2.1. Study Area, Sampling and Microbiological Analysis

Twenty wild-caught mullets, Mugil cephalus (n = 4), Chelon ramada (n = 6), Chelon labrosus (n = 8) and Chelon saliens (n = 2), destined for the local food market, were captured by professional fisheries on 27 September 2018 (autumn) and 10 February 2019 (winter). The study area was Santa Giusta lagoon, an 8 km² area with a mean depth of approximately 1 m (Sardinia, Italy; coordinates: Lat 39°52′N, Long 8°35′E). Water temperature (Temp: 12.5–28.0 °C), salinity (Sal: 28.0–44.0 ppm) and dissolved oxygen (DO: 7.0–11.5 mg L⁻¹) were measured in situ using a YSI 6600 v2 (YSI Inc., Yellow Springs, OH, USA) multi-parameter probe. This lagoon is peculiar for its recurring ecological instability due to different anthropogenic impacts since 2000 [29]. The fish (average size and weight, 30.1 ± 5.8 cm and 277.4 ± 183 g, respectively) were transported inside a refrigerated bag to the Bonassai laboratory within 6–8 h, weighed using a scale (d = 0.01 g) and measured in length (d = 0,1 cm). The entire intestine (mean weight 14.0 ± 6.0 g) was aseptically removed from each fish, diluted (10% w/v) in saline solution (0.9% NaCl) and homogenized in plastic bags by a Stomacher^® 400 (FermionX Ltd., Worthing, UK) at room temperature. Samples were made by mixing the guts of two individuals of each species in order to obtain five samples for each date, up to a total of ten samples. Serial dilutions of the homogenate were prepared, and 100 µL of each dilution were spread on Marine agar (MA, Himedia, Mumbay, India) plates in duplicate and incubated at 30 °C for 48 h, for the enumeration of heterotrophic marine bacteria.

Bacterial colonies were randomly isolated and streaked onto fresh medium four times to obtain pure cultures. The purified isolates were stored at −80 °C in a 15% (v/v) glycerol-Nutrient broth (NB, Conda Pronadisa, Madrid, Spain) solution. The strains were assayed for the BS production, as follows.