Buffered peptone water (bpw)

Upon arrival, samples were refrigerated at 4°C and sample processing was carried out within 3 days after collection, at the latest. Pooled fecal samples (25 g) were thoroughly mixed, diluted 1:10 in buffered peptone water (BPW, bioMérieux), and incubated at 37°C for 20 ± 2 h. For the isolation of ESBL-/AmpC-producing E. coli, two loops (20 μl) of BPW were subcultured onto MacConkey agar supplemented with 1 mg/l of cefotaxime and incubated at 37°C for 20 ± 2 h. Two morphologically different colonies per plate were harvested and confirmed as E. coli by species-specific real-time PCR detection of the uidA gene (Frahm and Obst, 2003 (link)).
For the isolation of CP-producing E. coli, two loops (20 μl) of BPW were subcultured onto MacConkey agar without antibiotics. A loopful of grown colonies was then harvested for DNA extraction and subjected to a real-time PCR amplification screening targeting the CP-coding genes bla_NDM, bla_VIM, bla_KPC, and bla_OXA-48 (Ellington et al., 2016 (link)). If any of these genes tested positive, a loopful of bacterial growth from the MacConkey agar was subcultured on ChromID^® Carba Smart selective agar plates (bioMérieux), and isolated colonies were identified by uidA gene detection as above.

The bivalve batch samples collected in 2016 (n=244) were only analysed quantitatively. Soft material and intravalvular fluid from 10 to 25 living bivalve individuals were first homogenized in a Stomacher (Interscience) at 185 r.p.m. for 2.5 min. From the homogenate, 25 g was diluted 1 : 10 with buffered peptone water (bioMérieux) further homogenized for 30 s, prior to enrichment at 37 °C±1 °C for 21±3 h. Aliquots of 1.5 ml from the enriched homogenate were mixed with 0.5 ml glycerol (85 %) prior to storage at −80 °C [27 (link)]. From the frozen homogenates, 0.5 ml was re-cultivated in 10 ml Streptococcus Broth at 37±1 °C for 48±2 h, and subsequently 10 µl was streaked on Enterococcus Agar (BD DifcoTM) and incubated in water bath at 44±1 °C for 48±2 h. One or several isolates in case of clear difference in morphology, were picked and grown into pure culture. See protocol overview in Fig. 2.

Isolation of Salmonella was carried out according to the PN-EN ISO 6579: 2003 + AC: 2014-11. Media used for incubations included:
1. Nonselective pre-enrichment medium—Buffered Peptone Water (Biomerieux— Marcy l’Etoile, France);
2. Selective enrichment medium—Modified Semisolid Rappaport Vassiliadis medium—MSRV (Graso—Starogard Gdański, Poland);
3. Solid selective media—Salmonella Chrom Agar SAL-P (Graso), Briliant Green Agar—BGA (Graso— Starogard Gdański, Poland).
Portion of collected material was added to plastic bags and then diluted in a ratio of 1:10 with Buffered Peptone Water. The inoculated bags were homogenized and incubated for 18+/−2 h at 36 °C+/−2 °C. After incubation, samples were cultured on a plate with MSRV medium by adding 100 µL bacterial culture in three drops. The plates were incubated at 41.5+/−1 °C for 48+/−2 h. Typical growth on the plates (grey-white turbid zone extending out from the inoculated drop) were cultured onto agar medium BGA and Chrom agar. Then, plates were incubated at 37+/−1 °C for 24+/−2 h.