High Clostridium difficile contamination rates of domestic and imported potatoes compared to some other vegetables in Slovenia.

Clostridium difficile, recently reclassified to Clostridioides difficile, is among most important causes of intestinal infections in humans. Zoonotic potential and foodborne transmissions are considered to be partially involved in C. difficile spread. Here we report prevalence of C. difficile in 142 retail and 12 homegrown vegetables in Slovenia between years 2014 and 2017. The overall prevalence of C. difficile on vegetables was 18,2% (28/154). A total of 115 isolates were obtained which belonged to 25 PCR ribotypes. Ten of those were toxigenic and PCR ribotype 014/020 was the most prevalent. Most of 25 determined PCR ribotypes were previously reported in humans, animals, soil or water in Slovenia. Among tested vegetables, potatoes had the highest positivity rate (28,0% vs. 6,7% and 9,4% for ginger and leaf vegetables). Altogether 66,7% of C. difficile positive potato samples were imported from 12 different countries of three different continents. The origin of contamination could be any point between production and retail store, however, our results suggest a possibility that potatoes represent a transnational and transcontinental way of C. difficile transmissions.

Comparison of Cultivable Acetic Acid Bacterial Microbiota in Organic and Conventional Apple Cider Vinegar.

Organic apple cider vinegar is produced from apples that go through very restricted treatment in orchard. During the first stage of the process, the sugars from apples are fermented by yeasts to cider. The produced ethanol is used as a substrate by acetic acid bacteria in a second separated bioprocess. In both, the organic and conventional apple cider vinegars the ethanol oxidation to acetic acid is initiated by native microbiota that survived alcohol fermentation. We compared the cultivable acetic acid bacterial microbiota in the production of organic and conventional apple cider vinegars from a smoothly running oxidation cycle of a submerged industrial process. In this way we isolated and characterized 96 bacteria from organic and 72 bacteria from conventional apple cider vinegar. Using the restriction analysis of the PCR-amplified 16S-23S rRNA gene ITS regions, we identified four different HaeIII and five different HpaII restriction profiles for bacterial isolates from organic apple cider vinegar. Each type of restriction profile was further analyzed by sequence analysis of the 16S-23S rRNA gene ITS regions, resulting in identification of the following species: Acetobacter pasteurianus (71.90%), Acetobacter ghanensis (12.50%), Komagataeibacter oboediens (9.35%) and Komagataeibacter saccharivorans (6.25%). Using the same analytical approach in conventional apple cider vinegar, we identified only two different HaeIII and two different HpaII restriction profiles of the 16S‒23S rRNA gene ITS regions, which belong to the species Acetobacter pasteurianus (66.70%) and Komagataeibacter oboediens (33.30%). Yeasts that are able to resist 30 g/L of acetic acid were isolated from the acetic acid production phase and further identified by sequence analysis of the ITS1-5.8S rDNA‒ITS2 region as Candida ethanolica, Pichia membranifaciens and Saccharomycodes ludwigii. This study has shown for the first time that the bacterial microbiota for the industrial production of organic apple cider vinegar is clearly more heterogeneous than the bacterial microbiota for the industrial production of conventional apple cider vinegar. Further chemical analysis should reveal if a difference in microbiota composition influences the quality of different types of apple cider vinegar.

Chryseobacterium limigenitum sp. nov., isolated from dehydrated sludge.

An intense yellow pigmented strain (SUR2(T)) isolated from dehydrated activated sludge was studied in detail to clarify its taxonomic assignment. Cells of the isolate showed a rod-shaped morphology and stained Gram-negative. Comparative 16S rRNA gene sequence analysis revealed highest similarities to the type strains of Chryseobacterium polytrichastri YG4-6(T) (98.6 %), Chryseobacterium aahli T68F(T) (97.9 %), Chryseobacterium daeguense K105(T) and Chryseobacterium gregarium DSM 79109(T) (both 97.4 %). 16S rRNA gene-sequence similarities to all other Chryseobacterium species were below 97.3 %. The fatty acid analysis of strain SUR2(T) revealed a Chryseobacterium typical profile composed mainly of the fatty acids C15:0 iso, C15:0 iso 2-OH, C17:1 iso ω9c, and C17:0 iso 3-OH. DNA-DNA hybridizations with the type strains of C. polytrichastri, C. aahli, C. daeguense and C. gregarium resulted in values below 70 %. Differentiating biochemical and chemotaxonomic properties showed differences to the most closely related species and suggest that the isolate SUR2(T) represents a novel species, for which the name Chryseobacterium limigenitum sp. nov. (type strain SUR2(T) = ZIM B1019(T) = CCM 8594(T) = LMG 28734(T)) is proposed.