Cryovials

Individuals of R. decussatus and R. philippinarum were collected in the Óbidos Lagoon (Caldas da Rainha, Portugal, 39°24′44.1″ N 9°12′54.0″ W, in November of 2019). And other individuals of R. decussatus were collected in Ria Formosa (Algarve, Portugal, 37°01′05.4″ N 8°00′11.6″ W, in January of 2020) (Figure 1). The clams were transported, under controlled temperature (10 °C), to the laboratory and immediately processed upon arrival.
Enumeration of Vibrionaceae and coliforms was performed by serial dilutions, of the molluscs edible part and inter-valve liquid, in saline solution (0.85% w/v) and plating in thiosulfate citrate bile salts sucrose agar (TCBS; VWR, Radnor, PA, USA) and Chromocult^® Coliform agar (Merck, Darmstadt, Germany), respectively.
Plates were incubated for 24 h at 28 °C for Vibrio spp. and at 37 °C for coliforms. Colony-Forming Units (CFUs) with distinct morphology on TCBS agar were inoculated onto Tryptic Soy Agar (TSA; Biolife, Milano, Italy) supplemented with NaCl (1.5% w/v), and incubated for 24 h at 28 °C. CFU that had distinct morphology on Chromocult^® were inoculated onto Nutrient Agar (NA; VWR, Radnor, PA, USA), and incubated for 24 h at 37 °C. The isolates were conserved at −80 °C in cryovials (VWR, Radnor, PA, USA).

The C. thermocellum DSM 1313 wild-type (WT) strain was purchased from the DSMZ microorganism collection (www.dsmz.de). All strains used or constructed in this study are listed in Table 5. Freezer stocks were prepared by growing strains in the complex medium CTFUD (described by Olson and Lynd [52 ]) at 55°C to an optical density (OD) of 0.6 to 2.0. After the addition of glycerol to 25% (vol/vol), 1 mL aliquots were stocked in cryo-vials (VWR, Stockholm, Sweden) at −80°C. Strain construction and stocking were performed in an anaerobic vinyl chamber from Coy Laboratory Products (TG Instruments, Helsingborg, Sweden) with an atmosphere of 5% H₂, 10% CO₂, and 85% N₂ (Strandmöllen AB, Ljungby, Sweden).

We used CSF sample from 26 patients with neurological symptoms. CSF samples were collected in accordance to the ethical agreement 3633-11/2012 (JUH Jena). From all participants 12 mL of CSF were collected in polypropylene tubes (Greiner Bio-One 115261) following standard protocol (of the 12, the first 4 mL were used for laboratory measurements). The samples were kept on ice and immediately centrifuged at the bed side for 10 min. at room temperature (3500 g). Supernatants were aliquoted into cryovials (VWR, 479–1237), shock frozen in liquid nitrogen and stored at −150 °C. The CSF samples were characterized using key laboratory parameters. For MS analysis we used only samples without either blood contamination or blood-brain barrier disturbance. We assessed blood contamination by visual inspection for erythrocyte sediment and barrier disturbance by checking for erythrocytes, total protein, cell count and CSF:serum albumin ratio). To ensure adequate amounts of protein for analysis we created 3 mixtures. Each mixture was made up of equal volumes of CSF samples from 7–10 individuals. These pools were concentrated ~70–95-fold by ultrafiltration (AMICON^™ Ultra, MWCO 3 kDa, Merck Millipore), followed by 2D fractionation as described below.