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The ability of Salmonella species to adhere to surfaces and form biofilms, leading to persistent environmental reservoirs, might represent a direct link between environmental contamination and food processing contamination. The purpose of this study was to investigate the biofilm-forming ability of 80 multidrug-resistant (MDR) and extended-spectrum beta-lactamase (ESBL) producing Salmonella enterica serovar Infantis strains isolated from the broiler food chain production through whole genome sequencing (WGS), PCR, and morphotype association assays. Biofilm formation was quantified by testing the strains at two different temperatures, using 96-well polystyrene plates. The rough and dry colony (rdar) morphotype was assessed visually on Congo red agar (CRA) plates. Based on our results, all tested S. Infantis strains produced biofilm at 22 °C with an rdar morphotype, while at 37 °C, all the isolates tested negative, except one positive. Most isolates (58.75%) exhibited strong biofilm production, while 36.25% showed moderate production. Only 5 out of 80 (6.25%) were weak biofilm producers. WGS analysis showed the presence of the fim cluster (fimADF) and the csg cluster (csgBAC and csgDEFG), also described in S. Typhimurium, which are responsible for fimbriae production. PCR demonstrated the presence of csgD, csgB, and fimA in all 80 S. Infantis strains. To our knowledge, this is the first study comparing the effects of two different temperatures on the biofilm formation capacity of ESBL producing S. Infantis from the broiler production chain. This study highlights that the initial biofilm components, such as curli and cellulose, are specifically expressed at lower temperatures. It is important to emphasize that within the broiler farm, the environmental temperature ranges between 18–22 °C, which is the optimum temperature for in vitro biofilm formation by Salmonella spp. This temperature range facilitates the expression of biofilm-associated genes, contributing to the persistence of S. Infantis in the environment. This complicates biosecurity measures and makes disinfection protocols on the farm and in the production chain more difficult, posing serious public health concerns.

ABSTRACT: The majority of poultry meat used to be sourced from intensively housed birds. However, consumer preference has since demanded poultry producers develop more sustainable farming systems. Although free-range farming is considered beneficial for animal welfare, it is not as easy to standardize as an intensive system, which makes the choice of bird genotype appear crucial for alternative systems. In this study, we aimed to evaluate the effect of conventional and free-range rearing systems on the immune status, stress parameters, intestinal morphology and mortality in commercial hybrids (Ross 308) and local poultry strains, Bionda Piemontese (BP), Robusta Maculata (RM), BP x Sasso (BPxS), and RM x Sasso (RMxS). RNA was extracted from the jejunum and spleen to assess the mRNA expression of IL-2, IL-6, IL-10, IL-18, IL-1β, inducible nitric oxide synthase (iNOS), toll-like receptor (TLR)-4, and interferon gamma (IFN-γ). The heterophil:lymphocyte (H/L) ratio and intestinal histomorphometric evaluation were also calculated. We found that compared to the conventional system, the rearing system significantly affected the jejunum expression of IL-10, iNOS, IL-2, and IL-6, where these genes were upregulated in free-range system. A significant interaction between the rearing system and the genotype was also shown. More specifically, local breeds showed a significantly higher expression (P < 0.001) of IL-6 in the free-range system compared to the same genotypes in the conventional system. Moreover, IL-6 is constantly upregulated in local breeds within the free-range system compared to Ross hybrids. We also found significantly increased H/L and mortality rates in the latter, compared to the local breeds in the free-range reared system. The jejunum morphology also demonstrated a significantly higher villus height in BP and BPxS compared to the Ross hybrids. Overall, the results of our study confirm that the intense selection for growth in broiler chickens may have reduced their ability to react to the environmental stimuli related to free-range systems, resulting in a lower adaptability to a free-range environment, thus making them inappropriate for any farming system other than the conventional one. On the contrary, local chicken breeds are able to adapt and survive in the free-range system of rearing, and represent a genetic resource especially when adaptability to free-range conditions is required.

The role of wildlife, including birds, in antimicrobial resistance is nowadays a speculative topic for the scientific community as they could be spreaders/sources of antimicrobial resistance genes. In this respect, we aimed to investigate the antimicrobial susceptibility of 100 commensal Escherichia coli strains, isolated from wild birds from an Umbrian rescue centre and admitted to the Veterinary Teaching Hospital of Perugia (Central Italy) mainly for traumatic injuries. The possible presence of Salmonella spp. and ESBL-producing E. coli was also estimated. The highest prevalence of resistance was observed for ampicillin (85%) and amoxicillin/clavulanic acid (47%), probably due to their extensive use in human and veterinary medicine. Seventeen out of the one hundred E. coli isolates (17%) displayed a multidrug-resistance profile, including the beta-lactam category, with the most common resistance patterns to three or four classes of antibiotics. Resistance to ciprofloxacin, cefotaxime and ceftazidime exhibited values of 18%, 17% and 15%, respectively. Eight out of the hundred E. coli isolates (8%) were ESBL and seven showed multidrug resistance profiles. Salmonella spp. was not isolated. Resistance to third-generation cephalosporins, also detected in long-distance migratory birds, suggests the need for monitoring studies to define the role of wild birds in antimicrobial resistance circuits.

Outer membrane vesicles (OMVs) are nanoparticles released by Gram-negative bacteria, which contain different cargo molecules and mediate several biological processes. Recent studies have shown that OMVs are involved in antibiotic-resistance (AR) mechanisms by including β-lactamase enzymes in their lumen. Since no studies have as yet been conducted on Salmonella enterica subs. enterica serovar Infantis’ OMVs, the aim of the work was to collect OMVs from five S. Infantis β-lactam resistant strains isolated from a broiler meat production chain and to investigate whether β-lactamase enzymes are included in OMVs during their biogenesis. OMVs were isolated by means of ultrafiltration and a Nitrocefin assay quantified the presence of β-lactamase enzymes in the OMVs. Transmission electron microscopy (TEM) and dynamic light scattering (DLS) were used to identify the OMVs. The results showed that all strains release spherical OMVs, ranging from 60 to 230 nm. The Nitrocefin assay highlighted the presence of β-lactamase enzymes within the OMVs. This suggests that β-lactamase enzymes also get packaged into OMVs from bacterial periplasm during OMV biogenesis. An investigation into the possible role played by OMVs in AR mechanisms would open the door for an opportunity to develop new, therapeutic strategies.

This work aimed to evaluate phenotypically and genotypically the colistin susceptibility of 85 Salmonella Infantis strains isolated in Italy from the broiler production chain, and to apply a whole-genome approach for the determination of genes conferring antimicrobial resistance (AMR). All isolates were tested by the broth microdilution method to evaluate the colistin minimum inhibitory concentrations (MICs). A multiplex PCR was performed in all isolates for the screening of mcr-1, mcr-2, mcr-3 mcr-4, mcr-5 genes and whole-genome sequencing (WGS) of six S. Infantis was applied. Three out of 85 (3.5%) S. Infantis strains were colistin resistant (MIC values ranged from 4 to 8 mg/L) and mcr-1 positive. The mcr-1.1 and mcr-1.2 variants located on the IncX4 plasmid were detected in three different colistin-resistant isolates. The two allelic variants showed identical sequences. All six isolates harbored blaCTXM-1, aac(6′)-Iaa and gyrA/parC genes, mediating, respectively, beta-lactam, aminoglycoside and quinolone resistance. The pESI-megaplasmid carrying tet(A) (tetracycline resistance), dfrA1, (trimethoprim resistance) sul1, (sulfonamide resistance) and qacE (quaternary ammonium resistance) genes was found in all isolates. To our knowledge, this is the first report of the mcr-1.2 variant described in S. Infantis isolated from broilers chickens. Our results also showed a low prevalence of colistin- resistance, probably due to a reduction in colistin use in poultry. This might suggest an optimization of biosecurity control both on farms and in slaughterhouses.