In silico and PCR Screening for a Live Attenuated Salmonella Typhimurium Vaccine Strain.

The application of live attenuated Salmonella Typhimurium vaccines has significantly helped control Salmonella in poultry products. Because the U.S. Department of Agriculture-Food Safety Inspection Service (USDA-FSIS) scores all Salmonella as positive, regardless of serovar, attenuated vaccine strains that are identified at processing contribute negatively toward Salmonella performance standards. This study was designed to determine the incidence of a live attenuated Salmonella serovar Typhimurium vaccine identified in broiler products by FSIS and to develop a PCR assay for screening of isolates. Salmonella Typhimurium short-read sequences from broiler samples uploaded to the National Center for Biotechnology Information (NCBI) Pathogen Detection database by the USDA-FSIS from 2016 to 2022 were downloaded and assembled. These were analyzed using the Basic Local Alignment Search Tool (BLAST) with a sequence unique to field strains, followed by a sequence unique to the vaccine strain. The PCR assays were developed against field and vaccine strains by targeting transposition events in the crp and cya genes and validated by screening Salmonella serovar Typhimurium isolates. Between 2016 and 2022, 1708 Salmonella Typhimurium isolates of chicken origin were found in the NCBI Pathogen Detection database, corresponding to 7.99% of all Salmonella identified. Of these, 104 (5.97%) were identified as the vaccine strain. The PCR assay differentiated field strains from the vaccine strain when applied to isolates and was also able to detect the vaccine strain from DNA isolated from mixed serovar overnight Salmonella enrichment cultures. Live attenuated Salmonella vaccines are a critical preharvest tool for Salmonella control and are widely used in industry. With forthcoming regulations that will likely focus on Salmonella Typhimurium, along with other serovars, there is a need to distinguish between isolates belonging to the vaccine strain and those that are responsible for causing human illness.

Detección in silico y por PCR de una cepa vacunal viva atenuada de Salmonella Typhimurium. La aplicación de vacunas vivas atenuadas contra Salmonella Typhimurium ha ayudado significativamente a controlar Salmonella en productos avícolas. Debido a que el Servicio de Inspección de Seguridad Alimentaria del Departamento de Agricultura de los Estados Unidos. (USDA-FSIS) califica todas las Salmonella como positivas, independientemente del serovar. Las cepas atenuadas de la vacuna que se identifican en el procesamiento contribuyen negativamente a los estándares de desempeño de Salmonella. Este estudio fue diseñado para determinar la incidencia de una vacuna viva atenuada de Salmonella serovar Typhimurium identificada en productos de pollo de engorde por el FSIS y para desarrollar un ensayo de PCR para la detección de aislados. Se recolectaron y ensamblaron secuencias de lectura corta de Salmonella Typhimurium de muestras de pollos de engorde introducidas en la plataforma de detección de patógenos del Centro Nacional de Información Biotecnológica (NCBI) por el USDA-FSIS entre los años 2016 al 2022. Estos se analizaron utilizando la herramienta de búsqueda de alineación local básica con una secuencia exclusiva para las cepas de campo, seguida de una secuencia exclusiva para la cepa vacunal. Los ensayos de PCR se desarrollaron contra cepas de campo y vacunales centrándose en eventos de transposición en los genes crp y cya y se validaron mediante la detección de aislados de Salmonella serovar Typhimurium. Entre 2016 y 2022, se encontraron 1708 aislados de Salmonella Typhimurium de origen avícola en el sistema de detección de patógenos del NCBI, lo que corresponde al 7.99 % de todas las Salmonellas identificadas. De ellas, 104 (5.97%) fueron identificadas como cepa vacunal. El ensayo de PCR diferenció las cepas de campo de la cepa de la vacuna cuando se aplicó a los aislados y también fue capaz de detectar la cepa de la vacuna a partir del ADN aislado de cultivos de enriquecimiento por toda la noche de Salmonella con serovares mixtos. Las vacunas vivas atenuadas contra Salmonella son una herramienta fundamental para el control de Salmonella y se utilizan ampliamente en la industria. Con las próximas regulaciones que probablemente se centrarán en Salmonella Typhimurium, junto con otros serovares, es necesario distinguir entre los aislados que pertenecen a la cepa vacunal y los que son responsables de causar enfermedades humanas.

Growth assessment of Salmonella enterica multi-serovar populations in poultry rinsates with commonly used enrichment and plating media.

Isolation of Salmonella from enrichment cultures of food or environmental samples is a complicated process. Numerous factors including fitness in various selective enrichment media, relative starting concentrations in pre-enrichment, and competition among multi-serovar populations and associated natural microflora, come together to determine which serovars are identified from a given sample. A recently developed approach for assessing the relative abundance (RA) of multi-serovar Salmonella populations (CRISPR-SeroSeq or Deep Serotyping, DST) is providing new insight into how these factors impact the serovars observed, especially when different selective enrichment methods are used to identify Salmonella from a primary enrichment sample. To illustrate this, we examined Salmonella-positive poultry pre-enrichment samples through the selective enrichment process in Tetrathionate (TT) and Rappaport Vassiliadis (RVS) broths and assessed recovery of serovars with each medium. We observed the RA of serovars detected post selective enrichment varied depending on the medium used, initial concentration, and competitive fitness factors, all which could result in minority serovars in pre-enrichment becoming dominant serovars post selective enrichment. The data presented provide a greater understanding of culture biases and lays the groundwork for investigations into robust enrichment and plating media combinations for detecting Salmonella serovars of greater concern for human health.

Poultry Processing Interventions Reduce Salmonella Serovar Complexity on Postchill Young Chicken Carcasses as Determined by Deep Serotyping.

Nearly 20% of salmonellosis cases are attributed to broilers, with renewed efforts to reduce Salmonella during broiler production and processing. A limitation to Salmonella culture is that often a single colony is picked for characterization, favoring isolation of the most abundant serovar found in a sample, while low abundance serovars can remain undetected. We used a deep serotyping approach, CRISPR-SeroSeq (serotyping by sequencing the clustered regularly interspaced palindromic repeats), to assess Salmonella serovar complexity during broiler processing and to determine the impact of antimicrobial interventions upon serovar population dynamics. Paired hot rehang and postchill young chicken carcasses were collected from establishments across the United States from August to November 2022. CRISPR-SeroSeq was performed on Salmonella culture-positive hot rehang (n = 153) and postchill (n = 38) samples, including 31 paired hot rehang and postchill samples. Multiple serovars were detected in 48.4% (74/153) and 7.9% (3/38) of hot rehang and postchill samples, respectively. On average, hot rehang carcasses contained 1.6 serovars, compared to 1.1 serovars at postchill (Mann Whitney U, p = 0.00018). Nineteen serovars were identified with serovar Kentucky the most common at hot rehang (72.5%; 111/153) and postchill (73.7%; 28/38). Serovar Infantis prevalence was higher at hot rehang (39.9%; 61/153) than in postchill (7.9%; 3/38). At hot rehang, serovar Enteritidis was outnumbered by other serovars 81.3% (13/16) of the time but was always the single or most abundant serovar detected when it was present at postchill (n = 5). We observed 98.4% (188/191) concordance between traditional isolation with serotyping and CRISPR-SeroSeq. Deep serotyping was able to explain serovar discrepancies between paired hot rehang and postchill samples when only traditional isolation and serotyping methods were used. These data demonstrate that processing interventions are effective in reducing Salmonella serovar complexity.

Combined Quantification and Deep Serotyping for Salmonella Risk Profiling in Broiler Flocks.

Despite a reduction of Salmonella contamination on final poultry products, the level of human salmonellosis cases attributed to poultry has remained unchanged over the last few years. There needs to be improved effort to target serovars which may survive antimicrobial interventions and cause illness, as well as to focus on lessening the amount of contamination entering the processing plant. Advances in molecular enumeration approaches allow for the rapid detection and quantification of Salmonella in pre- and postharvest samples, which can be combined with deep serotyping to properly assess the risk affiliated with a poultry flock. In this study, we collected a total of 160 boot sock samples from 20 broiler farms across four different integrators with different antibiotic management programs. Overall, Salmonella was found in 85% (68/80) of the houses, with each farm having at least one Salmonella-positive house. The average Salmonella quantity across all four complexes was 3.6 log10 CFU/sample. Eleven different serovars were identified through deep serotyping, including all three key performance indicators (KPIs; serovars Enteritidis, Infantis, and Typhimurium) defined by the U.S. Department of Agriculture-Food Safety and Inspection Service (USDA-FSIS). There were eight multidrug resistant isolates identified in this study, and seven which were serovar Infantis. We generated risk scores for each flock based on the presence or absence of KPIs, the relative abundance of each serovar as calculated with CRISPR-SeroSeq (serotyping by sequencing the clustered regularly interspaced palindromic repeats), and the quantity of Salmonella organisms detected. The work presented here provides a framework to develop directed processing approaches and highlights the limitations of conventional Salmonella sampling and culturing methods. IMPORTANCE Nearly one in five foodborne Salmonella illnesses are derived from chicken, making it the largest single food category to cause salmonellosis and indicating a need for effective pathogen mitigation. Although industry has successfully reduced Salmonella incidence in poultry products, there has not been a concurrent reduction in human salmonellosis linked to chicken consumption. New efforts are focused on improved control at preharvest, which requires improved Salmonella surveillance. Here, we present a high-resolution surveillance approach that combines quantity and identity of Salmonella in broiler flocks prior to processing which will further support improved Salmonella controls in poultry. We developed a framework for this approach, indicating that it is possible and important to harness deep serotyping and molecular enumeration to inform on-farm management practices and to minimize risk of cross-contamination between flocks at processing. Additionally, this framework could be adapted to Salmonella surveillance in other food animal production systems.

Regional Salmonella Differences in United States Broiler Production from 2016 to 2020 and the Contribution of Multiserovar Populations to Salmonella Surveillance.

Poultry remains a considerable source of foodborne salmonellosis despite significant reduction of Salmonella incidence during processing. There are multiple entry points for Salmonella during production that can lead to contamination during slaughter, and it is important to distinguish the serovars present between the different stages to enact appropriate controls. National Salmonella data from the U.S. Department of Agriculture-Food Safety Inspection Service (USDA-FSIS) monitoring of poultry processing was analyzed from 2016 to 2020. The overall Salmonella incidence at processing in broiler carcasses and intact parts (parts) decreased from 9.00 to 6.57% over this period. The incidence in parts was higher (11.15%) than in carcasses (4.78%). Regional differences include higher proportions of serovars Infantis and Typhimurium in the Atlantic and higher proportion of serovar Schwarzengrund in the Southeast. For Georgia, the largest broiler-producing state, USDA-FSIS data were compared to Salmonella monitoring data from breeder flocks over the same period, revealing serovar Kentucky as the major serovar in breeders (67.91%) during production but not at processing, suggesting that it is more effectively removed during antimicrobial interventions. CRISPR-SeroSeq was performed on breeder samples collected between 2020 and 2021 to explain the incongruence between pre- and postharvest and showed that 32% of samples contain multiple serovars, with up to 11 serovars found in a single flock. High-resolution sequencing identifies serovar patterns at the population level and can provide insight to develop targeted controls. The work presented may apply to other food production systems where Salmonella is a concern, since it overcomes limitations associated with conventional culture. IMPORTANCE Salmonella is a leading cause of bacterial foodborne illness in the United States, with poultry as a significant Salmonella reservoir. We show the relative decrease in Salmonella over a 5-year period from 2016 to 2020 in processed chicken parts and highlight regional differences with respect to the prevalence of clinically important Salmonella serovars. Our results show that the discrepancy between Salmonella serovars found in pre- and postharvest poultry during surveillance are due in part by the limited detection depth offered by traditional culture techniques. Despite the reduction of Salmonella at processing, the number of human salmonellosis cases has remained stable, which may be attributed to differences in virulence among serovars and their associated risk. When monitoring for Salmonella, it is imperative to identify all serovars present to appropriately assess public health risk and to implement the most effective Salmonella controls.

Antimicrobial Resistance Hidden within Multiserovar Salmonella Populations.

Salmonella enterica can exist in food animals as multiserovar populations, and different serovars can harbor diverse antimicrobial resistance (AMR) profiles. Conventional Salmonella isolation assesses AMR only in the most abundant members of a multiserovar population, which typically reflects their relative abundance in the initial sample. Therefore, AMR in underlying serovars is an undetected reservoir that can readily be expanded upon antimicrobial use. CRISPR-SeroSeq profiling demonstrated that 60% of cattle fecal samples harbored multiple serovars, including low levels of Salmonella serovar Reading in 11% of samples, which were not found by culture-based Salmonella isolation. An in vitro challenge revealed that Salmonella serovar Reading was tetracycline resistant, while more abundant serovars were susceptible. This study highlights the importance of AMR surveillance in multiserovar populations.