Genomic characterisation of the population structure and antibiotic resistance of Salmonella enterica serovar Infantis in Chile, 2009-2022.

Background: Multidrug-resistant (MDR) Salmonella Infantis has disseminated worldwide, mainly linked to the consumption of poultry products. Evidence shows dissemination of this pathogen in Chile; however, studies are primarily limited to phenotypic data or involve few isolates. As human cases of Salmonella Infantis infections have substantially increased in recent years, this study aimed to characterise the genomic epidemiology and antimicrobial-resistance profiles of isolates obtained from different sources, aiming to inform effective surveillance and control measures. Methods: We sequenced 396 Salmonella Infantis genomes and analysed them with all publicly available genomes of this pathogen from Chile (440 genomes in total), representing isolates from environmental, food, animal, and human sources obtained from 2009 to 2022. Based on bioinformatic and phenotypic methods, we assessed the population structure, dissemination among different niches, and antimicrobial resistance (AMR) profiles of Salmonella Infantis in the country. Findings: The genomic and phylogenetic analyses showed that Salmonella Infantis from Chile comprised several clusters of highly related isolates dominated by sequence type 32. The HC20_343 cluster grouped an important proportion of all isolates. This was the only cluster associated with pESI-like megaplasmids, and up to 12 acquired AMR genes/mutations predicted to result in an MDR phenotype. Accordingly, antimicrobial-susceptibility testing revealed a strong concordance between the AMR genetic determinants and their matching phenotypic expression, indicating that a significant proportion of HC20_343 isolates produce extended-spectrum ß-lactamases and have intermediate fluoroquinolone resistance. HC20_343 Salmonella Infantis were spread among environmental, animal, food, and human niches, showing a close relationship between isolates from different years and sources, and a low intra-source genomic diversity. Interpretation: Our findings show a widespread dissemination of MDR Salmonella Infantis from the HC20_343 cluster in Chile. The high proportion of isolates with resistance to first-line antibiotics and the evidence of active transmission between the environment, animals, food, and humans highlight the urgency of improved surveillance and control measures in the country. As HC20_343 isolates predominate in the Americas, our results suggest a high prevalence of ESBL-producing Salmonella Infantis with intermediate fluoroquinolone resistance in the continent. Funding: Partially supported by the Food and Drug Administration (FDA) of the U.S. Department of Health and Human Services as part of an award, FDU001818, with 30% percent funded by FDA/HHS; and by Agencia de Investigación y Desarrollo de Chile (ANID) through FONDECYT de Postdoctorado Folio 3230796 and Folio 3210317, FONDECYT Regular Folio 1231082, and ANID-Millennium Science Initiative Program-ICN2021_044.

A review of the global emergence of multidrug-resistant Salmonella enterica subsp. enterica Serovar Infantis.

Salmonella enterica serovar Infantis is an emergent foodborne and zoonotic Salmonella serovar with critical implications for global health. In recent years, the prevalence of S. Infantis infections has increased in the United States, Europe, and Latin America, due to contaminated chicken and other foods. An essential trait of S. Infantis is its resistance to multiple antibiotics, including the critically important third-generation cephalosporins and quinolones, undermining effective medical treatment, particularly in low-resource settings. We describe the emergence of multidrug-resistant (MDR) S. Infantis, focusing on humans, animals, the environment, and food. We conducted a systematic review (1979-2021), selected 183 studies, and analyzed the origin, source, antimicrobial resistance, and presence of a conjugative plasmid of emerging S. Infantis (pESI) in reported isolates. S. Infantis has been detected worldwide, with a substantial increase since 2011. We found the highest number of isolations in the Americas (42.9 %), Europe (29.8 %), Western Pacific (17.2 %), Eastern Mediterranean (6.6 %), Africa (3.4 %), and South-East Asia (0.1 %). S. Infantis showed MDR patterns and numerous resistant genes in all sources. The primary source of MDR S. Infantis is broiler and their meat; however, this emerging pathogen is also present in other reservoirs such as food, wildlife, and the environment. Clinical cases of MDR S. Infantis have been reported in children and adults. The global emergence of S. Infantis is related to a plasmid (pESI) with antibiotic and arsenic- and mercury-resistance genes. Additionally, a new megaplasmid (pESI-like), carrying blaCTX-M-65 and antibiotic-resistant genes reported in an ancestral version, was detected in the broiler, human, and chicken meat isolates. Strains harboring pESI-like were primarily observed in the Americas and Europe. MDR S. Infantis has spread globally, potentially becoming a major public health threat, particularly in low- and middle-income countries.

Long-Term Interactions of Salmonella Enteritidis With a Lytic Phage for 21 Days in High Nutrients Media.

Salmonella spp. is a relevant foodborne pathogen with worldwide distribution. To mitigate Salmonella infections, bacteriophages represent an alternative to antimicrobials and chemicals in food animals and food in general. Bacteriophages (phages) are viruses that infect bacteria, which interact constantly with their host. Importantly, the study of these interactions is crucial for the use of phages as a mitigation strategy. In this study, experimental coevolution of Salmonella Enteritidis (S. Enteritidis) and a lytic phage was conducted in tryptic soy broth for 21 days. Transfer to fresh media was conducted daily and every 24 hours, 2 mL of the sample was collected to quantify Salmonella OD600 and phage titter. Additionally, time-shift experiments were conducted on 20 colonies selected on days 1, 12, and 21 to evaluate the evolution of resistance to past (day 1), present (day 12), and future (day 21) phage populations. The behavior of the dynamics was modeled and simulated with mathematical mass-action models. Bacteria and phage from days 1 and 21 were sequenced to determine the emergence of mutations. We found that S. Enteritidis grew for 21 days in the presence and absence of the phage and developed resistance to the phage from day 1. Also, the phage was also able to survive in the media for 21 days, however, the phage titer decreased in approx. 3 logs PFU/mL. The stability of the lytic phage population was consistent with the leaky resistance model. The time-shift experiments showed resistance to phages from day 1 of at least 85% to the past, present, and future phages. Sequencing of S. Enteritidis showed mutations in genes involved in lipopolysaccharide biosynthesis genes rfbP and rfbN at day 21. The phage showed mutations in the tail phage proteins responsible for recognizing the cell surface receptors. These results suggest that interactions between bacteria and phage in a rich resource media generate a rapid resistance to the infective phage but a fraction of the population remains susceptible. Interactions between Salmonella and lytic phages are an important component for the rational use of phages to control this important foodborne pathogen.

Isolation and engineering of a Listeria grayi bacteriophage.

The lack of bacteriophages capable of infecting the Listeria species, Listeria grayi, is academically intriguing and presents an obstacle to the development of bacteriophage-based technologies for Listeria. We describe the isolation and engineering of a novel L. grayi bacteriophage, LPJP1, isolated from farm silage. With a genome over 200,000 base pairs, LPJP1 is the first and only reported jumbo bacteriophage infecting the Listeria genus. Similar to other Gram-positive jumbo phages, LPJP1 appeared to contain modified base pairs, which complicated initial attempts to obtain genomic sequence using standard methods. Following successful sequencing with a modified approach, a recombinant of LPJP1 encoding the NanoLuc luciferase was engineered using homologous recombination. This luciferase reporter bacteriophage successfully detected 100 stationary phase colony forming units of both subspecies of L. grayi in four hours. A single log phase colony forming unit was also sufficient for positive detection in the same time period. The recombinant demonstrated complete specificity for this particular Listeria species and did not infect 150 non-L. grayi Listeria strains nor any other bacterial genus. LPJP1 is believed to be the first reported lytic bacteriophage of L. grayi as well as the only jumbo bacteriophage to be successfully engineered into a luciferase reporter.

Comparative Analysis of Felixounavirus Genomes Including Two New Members of the Genus That Infect Salmonella Infantis.

Salmonella spp. is one of the most common foodborne pathogens worldwide; therefore, its control is highly relevant for the food industry. Phages of the Felixounavirus genus have the characteristic that one phage can infect a large number of different Salmonella serovars and, thus, are proposed as an alternative to antimicrobials in food production. Here, we describe two new members of the Felixounavirus genus named vB_Si_35FD and vB_Si_DR94, which can infect Salmonella Infantis. These new members were isolated and sequenced, and a subsequent comparative genomic analysis was conducted including 23 publicly available genomes of Felixounaviruses that infect Salmonella. The genomes of vB_Si_35FD and vB_Si_DR94 are 85,818 and 85,730 bp large and contain 129 and 125 coding sequences, respectively. The genomes did not show genes associated with virulence or antimicrobial resistance, which could be useful for candidates to use as biocontrol agents. Comparative genomics revealed that closely related Felixounavirus are found in distinct geographical locations and that this genus has a conserved genomic structure despite its worldwide distribution. Our study revealed a highly conserved structure of the phage genomes, and the two newly described phages could represent promising biocontrol candidates against Salmonella spp. from a genomic viewpoint.