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Background: Helichrysum italicum is a Mediterranean plant with well-known anti-inflammatory activity, but our previous whole transcriptome analysis has found that H. italicum infusion (HII) can also affect cytoskeletal rearrangement and tight junctions. The goal of the present study was to determine if HII improves the intestinal barrier (IB) dysfunction and by what mechanism. Methods: Caco-2 cells on Transwell inserts were used as a model of IB permeability. Heat-killed (HKB) or live Salmonella Infantis bacteria were used to induce IB integrity disruption upon three different testing conditions: pre-, co-, and post-treatment with 0.2 v/v% HII. Transepithelial electrical resistance values were used as an indicator of monolayer integrity before and after all treatments, and RT-PCR was used to assess the expression of tight junction proteins (TJPs) and inflammatory cytokines known to regulate intestinal permeability. Results: We found that all three treatments with HII improved the HKB-induced integrity disruption and decreased the down-regulation of TJP1, OCLN, and CLDN1, with the greatest effect observed in the pre-treated cells. Treatment with HII also decreased the up-regulation of CLDN2, TNF-α, IL-1β, and IL-6. In addition, pre-treatment of Caco-2 cells with HII prevented translocation of S. Infantis but did not prevent adhesion and invasion. Conclusion: This study showed that HII can improve inflammation-disrupted IB function by indirect modulation of mRNA expression of TJPs, especially in a preventive manner.

Salmonella enterica serovar Infantis (S. Infantis) poses a growing issue in the poultry sector, with phage-based products emerging as a safe and effective control measure. This study investigated the emergence of reduced-phage-susceptibility variants (RPSV) of S. Infantis in PhagoVet-treated broilers, given that RPSV could undermine phage treatment efficacy. The bacteriophages in the PhagoVet product were characterized using transmission electron microscopy (TEM), genome sequencing, and infection profiling. Furthermore, two broiler trials were conducted: a challenge group (T1) and a challenge-and-treated group (T2). The S. Infantis infective dose was set at 104 and 106 colony-forming units (CFUs) per animal, with PhagoVet administration at 106 and 108 plaque-forming units (PFUs) per animal, in Trials 1 and 2, respectively. The results revealed that the four PhagoVet bacteriophages belonged to different genera. PhagoVet evidenced broad-spectrum efficacy against 271 strains representing 18 Salmonella serovars. In Trial 1, PhagoVet reduced bacterial counts in feces to nearly undetectable levels by day 42, with no RPSV detected. However, in Trial 2, three and five RPSVs were detected in feces and ceca, respectively. Consequently, PhagoVet demonstrated efficacy against S. Infantis in broilers, and the potential impact of RPSV is deemed unlikely to compromise its efficacy.

Salmonella Infantis is one of the most frequent serovars reported in broilers and is also regularly identified in human salmonellosis cases, representing a relevant public health problem. In the laboratories of the Istituto Zooprofilattico Sperimentale della Puglia e della Basilicata (IZSPB), six Salmonella Infantis strains with antigenic formula -:r:1,5 have been isolated from the litter and carcass of broilers between 2018 and 2022. The strains were investigated to evaluate their phenotype, antibiotic resistance and genomic profiles. Genomic analysis confirmed that the isolates belonged to the Infantis serotype and to the sequence type ST32. Moreover, all strains showed a multidrug-resistant (MDR) profile and were characterised by the presence of the IncFIB plasmid incompatibility group. Three strains had the blaCTX-M-1 gene, and one of them carried IncX1. The presence of this new variant of S. Infantis is particularly relevant because it could expand the landscape of the S. Infantis population. The absence of the somatic antigen could pose a problem in both isolation and serotyping and a consequent public health concern due to the spread of Salmonella infection.

Salmonella spp. has been globally recognized as one of the leading causes of acute human bacterial gastroenteritis resulting from the consumption of animal-derived products. Salmonella Enteritidis, S. Typhimurium, and its monophasic variant are the main serovars responsible for human disease. However, a serovar known as S. Infantis has emerged as the fourth most prevalent serovar associated with human disease. A total of 95% of isolated S. Infantis serovars originate from broilers and their derived products. This serovar is strongly associated with an elevated antimicrobial (AMR) and multidrug resistance, a resistance to disinfectants, an increased tolerance to environmental mercury, a heightened virulence, and an enhanced ability to form biofilms and attach to host cells. Furthermore, this serovar harbors genes that confer resistance to colistin, a last-resort antibiotic in human medicine, and it has the potential to acquire additional transferable AMR against other critically important antimicrobials, posing a new and significant challenge to global public health. This review provides an overview of the current status of the S. Infantis serovar in the poultry sector, focusing on its key virulence factors, including its virulence genes, antimicrobial resistance, and biofilm formation. Additionally, novel holistic strategies for controlling S. Infantis along the entire food chain are presented in this review.

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.

Salmonella enterica subsp. enterica serovar Infantis is the most prevalent serovar in broilers and broiler meat in the European Union. The aim of our study was to test the biofilm formation and antimicrobial effect of disinfectants on genetically characterized S. Infantis isolates from poultry, food, and humans. For the biofilm formation under various temperature conditions (8 °C, 20 °C, and 28 °C) and incubation times (72 h and 168 h), the crystal violet staining method was used. The evaluation of the in vitro antimicrobial effect of Ecocid® S, ethanol, and hydrogen peroxide was determined using the broth microdilution method. The antibiofilm effect of subinhibitory concentration (1/8 MIC) of disinfectants was then tested on S. Infantis 323/19 strain that had the highest biofilm formation potential. Our results showed that the biofilm formation was strain-specific; however, it was higher at 20 °C and prolonged incubation time. Moreover, strains carrying a pESI plasmid showed higher biofilm formation potential. The antibiofilm potential of disinfectants on S. Infantis 323/19 strain at 20 °C was effective after a shorter incubation time. As shown in our study, more effective precautionary measures should be implemented to ensure biofilm prevention and removal in order to control the S. Infantis occurrence.

Infantis has recently become one of the most common serotypes of Salmonella isolated in the U.S. from raw meat samples collected in processing facilities and in retail stores. Investigations have determined that the majority of these isolates contain the pESI plasmid, but there has not been a large-scale investigation of the chromosome of these isolates. Here, we investigated 3276 whole-genome sequences of Salmonella Infantis with and without the pESI plasmid to understand chromosomal differences between plasmid carriage groups. S. Infantis genomes arranged into multiple clades with a single clade containing the isolates carrying the plasmid. Fifty-eight SNPs were identified in complete linkage disequilibrium between isolates that did and did not carry the plasmid. However, there were no unique genes present only in the genomes of isolates containing the plasmid. On average, isolates with the plasmid did contain more insertion sequences than those without (p < 0.05). Given that S. Infantis isolates carrying pESI form a single clade, it can be inferred that the increase in carriage of this plasmid in the U.S. is due to rapid clonal expansion of a single strain rather than as a result of multiple transfer events. As this S. Infantis clone does not contain any unique chromosomal genes, its proliferation appears to be due to pESI plasmid-encoded genes that may be advantageous in the chickens and turkeys or in their environment.

Worldwide, poultry infections by Salmonella are the cause of significant economic losses, not only due to reduced production (due to fowl typhoid disease), but also considering the efforts and control measures that must be constantly applied, especially due to zoonotic serovars. Poultry is a common reservoir of Salmonella and its transmission into the food chain is a risk for humans. The vaccination of layers plays an important role in the overall efforts to prevent Salmonella infections. An inactivated trivalent vaccine was prepared with S. Enteritidis, S. Typhimurium, and S. Infantis strains. Infection trials were performed to evaluate the efficacy of three vaccination schedules using inactivated and live S. Gallinarum 9R vaccines. For this purpose, at week 5 of life, one subcutaneous dose of live S. Gallinarum 9R vaccine (1–5 × 107 CFU) was given to Groups 1 and 2. At weeks 8 and 11 of life, chickens were also vaccinated with one (Group 1) or two (Groups 2 and 3) intramuscular doses of the inactivated oil-adjuvant trivalent vaccine (1 × 108 CFU/dose of each antigen). Group 4 consisted of chickens that remained unvaccinated (control). At week 14 of life, the efficacy of the vaccination plans was evaluated in three separate inoculation trials with S. Enteritidis, S. Typhimurium, or S. Infantis. After vaccination with the inactivated vaccine, homologous antibody production was observed, and after challenge, a significant reduction in the faecal shedding, invasion, and colonization of S. Typhimurium and S. Infantis was achieved by all vaccination schedules, while the vaccination with at least one dose of the live S. Gallinarum 9R vaccine was necessary to obtain such a significant protection against S. Enteritidis infection.

Salmonella enterica is an important foodborne pathogen. Commercial poultry are the main reservoirs of Salmonella enterica, leading to the contamination of food and outbreaks in humans. The vaccination of chickens is one of the most important strategies to reduce the number of Salmonella in poultry farms. Unfortunately, commercial vaccines have not been fully effective in controlling the spread and do not contain all the Salmonella serovars that circulate on farms. In this study, we evaluate a new, cochleate-based, trivalent injectable vaccine against S. Enteritidis, S. Typhimurium and S. Infantis, describing the vaccine security, capacity to induce specific anti-Salmonella serovar IgY and the gene expression of immune markers related to CD4 and CD8 T-cell-mediated immunity. Efficacy was evaluated through oral challenges performed separately for each Salmonella serotype. The efficacy and safety of the trivalent vaccine was proven under controlled conditions. The vaccine has no local or systemic reactions or adverse effects on poultry performance related to the vaccine. The vaccine provided significantly increased serum IgY titer levels, significantly reduced Salmonella CFU/g present in the cecum and an increased CD4+/CD8+ ratio in vaccinated animals when challenged with S. Infantis, S. Enteritidis and S. Typhimurium. These results indicate that this new trivalent vaccine does not generate adverse effects in poultry and produces an increase in neutralizing antibodies against the three Salmonella serovars.

Human salmonellosis incidence is increasing in the European Union (EU). Salmonellaenterica subsp. enterica serovar Enteriditis, Salmonellaenterica subsp. enterica serovar Typhimurium (including its monophasic variant) and Salmonellaenterica subsp. enterica serovar Infantis represent targets in control programs due to their frequent association with human cases. This study aimed to detect the most prevalent serotypes circulating in Abruzzo and Molise Regions between 2015 and 2020 in the framework of the Italian National Control Program for Salmonellosis in Poultry (PNCS)]. A total of 332 flocks of Abruzzo and Molise Regions were sampled by veterinary services in the period considered, and 2791 samples were taken. Samples were represented by faeces and dust from different categories of poultry flocks: laying hens (n = 284), broilers (n = 998), breeding chickens (n = 1353) and breeding or fattening turkeys (n = 156). Breeding and fattening turkeys had the highest rate of samples positive for Salmonella spp. (52.6%; C.I. 44.8%–60.3%). Faeces recovered through boot socks represented the greatest number of positive samples (18.2%). Salmonellaenterica subsp. enterica serovar Infantis was the prevalent serotype in breeding and fattening turkeys (32.7%; C.I. 25.8%–40.4%) and in broiler flocks (16.5%; C.I. 14.4%–19.0%). Salmonellaenterica subsp. enterica serovar Typhimurium was detected at low levels in laying hens (0.7%; C.I. 0.2%–2.5%) followed by breeding and fattening turkeys (0.6%; C.I. 0.2%–2.5%). Salmonellaenterica subsp. enterica serovar Enteriditis was also detected at low levels in laying hens (2.5%; C.I. 1.2%–5.0%). These findings highlight the role of broilers and breeding/fattening turkeys as reservoirs of Salmonella spp. and, as a consequence, in the diffusion of dangerous serotypes as Salmonellaenterica subsp. enterica serovar Infantis. This information could help veterinary services to analyze local trends and to take decisions not only based on indications from national control programs, but also based on real situations at farms in their own competence areas.

Non-typhoidal Salmonella infections remain a significant public health problem worldwide. In this study, we present the first detailed genomic analysis report based on short-read (Illumina) whole-genome sequencing (WGS) of 45 multidrug-resistant (MDR) Salmonella enterica subsp. enterica serotype Infantis isolates from poultry and meat product samples obtained in Russia during 2018–2020, and long-read (MinION) WGS of five more representative isolates. We sought to determine whether foodborne S. Infantis have acquired new characteristics, traits, and dynamics in MDR growth in recent years. All sequenced isolates belonged to the sequence type ST32 and more than the half of isolates was characterized by six similar antimicrobial susceptibility profiles, most of which corresponded well with the antimicrobial resistance determinants to aminoglycosides, sulphonamides, tetracycline, and chloramphenicol revealed in silico. Some of the isolates were characterized by the presence of several types of plasmids simultaneously. Plasmid typing using WGS revealed Col440I, ColpVC, ColRNAI, IncFIB, IncFII, IncX1, IncHI2, IncHI2A, and IncN replicons. The identified virulence genes for 45 whole genomes of S. Infantis were similar and included 129 genes encoding structural components of the cell, factors responsible for successful invasion of the host, and secreted products. These data will be a valuable contribution to further comparative genomics of S. Infantis circulating in Russia, as well as to epidemiological surveillance of foodborne Salmonella isolates and investigations of Salmonella outbreaks.

Salmonella comprises over 2500 serotypes and foodborne contamination associated with this pathogen remains an important health concern worldwide. During the last decade, a shift in serotype prevalence has occurred as traditionally less prevalent serotypes are increasing in frequency of infections, especially those related to poultry meat contamination. S. Infantis is one of the major emerging serotypes, and these strains commonly display antimicrobial resistance and can persist despite cleaning protocols. Thus, this work aimed to isolate S. Infantis strains from a poultry meat farm in Santiago, Chile and to characterize genetic variations present in them. We determined their genomic and phenotypic profiles at different points along the production line. The results indicate that the strains encompass 853 polymorphic sites (core-SNPs) with isolates differing from one another by 0–347 core SNPs, suggesting variation among them; however, we found discrete correlations with the source of the sample in the production line. Furthermore, the pan-genome was composed of 4854 total gene clusters of which 2618 (53.9%) corresponds to the core-genome and only 181 (3.7%) are unique genes (those present in one particular strain). This preliminary analysis will enrich the surveillance of Salmonella, yet further studies are required to assess their evolution and phylogeny.

The aim of this study was to develop and characterize Salmonellaenterica serovar Infantis (S. Infantis) cochleates protected by encapsulation technology as a potential vaccine and to determine its safety in pullets. Cochleates were encapsulated by two technologies, spray drying and ionotropic gelation at different concentrations (0–15% v/v), and were characterized by physicochemical properties, protein content and Fourier Transform Infrared Spectroscopy (FTIR). The cochleates were white liquid suspensions with tubular shapes and a protein content of 1.0–2.1 mg/mL. After encapsulation by spray drying, microparticles ranged in size from 10.4–16.9 µm, were spherical in shape, and the protein content was 0.7–1.8 mg/g. After encapsulation by ionotropic gelation, beads ranged in size from 1620–1950 µm and were spherical in shape with a protein content of 1.0–2.5 mg/g. FTIR analysis indicated that both encapsulation processes were efficient. The cochleates encapsulated by ionotropic gelation were then tested for safety in pullets. No ill effect on the health of animals was observed upon physical or postmortem examination. In conclusion, this study was the first step in developing a potential oral S. Infantis vaccine safe for poultry using a novel cochleate encapsulation technology. Future studies are needed to determine the effectiveness of the vaccine.

Salmonella Infantis, a common contaminant of poultry products, is known to harbor mobile genetic elements that confer multi-drug resistance (MDR) and have been detected in many continents. Here, we report four MDR S. Infantis strains recovered from poultry house environments in Santa Cruz Island of the Galapagos showing extended-spectrum β-lactamase (ESBL) resistance and reduced fluoroquinolone susceptibility. Whole-genome sequencing (WGS) revealed the presence of the ESBL-conferring blaCTX-M-65 gene in an IncFIB-like plasmid in three S. Infantis isolates. Multi-locus sequence typing (MLST) and single nucleotide variant/polymorphism (SNP) SNVPhyl analysis showed that the S. Infantis isolates belong to sequence type ST32, likely share a common ancestor, and are closely related (1–3 SNP difference) to blaCTX-M-65-containing clinical and veterinary S. Infantis isolates from the United States and Latin America. Furthermore, phylogenetic analysis of SNPs following core-genome alignment (i.e., ParSNP) inferred close relatedness between the S. Infantis isolates from Galapagos and the United States. Prophage typing confirmed the close relationship among the Galapagos S. Infantis and was useful in distinguishing them from the United States isolates. This is the first report of MDR blaCTX-M-65-containing S. Infantis in the Galapagos Islands and highlights the need for increased monitoring and surveillance programs to determine prevalence, sources, and reservoirs of MDR pathogens.

In recent years, Salmonella Infantis has become a predominant serovariant in clinical and poultry isolates, thereby imposing a substantial economic burden on both public health and the livestock industry. With the aim of coping with the steep increase in serovar Infantis prevalence, a polymerase chain reaction (PCR)-based rapid and accurate diagnostic assay was developed in this study through pangenome profiling of 60 Salmonella serovars. A gene marker, SIN_02055, was identified, which is present in the S. Infantis genome but not in the pangenome of the other serovars. Primers specific to SIN_02055 were used to accurately detect serovar Infantis, and to successfully differentiate Infantis from the other 59 serovars in real-time PCR with a R2 of 0.999 and an efficiency of 95.76%. The developed method was applied to 54 Salmonella strains belonging to eight dominant serovars, and distinguished Infantis from the other seven serovars with an accuracy of 100%. The diagnostic primer set also did not show false positive amplification with 32 strains from eight non-Salmonella bacterial species. This cost-effective and rapid method can be considered an alternative to the classic serotyping using antisera.

Salmonella Infantis is a zoonotic pathogen that causes gastroenteritis in humans and animals, with poultry being its main reservoir. In Chile, there are no data to characterize S. Infantis strains in poultry production. In this study, 87 S. Infantis strains were isolated from chicken meat for sale in supermarkets in Santiago, Chile, and characterized according to their virulence genes, biofilm formation abilities, antibiotic susceptibility, and resistance genes. Through polymerase chain reaction or PCR, the strains were analyzed to detect the presence of 11 virulence genes, 12 antibiotic resistance genes, and integrase genes. Moreover, disc diffusion susceptibility to 18 antimicrobials and the ability to form biofilm in vitro were evaluated. Results demonstrated six different virulence gene profiles. Ninety-four percent of the strains were multi-resistant to antibiotics with weak biofilm formation abilities, 63.2% of the strains were broad spectrum β- lactam resistant, and the bla CTX-M-65 gene was amplified in 13 strains. Only 3.4% of the strains were fluoroquinolone resistant, and the qnrB gene was amplified in two strains. Colistin resistance was exhibited in 28.7% of the strains, but mrc genes were not amplified in any strain under study. The isolated S. Infantis strains are pathogenic and antibiotic multi-resistant, and thus, this Salmonella serotype should be under surveillance in the poultry food production chain with the aim of protecting public health.

Salmonella Infantis is one of the most frequent serovars reported in broilers and is also regularly identified in human salmonellosis cases, representing a relevant public health problem. In the laboratories of the Istituto Zooprofilattico Sperimentale della Puglia e della Basilicata (IZSPB), six Salmonella Infantis strains with antigenic formula -:r:1,5 have been isolated from the litter and carcass of broilers between 2018 and 2022. The strains were investigated to evaluate their phenotype, antibiotic resistance and genomic profiles. Genomic analysis confirmed that the isolates belonged to the Infantis serotype and to the sequence type ST32. Moreover, all strains showed a multidrug-resistant (MDR) profile and were characterised by the presence of the IncFIB plasmid incompatibility group. Three strains had the bla CTX-M-1 gene, and one of them carried IncX1. The presence of this new variant of S . Infantis is particularly relevant because it could expand the landscape of the S . Infantis population. The absence of the somatic antigen could pose a problem in both isolation and serotyping and a consequent public health concern due to the spread of Salmonella infection.

Salmonella Infantis is considered in recent years an emerging Salmonella serovar, as it has been associated with several outbreaks and multidrug resistance phenotypes. Phages appear as a possible alternative strategy to control Salmonella Infantis (SI). The aims of this work were to characterize two phages of the Felixounavirus genus, isolated using the same strain of SI, and to expose them to interact in challenge assays to identify genetic and phenotypic changes generated from these interactions. These two phages have a shared nucleotide identity of 97% and are differentiated by their host range: one phage has a wide host range (lysing 14 serovars), and the other has a narrow host range (lysing 6 serovars). During the 12 h challenge we compared: (1) optical density of SI, (2) proportion of SI survivors from phage-infected cultures, and (3) phage titer. Isolates obtained through the assays were evaluated by efficiency of plating (EOP) and by host-range characterization. Genomic modifications were characterized by evaluation of single nucleotide polymorphisms (SNPs). The optical density (600 nm) of phage-infected SI decreased, as compared to the uninfected control, by an average of 0.7 for SI infected with the wide-host-range (WHR) phage and by 0.3 for SI infected with the narrow-host-range (NHR) phage. WHR phage reached higher phage titer (7 × 1011 PFU/mL), and a lower proportion of SI survivor was obtained from the challenge assay. In SI that interacted with phages, we identified SNPs in two genes (rfaK and rfaB), which are both involved in lipopolysaccharide (LPS) polymerization. Therefore, mutations that could impact potential phage receptors on the host surface were selected by lytic phage exposure. This work demonstrates that the interaction of Salmonella phages (WHR and NHR) with SI for 12 h in vitro leads to emergence of new phenotypic and genotypic traits in both phage and host. This information is crucial for the rational design of phage-based control strategies.

Salmonella is important as both a cause of clinical disease in swine and as a source of food-borne transmission of disease to humans. Lactobacillus and Bacillus are often used as antibiotic substitutes to prevent Salmonella infection. In this study, we evaluated the effects of a select mixture of Lactobacillus johnsonii L531, Bacillus licheniformis BL1721 and Bacillus subtilis BS1715 (LBB-mix) in prevention of Salmonella enterica serovar Infantis infection in a pig model. LBB-mix was orally administered to newly weaned piglets for seven days before S. Infantis challenge. LBB-mix pretreatment ameliorated S. Infantis-induced fever, leukocytosis, growth performance loss, and ileal inflammation. Pre-administration of LBB-mix reduced the number of Salmonella in the feces but increased the number of goblet cells in the ileum. S. Infantis infection resulted in an increase in cell death in the ileum, this increase was attenuated by LBB-mix consumption. Claudin 1 and cleaved caspase-1 expression was decreased in the ileum of pigs challenged with S. Infantis, but not in pigs pretreated with LBB-mix. In conclusion, our data indicate that a select LBB-mix has positive effects on controlling S. Infantis infection via alleviating inflammation and maintaining the intestinal mucosal barrier integrity in pigs.

Salmonella spp. has been globally recognized as one of the leading causes of acute human bacterial gastroenteritis resulting from the consumption of animal-derived products. Salmonella Enteritidis, S. Typhimurium, and its monophasic variant are the main serovars responsible for human disease. However, a serovar known as S . Infantis has emerged as the fourth most prevalent serovar associated with human disease. A total of 95% of isolated S . Infantis serovars originate from broilers and their derived products. This serovar is strongly associated with an elevated antimicrobial (AMR) and multidrug resistance, a resistance to disinfectants, an increased tolerance to environmental mercury, a heightened virulence, and an enhanced ability to form biofilms and attach to host cells. Furthermore, this serovar harbors genes that confer resistance to colistin, a last-resort antibiotic in human medicine, and it has the potential to acquire additional transferable AMR against other critically important antimicrobials, posing a new and significant challenge to global public health. This review provides an overview of the current status of the S . Infantis serovar in the poultry sector, focusing on its key virulence factors, including its virulence genes, antimicrobial resistance, and biofilm formation. Additionally, novel holistic strategies for controlling S. Infantis along the entire food chain are presented in this review.

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.

Salmonella enterica subsp. enterica serovar Infantis is the most prevalent serovar in broilers and broiler meat in the European Union. The aim of our study was to test the biofilm formation and antimicrobial effect of disinfectants on genetically characterized S. Infantis isolates from poultry, food, and humans. For the biofilm formation under various temperature conditions (8 °C, 20 °C, and 28 °C) and incubation times (72 h and 168 h), the crystal violet staining method was used. The evaluation of the in vitro antimicrobial effect of Ecocid ® S, ethanol, and hydrogen peroxide was determined using the broth microdilution method. The antibiofilm effect of subinhibitory concentration (1/8 MIC) of disinfectants was then tested on S. Infantis 323/19 strain that had the highest biofilm formation potential. Our results showed that the biofilm formation was strain-specific; however, it was higher at 20 °C and prolonged incubation time. Moreover, strains carrying a pESI plasmid showed higher biofilm formation potential. The antibiofilm potential of disinfectants on S. Infantis 323/19 strain at 20 °C was effective after a shorter incubation time. As shown in our study, more effective precautionary measures should be implemented to ensure biofilm prevention and removal in order to control the S. Infantis occurrence.

Infantis has recently become one of the most common serotypes of Salmonella isolated in the U.S. from raw meat samples collected in processing facilities and in retail stores. Investigations have determined that the majority of these isolates contain the pESI plasmid, but there has not been a large-scale investigation of the chromosome of these isolates. Here, we investigated 3276 whole-genome sequences of Salmonella Infantis with and without the pESI plasmid to understand chromosomal differences between plasmid carriage groups. S. Infantis genomes arranged into multiple clades with a single clade containing the isolates carrying the plasmid. Fifty-eight SNPs were identified in complete linkage disequilibrium between isolates that did and did not carry the plasmid. However, there were no unique genes present only in the genomes of isolates containing the plasmid. On average, isolates with the plasmid did contain more insertion sequences than those without (p < 0.05). Given that S. Infantis isolates carrying pESI form a single clade, it can be inferred that the increase in carriage of this plasmid in the U.S. is due to rapid clonal expansion of a single strain rather than as a result of multiple transfer events. As this S. Infantis clone does not contain any unique chromosomal genes, its proliferation appears to be due to pESI plasmid-encoded genes that may be advantageous in the chickens and turkeys or in their environment.

Worldwide, poultry infections by Salmonella are the cause of significant economic losses, not only due to reduced production (due to fowl typhoid disease), but also considering the efforts and control measures that must be constantly applied, especially due to zoonotic serovars. Poultry is a common reservoir of Salmonella and its transmission into the food chain is a risk for humans. The vaccination of layers plays an important role in the overall efforts to prevent Salmonella infections. An inactivated trivalent vaccine was prepared with S. Enteritidis, S. Typhimurium, and S. Infantis strains. Infection trials were performed to evaluate the efficacy of three vaccination schedules using inactivated and live S. Gallinarum 9R vaccines. For this purpose, at week 5 of life, one subcutaneous dose of live S. Gallinarum 9R vaccine (1-5 × 10 7 CFU) was given to Groups 1 and 2. At weeks 8 and 11 of life, chickens were also vaccinated with one (Group 1) or two (Groups 2 and 3) intramuscular doses of the inactivated oil-adjuvant trivalent vaccine (1 × 10 8 CFU/dose of each antigen). Group 4 consisted of chickens that remained unvaccinated (control). At week 14 of life, the efficacy of the vaccination plans was evaluated in three separate inoculation trials with S. Enteritidis, S. Typhimurium, or S. Infantis. After vaccination with the inactivated vaccine, homologous antibody production was observed, and after challenge, a significant reduction in the faecal shedding, invasion, and colonization of S. Typhimurium and S. Infantis was achieved by all vaccination schedules, while the vaccination with at least one dose of the live S. Gallinarum 9R vaccine was necessary to obtain such a significant protection against S. Enteritidis infection.

Salmonella enterica is an important foodborne pathogen. Commercial poultry are the main reservoirs of Salmonella enterica, leading to the contamination of food and outbreaks in humans. The vaccination of chickens is one of the most important strategies to reduce the number of Salmonella in poultry farms. Unfortunately, commercial vaccines have not been fully effective in controlling the spread and do not contain all the Salmonella serovars that circulate on farms. In this study, we evaluate a new, cochleate-based, trivalent injectable vaccine against S. Enteritidis, S. Typhimurium and S. Infantis, describing the vaccine security, capacity to induce specific anti- Salmonella serovar IgY and the gene expression of immune markers related to CD4 and CD8 T-cell-mediated immunity. Efficacy was evaluated through oral challenges performed separately for each Salmonella serotype. The efficacy and safety of the trivalent vaccine was proven under controlled conditions. The vaccine has no local or systemic reactions or adverse effects on poultry performance related to the vaccine. The vaccine provided significantly increased serum IgY titer levels, significantly reduced Salmonella CFU/g present in the cecum and an increased CD4+/CD8+ ratio in vaccinated animals when challenged with S. Infantis, S. Enteritidis and S. Typhimurium. These results indicate that this new trivalent vaccine does not generate adverse effects in poultry and produces an increase in neutralizing antibodies against the three Salmonella serovars.

Human salmonellosis incidence is increasing in the European Union (EU). Salmonella enterica subsp. enterica serovar Enteriditis, Salmonella enterica subsp. enterica serovar Typhimurium (including its monophasic variant) and Salmonella enterica subsp. enterica serovar Infantis represent targets in control programs due to their frequent association with human cases. This study aimed to detect the most prevalent serotypes circulating in Abruzzo and Molise Regions between 2015 and 2020 in the framework of the Italian National Control Program for Salmonellosis in Poultry (PNCS)]. A total of 332 flocks of Abruzzo and Molise Regions were sampled by veterinary services in the period considered, and 2791 samples were taken. Samples were represented by faeces and dust from different categories of poultry flocks: laying hens ( n = 284), broilers ( n = 998), breeding chickens ( n = 1353) and breeding or fattening turkeys ( n = 156). Breeding and fattening turkeys had the highest rate of samples positive for Salmonella spp. (52.6%; C.I. 44.8%-60.3%). Faeces recovered through boot socks represented the greatest number of positive samples (18.2%). Salmonella enterica subsp. enterica serovar Infantis was the prevalent serotype in breeding and fattening turkeys (32.7%; C.I. 25.8%-40.4%) and in broiler flocks (16.5%; C.I. 14.4%-19.0%). Salmonella enterica subsp. enterica serovar Typhimurium was detected at low levels in laying hens (0.7%; C.I. 0.2%-2.5%) followed by breeding and fattening turkeys (0.6%; C.I. 0.2%-2.5%). Salmonella enterica subsp. enterica serovar Enteriditis was also detected at low levels in laying hens (2.5%; C.I. 1.2%-5.0%). These findings highlight the role of broilers and breeding/fattening turkeys as reservoirs of Salmonella spp. and, as a consequence, in the diffusion of dangerous serotypes as Salmonella enterica subsp. enterica serovar Infantis. This information could help veterinary services to analyze local trends and to take decisions not only based on indications from national control programs, but also based on real situations at farms in their own competence areas.

Non-typhoidal Salmonella infections remain a significant public health problem worldwide. In this study, we present the first detailed genomic analysis report based on short-read (Illumina) whole-genome sequencing (WGS) of 45 multidrug-resistant (MDR) Salmonella enterica subsp. enterica serotype Infantis isolates from poultry and meat product samples obtained in Russia during 2018-2020, and long-read (MinION) WGS of five more representative isolates. We sought to determine whether foodborne S. Infantis have acquired new characteristics, traits, and dynamics in MDR growth in recent years. All sequenced isolates belonged to the sequence type ST32 and more than the half of isolates was characterized by six similar antimicrobial susceptibility profiles, most of which corresponded well with the antimicrobial resistance determinants to aminoglycosides, sulphonamides, tetracycline, and chloramphenicol revealed in silico. Some of the isolates were characterized by the presence of several types of plasmids simultaneously. Plasmid typing using WGS revealed Col440I, ColpVC, ColRNAI, IncFIB, IncFII, IncX1, IncHI2, IncHI2A, and IncN replicons. The identified virulence genes for 45 whole genomes of S. Infantis were similar and included 129 genes encoding structural components of the cell, factors responsible for successful invasion of the host, and secreted products. These data will be a valuable contribution to further comparative genomics of S. Infantis circulating in Russia, as well as to epidemiological surveillance of foodborne Salmonella isolates and investigations of Salmonella outbreaks.

Salmonella comprises over 2500 serotypes and foodborne contamination associated with this pathogen remains an important health concern worldwide. During the last decade, a shift in serotype prevalence has occurred as traditionally less prevalent serotypes are increasing in frequency of infections, especially those related to poultry meat contamination. S. Infantis is one of the major emerging serotypes, and these strains commonly display antimicrobial resistance and can persist despite cleaning protocols. Thus, this work aimed to isolate S . Infantis strains from a poultry meat farm in Santiago, Chile and to characterize genetic variations present in them. We determined their genomic and phenotypic profiles at different points along the production line. The results indicate that the strains encompass 853 polymorphic sites (core-SNPs) with isolates differing from one another by 0-347 core SNPs, suggesting variation among them; however, we found discrete correlations with the source of the sample in the production line. Furthermore, the pan-genome was composed of 4854 total gene clusters of which 2618 (53.9%) corresponds to the core-genome and only 181 (3.7%) are unique genes (those present in one particular strain). This preliminary analysis will enrich the surveillance of Salmonella , yet further studies are required to assess their evolution and phylogeny.

Salmonella Infantis, a common contaminant of poultry products, is known to harbor mobile genetic elements that confer multi-drug resistance (MDR) and have been detected in many continents. Here, we report four MDR S . Infantis strains recovered from poultry house environments in Santa Cruz Island of the Galapagos showing extended-spectrum β-lactamase (ESBL) resistance and reduced fluoroquinolone susceptibility. Whole-genome sequencing (WGS) revealed the presence of the ESBL-conferring bla CTX-M-65 gene in an IncFIB-like plasmid in three S . Infantis isolates. Multi-locus sequence typing (MLST) and single nucleotide variant/polymorphism (SNP) SNVPhyl analysis showed that the S . Infantis isolates belong to sequence type ST32, likely share a common ancestor, and are closely related (1-3 SNP difference) to bla CTX-M-65 -containing clinical and veterinary S . Infantis isolates from the United States and Latin America. Furthermore, phylogenetic analysis of SNPs following core-genome alignment (i.e., ParSNP) inferred close relatedness between the S . Infantis isolates from Galapagos and the United States. Prophage typing confirmed the close relationship among the Galapagos S . Infantis and was useful in distinguishing them from the United States isolates. This is the first report of MDR bla CTX-M-65 -containing S . Infantis in the Galapagos Islands and highlights the need for increased monitoring and surveillance programs to determine prevalence, sources, and reservoirs of MDR pathogens.

In recent years, Salmonella Infantis has become a predominant serovariant in clinical and poultry isolates, thereby imposing a substantial economic burden on both public health and the livestock industry. With the aim of coping with the steep increase in serovar Infantis prevalence, a polymerase chain reaction (PCR)-based rapid and accurate diagnostic assay was developed in this study through pangenome profiling of 60 Salmonella serovars. A gene marker, SIN&#95;02055, was identified, which is present in the S . Infantis genome but not in the pangenome of the other serovars. Primers specific to SIN&#95;02055 were used to accurately detect serovar Infantis, and to successfully differentiate Infantis from the other 59 serovars in real-time PCR with a R 2 of 0.999 and an efficiency of 95.76%. The developed method was applied to 54 Salmonella strains belonging to eight dominant serovars, and distinguished Infantis from the other seven serovars with an accuracy of 100%. The diagnostic primer set also did not show false positive amplification with 32 strains from eight non- Salmonella bacterial species. This cost-effective and rapid method can be considered an alternative to the classic serotyping using antisera.

This work aimed to evaluate the antimicrobial susceptibility of 87 Salmonella Infantis strains isolated in Italy from 2016 to 2019 along the food chain of broiler meat production and in humans and to determine the genetic profiles of the strains in order to establish a possible correlation with the antimicrobial pattern. All isolates were tested by the disk diffusion method to evaluate antimicrobial susceptibility toward sixteen antimicrobials, and the broth microdilution method was used to confirm extended spectrum β-lactamase (ESBL) production. PCR and pulsed field gel electrophoresis (PFGE) were applied to characterize ESBL-encoding and AmpC β-lactamase genes and to analyze the S . Infantis strains genetic profiles respectively. S . Infantis isolates showed high prevalence of resistance, in particular toward nalidixic acid (97.7%), tetracycline (96.5%), sulphamethoxazole/trimethoprim (91%) and cefepime (72.4%). The 80.5% of isolates were ESBL, cefotaxime-resistant, carrying the bla CTX-M1 gene. The most prevalent PFGE profile was XbaI.0126 (35.6%). The remaining strains had a genetic homology from 81% to 97% with the XbaI.0126 profile. The strains belonging to these profiles were isolated from different matrices collected along the broiler food chain independently on the year and from the region and there was no correlation between the PFGE profiles and resistance patterns. We found two ESBL-producing S. Infantis strains with the same XbaI.2621 profile isolated from humans and from poultry feces, not yet reported in Italy. Our findings confirmed the diffusion of ESBL-multi drug resistant (MDR) S . Infantis along the broiler food chain and in humans and underlined the importance of continuous monitoring to control and to reduce the prevalence of this bacterium, applying a global One Health approach.

Salmonella is important as both a cause of clinical disease in swine and as a source of food-borne transmission of disease to humans. Lactobacillus and Bacillus are often used as antibiotic substitutes to prevent Salmonella infection. In this study, we evaluated the effects of a select mixture of Lactobacillus johnsonii L531, Bacillus licheniformis BL1721 and Bacillus subtilis BS1715 (LBB-mix) in prevention of Salmonella enterica serovar Infantis infection in a pig model. LBB-mix was orally administered to newly weaned piglets for seven days before S . Infantis challenge. LBB-mix pretreatment ameliorated S . Infantis-induced fever, leukocytosis, growth performance loss, and ileal inflammation. Pre-administration of LBB-mix reduced the number of Salmonella in the feces but increased the number of goblet cells in the ileum. S . Infantis infection resulted in an increase in cell death in the ileum, this increase was attenuated by LBB-mix consumption. Claudin 1 and cleaved caspase-1 expression was decreased in the ileum of pigs challenged with S . Infantis, but not in pigs pretreated with LBB-mix. In conclusion, our data indicate that a select LBB-mix has positive effects on controlling S . Infantis infection via alleviating inflammation and maintaining the intestinal mucosal barrier integrity in pigs.