Accublock

The production of Stx1 and Stx2 was detected with an immunochromatographic test (Duopath Verotoxin test; Merck) (Park et al., 2003 (link)). The detection limit of this test is 25 ng/ml for Stx1 and 62.5 ng/ml for Stx2. Each isolate was inoculated in blood broth and simultaneously in 2 ml of Caye broth with Caye supplement, then incubated for 24 h at 37°C and 6 h at 37°C, respectively, following the recommendations of the supplier. Subsequently, cultures were treated with polymyxin B sulfate in a ratio of 9:1 (360 μl broth + 40 μl antibiotic) and incubated in a dry bath (Accu Block, Labnet) for 10 min at 37°C. An aliquot of the inoculum was transferred to the sample port, the test device was incubated at room temperature, and the result was interpreted after 10 min.

Bacterial DNA was extracted by boiling method as described by Momtaz et al. [23 (link)] with little modification. Presumptive E. coli in glycerol stocks stored at −80 °C were resuscitated in Luria-Bertani broth, incubated for 24 h at 37 °C followed by DNA extraction as follows. For the bacteria DNA extraction, 200 µL of the overnight broth was transfer into DNase free 2 mL microcentrifuge tubes, centrifuged at 15,000 rpm for 10 min and the supernatant was decanted. The pellet was re-suspended in 200 µL sterile distilled water; vortexed and the cells were lysed by boiling on AccuBlock (Digital dry bath, Labnet, Staffordshire, UK) for 15 min at 100 °C and thereafter centrifuge at 15,000 rmp for 5 min. DNA-containing supernatants were transferred into another DNase free microcentrifuge tubes and were stored at −20 °C for future analyses.

The isolates' genomic DNA extraction was carried out using the boiling technique^{6 (link)}. Specifically, 18–24 h old pure colonies of presumptive Vibrio spp. were suspended in sterile 200 μl distilled water, then lysed by heating for 15 min at 100 °C temperature using an AccuBlock (Labnet). A Mini Spin microcentrifuge was used to decant cell debris by centrifugation for 10 min at rate of 15,000 rpm. The DNA template from the recovered supernatants was employed in subsequent PCR tests.

PHB quantification was done using the procedure described by Schlebusch and Forchhammer (2010 (link)) and Taroncher-Oldenburg et al. (2000 (link)). Pre-weighed dried cells (5–10 mg) were boiled with 1 mL conc. H₂SO₄ at 100 °C on a heating block (Accublock™, Labnet, USA) for one hour to convert PHB to crotonic acid. Samples were allowed to cool down and subsequently diluted 20 times using 0.014 M H₂SO₄. Crotonic acid was determined using a high-performance liquid chromatography system (Thermo-Fischer Scientific, USA) with a Nucleosil C8 column (Macherey–Nagel, Germany) using an isocratic method. The mobile phase used was 20 mM NaH₂PO₄ buffer; pH 2.5 and acetonitrile (70:30) with a flow rate of 0.85 mL min⁻¹ and a column temperature of 30 °C. Detection of crotonic acid was done using a diode array detector (DAD) detector (Thermo-Fischer Scientific, USA) at 210 nm. For calibration, pure PHB (Sigma-Aldrich, USA) was treated accordingly and analyzed in parallel with samples. Instrument control and peak evaluation were done with Chromeleon 7.2 (Thermo-Fischer Scientific, USA). The percentage (dcw) PHB was determined with the amount of PHB obtained from HPLC analysis and the cell dry weight of biomass used for the analysis using Eq. (2): $$\%\text{dcw}PHB=\frac{mg(\text{PHB})}{mg(dcw)}\ast 100.$$

30 μg of whole cell lysate, determined using a BCA assay were mixed with an equal volume of 2x Laemmli sample buffer concentrate and 10% 2‐mercaptoethanol (Sigma, S3041‐1VL) and heated at 95°C for 5 min in an AccuBlock™ digital dry bath (Labnet International, D1200). Protein separation was carried out by SDS‐PAGE and then transferred to a nitrocellulose membrane (Bio‐rad, 1620094). Non‐specific binding sites were blocked with 5% milk (Marvel) in tris‐buffered saline TWEEN®20 (TBS‐T) rocking for 60 min at room temperature. Membranes were incubated with specific primary antibodies diluted in fresh blocking solution overnight at 4°C: α‐tubulin (Millipore, 05‐829; 1:5000), IGFBP‐2 (Abcam, 109284; 1:1000), GRP78 (BD Transduction Laboratories™, 610979). Membranes were washed with TBS‐T and incubated with secondary antibodies, (Sigma, A0545 or A4416; 1:2000). Proteins were visualised exposing with the ChemiDoc MP Imaging System with Image Lab software (Bio‐Rad) once treated with Clarity Western ECL Substrate (Bio‐Rad, 1705061). Densiometric analysis of protein bands was carried out using Image J (NIH).

DNA was extracted from samples using a QIAGEN DNeasy UltraClean Microbial Extraction Kit. Frozen mat samples were thawed at room temperature for 0.5 h, and ~ 0.15 g of mat was removed for DNA extraction. DNA extraction followed the manufacturer’s protocol, except for a modified cell lysis step. The samples were pretreated to facilitate cell rupture, which consisted of three freeze/heat cycles with liquid nitrogen and heating in an AccuBlock (Labnet International Inc.) at 65 °C. Between each cycle, a drill and a plastic pistil were used to mechanically break up the mat. The presence of DNA was confirmed by electrophoresis (0.8% agarose gel), and DNA concentrations were measured in a microplate spectrophotometer (Epoch: BioTek Instruments Inc., USA).