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.

Brucella abortus is a highly pathogenic zoonotic agent, tempting for the development of a rapid diagnostic method to enable adequate treatment and prevent further spread. Enrichment of the bacteria is often used as a first step in diagnostics to increase the bacterial number above the detection limit of the real-time PCR. The enrichment of Brucella spp. takes at least 3 days, which might be avoidable if sensitive PCR methods can be used. Since many matrices contain PCR inhibitors, the limit of detection (LOD) must be determined for each separate matrix. Another aim of this study was the determination of survival of Brucella abortus in the analyzed matrices. The LOD for the detection of B. abortus in 14 matrices, relevant for human medicine, veterinary medicine and food and feed safety, was determined to evaluate the need of a pre-enrichment step prior to real-time PCR. The survival of B. abortus in the spiked matrices was tested by plate count in a 7-day interval for 132 days. The limit of detection for B. abortus in most matrices was in the range of 103–104 CFU/g for cultivation and 104–105 CFU/g for direct real-time PCR. The survival time of B. abortus was less than 21 days in apple purée and stomach content and 28 days in water while B. abortus remained viable at day 132 in milk, blood, spinach and minced meat. A direct PCR analysis without enrichment of bacteria saves at least 3 days. However, the limit of detection between direct PCR and plate count differs in a 10 fold range. We conclude that this lower sensitivity is acceptable in most cases especially if quick analysis are required.

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 : 1ω7c and/or iso-C_15 : 0 2-OH, C_16 : 0, and C_18 : 1ω7c. 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 ( = CCUG 57711^T = ATCC BAA-1852^T = DSM 22694^T). 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.

Brucellosis 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. A 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. In this study we developed a single real-time PCR for the specific detection of all biovars of Brucella melitensis. This 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.

In 1977, a sample of diseased adult honeybees (Apis mellifera) from Egypt was found to contain large amounts of a previously unknown virus, Egypt bee virus, which was subsequently shown to be serologically related to deformed wing virus (DWV). By sequencing the original isolate, we demonstrate that Egypt bee virus is in fact a fourth unique, major variant of DWV (DWV-D): more closely related to DWV-C than to either DWV-A or DWV-B. DWV-A and DWV-B are the most common DWV variants worldwide due to their close relationship and transmission byVarroa destructor. However, we could not find any trace of DWV-D in several hundred RNA sequencing libraries from a worldwide selection of honeybee, varroa and bumblebee samples. This means that DWV-D has either become extinct, been replaced by other DWV variants better adapted to varroa-mediated transmission, or persists only in a narrow geographic or host range, isolated from common bee and beekeeping trade routes.

A Gram-negative, rod-shaped and motile bacterial isolate, designated strain NS9^T, isolated from air of the Sainsbury Centre for Visual Arts in Norwich, UK, was subjected to a polyphasic taxonomic study including phylogenetic analyses based on partial 16S rRNA, gyrB and lepA gene sequences and phenotypic characterization. The 16S rRNA gene sequence of NS9^T identified Massilia haematophila CCUG 38318^T, M. niastensis 5516S-1^T (both 97.7 % similarity), M. aerilata 5516S-11^T (97.4 %) and M. tieshanensis TS3^T (97.4 %) as the next closest relatives. In partial gyrB and lepA sequences, NS9^T shared the highest similarities with M. haematophila CCUG 38318^T (94.5 %) and M. aerilata 5516-11^T (94.3 %), respectively. These sequence data demonstrate the affiliation of NS9^T to the genus Massilia . The detection of the predominant ubiquinone Q-8, a polar lipid profile consisting of the major compounds diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol and a polyamine pattern containing 2-hydroxyputrescine and putrescine were in agreement with the assignment of strain NS9^T to the genus Massilia . Major fatty acids were summed feature 3 (C_16 : 1ω7c and/or iso-C_15 : 0 2-OH), C_16 : 0, C_18 : 1ω7c and C_10 : 0 3-OH. Dissimilarities in partial lepA and gyrB gene sequences as well as results from DNA–DNA hybridizations demonstrate that strain NS9^T is a representative of an as-yet undescribed species of the genus Massilia that is also distinguished from its close relatives based on physiological and biochemical traits. Hence, we describe a novel species, for which we propose the name Massilia norwichensis sp. nov., with the type strain NS9^T ( = CCUG 65457^T = LMG 28164^T).