We tested the predation of B. bacteriovorus against Escherichia coli in milk samples in three different experiments. In Experiment 1, the growth and predatory activity of B. bacteriovorus against E. coli in milk stored at 4°C were evaluated. In Experiment 2, the predatory activity of B. bacteriovorus against E. coli in the milk matrix was compared to the optimal one in the medium of choice. In Experiment 3, the influence of the native milk microbial community on the predation of B. bacteriovorus against E. coli experimentally added or indigenous grown at 4°C was tested. The predator increased at 4°C by about 1 Log in the first 48 hours and caused E. coli decrease by about 2 Log after 24 hours. The predator at 30°C reduced E. coli faster (3 Log after 6 hours) than at 4°C (2 Log after 24 hours). B. bacteriovorus at 30°C preyed on E. coli more in the nutrient broth than in the milk, with the most significant difference by about 4 Log after 48 hours. In raw milk contaminated only by the predator, it increased by about 1 Log after 48 hours at 4°C, suggesting that it preyed on indigenous microorganisms. B. bacteriovorus could find application in raw milk used as food or raw material during storage at 4°C to reduce the microbial load of spoilage and Shigatoxin-producing E. coli (STEC) strains of E. coli and therefore increase its shelf-life and healthiness.

The spread of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli is a major public health issue. Bivalves are filter-feeder animals capable of bioaccumulating the microorganisms present in water. This physiological characteristic makes them both good indicators of environmental contamination and possible carriers of pathogenic bacteria, including those resistant to antimicrobials. The aim of this study was to investigate the occurrence of ESBL-producing E. coli in clams (n = 308) collected from harvesting areas of the Central Adriatic Sea between 2018 and 2019. ESBL- /class C β-lactamase (AmpC)- producing E. coli and Escherichia spp. were isolated by streaking over the surface of MacConkey agar plates supplemented with cefotaxime enriched broths of the initial shellfish suspension. E. coli and Escherichia spp. resistant to cefotaxime were screened for ESBL production by using the double disk synergy test. Susceptibility to different antimicrobials and confirmation of ESBL-production were determined by the minimum inhibitory concentration (MIC) test. Isolates were further characterized by whole genome sequencing (WGS) and bioinformatic analysis of genomes with different tools. Overall, ESBL-producing E. coli were isolated from 3% of the samples. Of 13 ESBL- and ESBL−/AmpC-producing Escherichia spp. (n = 11 E. coli, n = 1 E. marmotae, n = 1 E. ruysiae) isolates, 13 were resistant to ampicillin and cefotaxime, 9 to sulfamethoxazole, 6 to tetracycline and nalidixic acid, 4 to trimethoprim, and 3 to ceftazidime, cefoxitin, ciprofloxacin, and chloramphenicol. Moreover, the majority (8/11) of the ESBL-producing E. coli isolates were multidrug-resistant. WGS showed that the isolates predominantly carried the bla_CTX-M-15 gene (3/11) and bla_CTX-M-14 and bla_CTX-M-1 (2/11 each). The AmpC β-lactamase CMY-2 was found in two isolates. Phylogroup A was the most prevalent (5/11), followed by phylogroups D (4/11), F (1/11), and B2 (1/11). Ten different sequence types (STs) were identified. Occurrence at sampling sites ranged between 0 and 27%. To identify associations between the occurrence of ESBL-producing E. coli and E. coli levels, samples were divided into two groups, with E. coli at >230 MPN/100 g and E. coli at ≤230 MPN/100 g. ESBL-producing E. coli isolates were significantly more commonly recovered in samples with higher E. coli levels (14%) than in those with lower levels of E. coli (2%). Moreover, the majority (3/4) of the potentially pathogenic strains were isolated in samples with higher E. coli levels. These findings provided evidence for the bacterial indicator of fecal contamination, E. coli, as an index organism for ESBL-producing E. coli isolates in bivalves.

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