene sequencing was also performed for those type strains of Enterococcus species without available sequences in the database. The pristine brook isolates formed a novel species, for which the name Enterococcus rivorum sp. nov. (type strain S299(T) = HAMBI 3055(T) = LMG 25899(T) = CCM 7986(T)) is proposed. On the basis of 16S rRNA gene sequence similarity, E. rivorum sp. nov. is related to the Enterococcus faecalis genogoup. It is distinguished from described Enterococcus species on the basis of 16S rRNA, atpA and pheS gene sequences and whole-cell protein and (GTG)(5)-PCR fingerprints. It is most closely related to E. faecalis, but DNA-DNA hybridization confirms it to represent a novel species.

Description of Pseudomonas gregormendelii sp. nov., a Novel Psychrotrophic Bacterium from James Ross Island, Antarctica.

During the microbiological research performed within the scope of activities of Czech expeditions based at the Johann Gregor Mendel Station at James Ross Island, Antarctica, two psychrotrophic gram-stain negative non-fluorescent strains CCM 8506T and CCM 8507 from soil were extensively characterized using genotypic and phenotypic methods. Initial characterization using ribotyping with HindIII restriction endonuclease and phenotyping implies that both isolates belong to a single Pseudomonas species. Sequencing of rrs, rpoB, rpoD and glnA genes of strain CCM 8506T confirmed affiliation of investigated strains within the genus Pseudomonas. Further investigation using automated ribotyping with EcoRI (RiboPrinter® Microbial Characterisation System), whole-cell protein profiling using the Agilent 2100 Bioanalyzer system, extensive biochemical testing and DNA–DNA hybridization experiments confirmed that both investigated strains are members of a single taxon which is clearly separated from all hitherto described Pseudomonas spp. Based on all findings, we describe a novel species Pseudomonas gregormendelii sp. nov. with the type strain CCM 8506T (=LMG 28632T).

A proteomic approach to the development of DIVA ELISA distinguishing pigs infected with Salmonella Typhimurium and pigs vaccinated with a Salmonella Typhimurium-based inactivated vaccine

BackgroundSalmonella enterica serovar Typhimurium is one of the most common enteropathogenic bacteria found in pigs in Europe. In our previous work, we demonstrated the protective effects in suckling piglets when their dams had been vaccinated with an S. Typhimurium-based inactivated vaccine. This study is focused on a procedure leading to serological discrimination between vaccinated and infected pigs. As we supposed, distinct environment during natural infection and in bacterial cultures used for vaccine preparation led to a slightly different spectrum of expressed S. Typhimurium proteins. The examination of porcine antibodies produced after the experimental infection with S. Typhimurium or after vaccination with S. Typhimurium-based inactivated vaccine by affinity chromatography and mass spectrometry revealed differences in antibody response applicable for serological differentiation of infected from vaccinated animals.ResultsAntibodies against Salmonella SipB, SipD and SseB proteins were detected at much higher levels in post-infection sera in comparison with control and post-vaccination sera. On the other hand, proteins BamB, OppA and a fragment of FliC interacted with antibodies from post-vaccination sera with a much higher intensity than from control and post-infection sera. In addition, we constructed ELISA assays using post-infection antigen - SipB protein and post-vaccination antigen - FliC-fragment and evaluated them on a panel of individual porcine sera.ConclusionsThe analysis of antibody response of infected and vaccinated pigs by proteomic tools enabled to identify S. Typhimurium antigens useful for distinguishing infected from vaccinated animals. This approach can be utilized in other challenges where DIVA vaccine and a subsequent serological assay are required, especially when genetic modification of a vaccine strain is not desirable.

Mortality of Goshawks (Accipiter gentilis) Due to West Nile Virus Lineage 2

In 2017, we isolated and identified West Nile virus (WNV) lineage 2 from two dead captive goshawks (Accipiter gentilis), for the first time in the Czech Republic. Goshawk might serve as an early indicator species for the ongoing WNV emergence in several European countries.