Six Helicobacter -like isolates were recovered from 15 gastric mucosa samples of red foxes (Vulpes vulpes) shot by hunters in the surroundings of Ljubljana, Slovenia. Gram-negative, tightly coiled, intensely motile, 7–15 µm long and ≤1 µm wide bacteria grew on the biphasic blood agar plates. By using a genus-specific polymerase chain reaction (PCR), all isolates were confirmed as Helicobacter sp. and subsequently subjected to whole-genome sequencing (WGS). Five isolates showed a genome-wide average nucleotide identity (ANI) value of <95 % to the previously described Helicobacter species and one isolate was classified as Helicobacter felis . In the five unidentified isolates, the 16S rRNA gene sequence similarity to the type strains of all Helicobacter species ranged from 98.6 to 98.9 %. Their taxonomic status was established using a polyphasic taxonomic approach comprising the core genome-based phylogeny, morphological and phenotypic characteristics, including an analysis of matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectra. Phylogeny revealed the existence of three novel and well-supported clusters, with Helicobacter bizzozeronii and Helicobacter baculiformis being the most closely related species. The isolates also differed from the previously described species in their MALDI-TOF profiles and some biochemical characteristics. In conclusion, the data presented herein indicate that the obtained isolates, excluding H. felis isolate, represent three novel Helicobacter species, for which the names Helicobacter labacensis sp. nov., Helicobacter mehlei sp. nov., and Helicobacter vulpis sp. nov. are proposed, with isolates L9^T (=DSM 108823^T=CRBIP 111719^T), L15^T (=DSM 108730^T=CCUG 72910^T) and L2^T (=DSM 108727^T=CCUG 72909^T) as type strains, respectively.

Strains of Pseudomonas savastanoi pv. savastanoi (Pss), isolated from infected olive trees (Olea europaea L.) in three European countries (Croatia, Slovenia and Portugal) were identified and characterised according to their colony morphology, physiological and biochemical features. According to the LOPAT scheme, 38.6% of Pss isolates were grouped in the Ib cluster. The Portuguese Pss strains were fully consistent with the typical LOPAT profile for this bacterium. Conversely, most Slovenian Pss strains showed delayed oxidase activity, whilst Croatian Pss strains did not produce any fluorescent pigment when grown in vitro. For Pss molecular identification, both end-point and real-time PCR were used, as well as MALDI–TOF, which was additionally used for proteomic analysis and the subsequent species identification of a number of strains that showed deviations from expected LOPAT results. Pss was confirmed as a causal agent of olive knot disease in 46.6% of olive orchards screened. Overall, these data suggests a possible correlation of certain Pss features with the geographical origin and the ecological niche of Pss isolates.

During the 2021 European Food Safety Authority coordinated harmonized monitoring of antimicrobial resistance in Campylobacter species in Slovenia, five Campylobacter -like strains were cultured from caeca of a total of 104 domestic pigs that could not be identified using the standard-prescribed biochemical tests or MALDI-TOF MS. The isolates were obtained using the standard ISO 10272 procedure for the isolation of thermotolerant Campylobacter with prolonged cultivation time. Small Campylobacter -like colonies were observed on mCCDA and CASA agar plates after 2–4 days of incubation; dark-field microscopy revealed relatively big spirilli-shaped bacteria exhibiting characteristic Campylobacter -like motility. The cells were 1.5–3 µm long and 0.5–0.7 µm wide, Gram-negative, oxidase-positive and catalase-positive. MALDI-TOF mass spectra were distinctive and consistent, but with low MALDI-TOF MS log scores and the closest matches being those of Campylobacter hyointestinalis and Campylobacter fetus . All five strains underwent whole-genome sequencing. Analysis of 16S rRNA gene sequences revealed that the isolates were most similar (98.3–98.4 % identity) to Campylobacter lanienae . Pairwise average nucleotide identity (ANI) values revealed that the five studied strains shared pairwise ANI of 96.2–96.5 % but were clearly distinct from the previously described Campylobacter species (ANI ≤72.8 %). The core genome-based phylogeny confirmed that the new strains form a distinct and well-supported clade within the genus Campylobacter . The conducted polyphasic taxonomic analysis confirmed that the five strains represent a novel Campylobacter species for which the name Campylobacter magnus sp. nov. is suggested, with strain 46386^T (=DSM 115534^T=CCUG 76865^T) as the type strain.

Seven Helicobacter -like isolates were cultured from caecal contents of 100 domestic pigs (Sus scrofa domesticus) sampled as part of the EFSA-coordinated harmonized monitoring of antimicrobial resistance in Campylobacter sp. in 2015. The bacteria were isolated using the standard ISO 10272 procedure for the isolation of thermotolerant Campylobacter with extended incubation time and formed small, grey, moist and flat colonies with a metallic sheen (small Campylobacter -like colonies) on modified Charcoal-Cefoperazone-Deoxycholate Agar (mCCDA) and Skirow agar plates. Morphologically, the bacterial cells were spirilli-shaped and highly motile, 1–2 µm long and ≤0.5 µm wide, Gram-negative, oxidase-positive and catalase-positive. They could not be identified using the standard-prescribed biochemical tests and had uniform, unique and reproducible MALDI-TOF mass spectra that most closely matched those of Helicobacter pullorum . Three strains (11154-15^T, 14348–15 and 16470–15) underwent whole-genome sequencing. Analysis of 16S rRNA gene sequences revealed a high similarity (≥99.8 % identity) to Helicobacter canadensis . Pairwise average nucleotide identity (ANI) values revealed that the three studied strains were closely related (ANI ≥98.9 %), but distinct from the previously described Helicobacter species (ANI ≤90.6 %). The core genome-based phylogeny confirmed that the new strains form a distinct clade most closely related to H. canadensis . The conducted polyphasic taxonomic analysis confirmed that the three strains represent a novel Helicobacter species for which the name Helicobacter colisuis sp. nov. is suggested, with strain 11154-15^T (= DSM 113688^T = CCUG 76053^T) as the type strain.