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Vancomycin-resistant enterococci (VRE) are a challenge to the health-care system regarding transmission rate and treatment of infections. VRE outbreaks have to be controlled from the first cases which means that appropriate and sensitive genotyping methods are needed. The aim of this study was to investigate the applicability of whole genome sequencing based analysis compared to Pulsed-Field Gel Electrophoresis (PFGE) and Multi-Locus Sequence Typing (MLST) in epidemiological investigations as well as the development of a user friendly method for daily laboratory use. Out of 14,000 VRE - screening samples, a total of 60 isolates positive for either vanA or vanB gene were isolated of which 38 were from patients with epidemiological links from three suspected outbreaks at Uppsala University Hospital. The isolates were genotypically characterised with PFGE, MLST, and WGS based core genome Average Nucleotide Identity analysis (cgANI). PFGE was compared to WGS and MLST regarding reliability, resolution, and applicability capacity. The PFGE analysis of the 38 isolates confirmed the epidemiological investigation that three outbreaks had occurred but gave an unclear picture for the largest cluster. The WGS analysis could clearly distinguish six ANI clusters for those 38 isolates. As result of the comparison of the investigated methods, we recommend WGS-ANI analysis for epidemiological issues with VRE. The recommended threshold for Enterococcus faecium VRE outbreak strain delineation with core genome based ANI is 98.5%. All referred sequences of this study are available from the NCBI BioProject number PRJNA301929.

BackgroundBrucellosis is a zoonosis that occurs worldwide. The disease has been completely eradicated in livestock in Sweden in 1994, and all cases of confirmed human brucellosis are imported into Sweden from other countries. However, due to an increase in the number of refugees and asylum seekers from the middle-east to Sweden, there is a need to improve the current diagnostic methodology for Brucella melitensis. Whilst culture of Brucella species can be used as a diagnostic tool, real-time PCR approaches provide a much faster result. The aim of this study was to set up a species-specific real-time PCR for the detection of all biovars of Brucella melitensis, which could be used routinely in diagnostic laboratories.MethodsA Brucella melitensis real-time PCR assay was designed using all available genomes in the public database of Brucella (N = 96) including all complete genomes of Brucella melitensis (N = 17). The assay was validated with a collection of 37 Brucella species reference strains, 120 Brucella melitensis human clinical isolates, and 45 clinically relevant non-Brucella melitensis strains.ResultsIn this study we developed a single real-time PCR for the specific detection of all biovars of Brucella melitensis.ConclusionsThis new real-time PCR method shows a high specificity (100%) and a high sensitivity (1.25 GE/μl) and has been implemented in the laboratories of four governmental authorities across Sweden.

Rhodoferax saidenbachensis sp. nov., a psychrotolerant, very slowly growing bacterium within the family Comamonadaceae, proposal of appropriate taxonomic position of Albidiferax ferrireducens strain T118T in the genus Rhodoferax and emended description of the genus Rhodoferax A Gram-stain-negative, oxidase and phosphatase-positive and catalase-negative, short rod-shaped bacterium was isolated from sediment of a drinking water reservoir in Germany. Based on 16S rRNA gene sequence and phenotypic properties, the bacterium belongs to the genus Rhodoferax within the family Comamonadaceae. The new taxon differed from related species mainly with respect to its fatty acid composition, low growth temperature, lack of pigments in young cultures and ability to utilize glycerol and D-mannose but not urea. The major fatty acids were C 16 : 1 v7c and/or iso-C 15 : 0 2-OH, C 16 : 0 , and C 18 : 1 v7c. The only ubiquinone detected was ubiquinone Q-8. The DNA G+C content was 60.3-61 mol%. Because of the phenotypic and genotypic differences from the most closely related taxa, the new strain represents a novel species for which the name Rhodoferax saidenbachensis sp. nov. is proposed. The type strain is ED16 T (5CCUG 57711). An emended description of the genus Rhodoferax is proposed. Based on the results of this study, strain T118 T (Albidiferax ferrireducens) is properly placed in the genus Rhodoferax as Rhodoferax ferrireducens.The oligotrophic Saidenbach reservoir (50 u 449 90 N 13 u 149 30 E) with a retaining capacity of 22.3 million m 3 and a watershed of 60.7 km 2 is one of the largest drinking water reservoirs in Saxony, Germany. During microbial screening of the sediment and pelagial, with the aim of displaying temporal differences in the composition of the population during one year, an unknown organism related to the family Comamonadaceae was isolated and cultured. The environmental sampling parameters in the hypolimnion were 8.38 mg O 2 l 21 , a conductivity of 220 mS cm 21 , a temperature of 3. (Hiraishi et al., 1991). All bacteria of this genus described to date are able to grow in a phototrophic manner and produce a peach-brown pigment. The only species of the most closely related genus, Albidiferax ferrireducens, was isolated from brackish water and might, therefore, be tolerant of salinity.Abbreviation: ANI, average nucleotide identity.The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequence and the whole genome sequence of strain ED16T are FJ755906 and AWQR00000000, respectively.