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Salmonella enterica serovar Infantis is one of the five main causes of human salmonellosis in the European Union (EU) and in recent years, has been increasingly reported to carry multiple antimicrobial resistance determinants, including extended-spectrum beta-lactamase (ESBL) genes. In our study, we used WGS-based tools to characterize S. Infantis strains circulating in the Abruzzo and Molise regions of Italy between 2017 and 2020 and compared this local dataset to the S. Infantis population present in Italy over the last two decades. Phylogenetic analyses demonstrated that the majority of strains isolated from poultry and turkeys from Abruzzo and Molise were closely related and belonged to one of the two main genetic clusters present in Italy, which were grouped predominantly as ESBL-producing strains that harbored pESI-like plasmid. We showed that 60% of the local strains carried multiple antibiotic resistance genes, including ESBL gene bla CTX-M-1 as well as aad A1, dfr A1, dfr A14, sul1 , and tet (A) genes present on the pESI-like megaplasmid. The analysis of strains from Abruzzo and Molise and the publicly available Italian S. Infantis sequences revealed a dramatic increase in the number of identified AMR genes in the strains isolated after 2011. Moreover, the number of strains resistant to five or more antibiotic classes increased from 20-80% in the last decade likely due to the acquisition of the megaplasmid. The persistence of the ESBL-producing and the multidrug-resistant (MDR) clone of S. Infantis in poultry populations in Italy and in Europe requires rapid and efficient intervention strategies to prevent further expansion of the clone., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Di Marcantonio, Romantini, Marotta, Chiaverini, Zilli, Abass, Di Giannatale, Garofolo and Janowicz.)

Monoclonal antibodies (MAbs) specific for the lipopolysaccharide (LPS) of Escherichia coli O104:H4 were produced by fusion of Sp2/O-Ag-14 mouse myeloma cells with spleen cells of Balb/c mice, immunized with heat-inactivated and sonicated E. coli O104:H4 bacterial cells. Four MAbs specific for the E. coli O104:H4 LPS (1E6G6, 1F4C9, 3G6G7, and 4G10D2) were characterized and evaluated for the use in a method for the detection of E. coli O104:H4 in milk samples that involves antibody conjugation to magnetic microbeads to reduce time and increase the efficiency of isolation. MAb 1E6G6 was selected and coupled to microbeads, then used for immuno-magnetic separation (IMS); the efficiency of the IMS method for E. coli O104:H4 isolation from milk was evaluated and compared to that of the EU RL VTEC conventional culture-based isolation procedure. Milk suspensions also containing other pathogenic bacteria that could potentially be found in milk (Campylobacter jejuni, Listeria monocytogenes, and Staphylococcus aureus) were also tested to evaluate the specificity of MAb-coated beads. Beads coated with MAb 1E6G6 showed a good ability to capture the E. coli O104:H4, even in milk samples contaminated with other bacteria, with a higher number of E. coli O104:H4 CFU reisolated in comparison with the official method (121 and 41 CFU, respectively, at 10(3) E. coli O104:H4 initial load; 19 and 6 CFU, respectively, at 10(2) E. coli O104:H4 initial load; 1 and 0 CFU, respectively, at 10(1) E. coli O104:H4 initial load). The specificity was 100%.