While the relevance of Bacillus (B.) cereus as a major cause of gastroenteritis is undisputed, the role of the closely related B. thuringiensis in foodborne disease is unclear. B. thuringiensis strains frequently harbor enterotoxin genes. However, the organism has only very rarely been associated with foodborne outbreaks, possibly due to the fact that during outbreak investigations, B. cereus is routinely not differentiated from B. thuringiensis. A recent EFSA scientific opinion stresses the urgent need for further data allowing for improved risk assessment, in particular as B. thuringiensis is a commonly used biopesticide. Therefore, the aim of this study was to gain further insights into the hazardous potential of B. thuringiensis. To this end, 39 B. thuringiensis isolates obtained from commercially used biopesticides, various food sources, as well as from foodborne outbreaks were characterized by panC typing, panC-based SplitsTree analysis, toxin gene profiling, FTIR spectroscopic analysis, a cytotoxicity assay screening for enterotoxic activity, and a sphingomyelinase assay. The majority of the tested B. thuringiensis isolates exhibited low (23%, n = 9) or mid level enterotoxicity (74%, n = 29), and produced either no (59%, n = 23) or low levels (33%, n = 13) of sphingomyelinase, which is reported to act synergistically with enterotoxins Nhe and Hbl. One strain isolated from rosemary was however classified as highly enterotoxic surpassing the cytotoxic activity of the high-level reference strain by a factor of 1.5. This strain also produced vast amounts of sphingomyelinase. Combining all results obtained in this study into a fingerprint pattern, several enterotoxic biopesticide strains were indistinguishable from those of isolates from foods or collected in association with outbreaks. Our study shows that many B. thuringiensis biopesticide strains exhibit mid-level cytotoxicity in a Vero cell assay and that some of these strains cannot be differentiated from isolates collected from foods or in association with outbreaks. Thus, we demonstrate that the use of B. thuringiensis strains as biopesticides can represent a food safety risk, underpinning the importance of assessing the hazardous potential of each strain and formulation used.

IntroductionWater supply and air-conditioner cooling towers (ACCT) are potential sources of Legionella pneumophila infection in people. During outbreaks, traditional typing methods cannot sufficiently segregate L. pneumophila strains to reliably trace back transmissions to these artificial water systems. Moreover, because multiple L. pneumophila strains may be present within these systems, methods to adequately distinguish strains are needed. Whole genome sequencing (WGS) and core genome multilocus sequence typing (cgMLST), with their higher resolution are helpful in this respect. In summer 2017, the health administration of the city of Basel detected an increase of L. pneumophila infections compared with previous months, signalling an outbreak.AimWe aimed to identify L. pneumophila strains populating suspected environmental sources of the outbreak, and to assess the relations between these strains and clinical outbreak strains.MethodsAn epidemiological and WGS-based microbiological investigation was performed, involving isolates from the local water supply and two ACCTs (n = 60), clinical outbreak and non-outbreak related isolates from 2017 (n = 8) and historic isolates from 2003–2016 (n = 26).ResultsIn both ACCTs, multiple strains were found. Phylogenetic analysis of the ACCT isolates showed a diversity of a few hundred allelic differences in cgMLST. Furthermore, two isolates from one ACCT showed no allelic differences to three clinical isolates from 2017. Five clinical isolates collected in the Basel area in the last decade were also identical in cgMLST to recent isolates from the two ACCTs.ConclusionCurrent outbreak-related and historic isolates were linked to ACCTs, which form a complex environmental habitat where strains are conserved over years.

In a broad market survey, 145 samples of tattoo-and permanent make-up pigments were bacteriologically analysed. The pigments originated from 39 unopened vials and 106 opened vials in use. The samples were collected from 33 tattoo shops and 22 beauty studios and the analyses were effected by direct plating on four agar media and by enrichment in a non-selective broth. None of the used media could detect all contaminated samples. The best performance was obtained with sheep blood agar (BA), which detected bacterial counts in 7 of 39 (17.9 %) of unopened vials and in 22 of 106 (20.8 %) opened vials. In 12 of 145 samples (8.3 %), bacterial growth was observed in the enrichment broth but not on sheep BA. In most cases, contaminations were in a low range of 10 1 -10 2 cfu / mL. Counts in the range of 10 3 -10 8 cfu / mL were registered in only four samples that were from different tattoo shops and from different manufacturers. Among the 31 bacterial species that were detected in the examined pigments, 20 (64.5 %) belonged to Gram positive rods or to Bacillus or related genera, respectively, 8 (25.8 %) to Gram positive cocci and only 3 (9.7 %) to Gram negative rods. Bacterial agents, known to cause dermal infections, were never detected. Based on the obtained data, a simple risk assessment was made, and microbiological criteria for future legal regulation proposed.Zusammenfassung In einer breit angelegten Marktüberwachung wurden insgesamt 145 Proben von Tätowier-und Permanent-Make-Up Farben bakteriologisch analysiert. Die Farben aus 39 ungeöffne-ten und 106 geöffneten Behältnissen stammten aus 33 Tätowierstudios und 22 Kosmetiksalons. Die Analysen erfolgten durch direktes Ausplattieren auf vier Agarmedien und durch Anreicherung in einem Flüssig-medium. Keines der eingesetzten Medien konnte alle kontaminierten Proben erfassen. Die höchste Positivrate wurde mit Schafblutagar erzielt, der in 7 von 39 (17.9 %) ungeöffneter und in 22 von 106 (20.8 %) geöff-neter Farben Keime nachwies. In 12 von insgesamt 145 Proben (8.3 %) zeigte sich in der Anreicherung ein Wachstum, nicht aber auf Schafblutagar. In den allermeisten Fällen lagen die Kontaminationen im quantitativ tiefen Bereich von 10 1 -10 2 KBE pro mL. Nur bei vier Proben aus unterschiedlichen Studios und von verschiedenen Herstellern wurden Keimzahlen im Bereich von 10 3 -10 8 KBE pro mL registriert. Unter den aus Farben isolierten 31 Keimarten gehörten 20 (64.5 %) zu den Gram positiven Stäbchen beziehungsweise zu Bacillus oder verwandten Genera, 8 (25.8 %)

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