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.

Genomic and phenotypic characterization of Salmonella enterica serovar Kentucky.

Non-typhoidal Salmonella are extremely diverse and different serovars can exhibit varied phenotypes, including host adaptation and the ability to cause clinical illness in animals and humans. In the USA, Salmonella enterica serovar Kentucky is infrequently found to cause human illness, despite being the top serovar isolated from broiler chickens. Conversely, in Europe, this serovar falls in the top 10 serovars linked to human salmonellosis. Serovar Kentucky is polyphyletic and has two lineages, Kentucky-I and Kentucky-II; isolates belonging to Kentucky-I are frequently isolated from poultry in the USA, while Kentucky-II isolates tend to be associated with human illness. In this study, we analysed whole-genome sequences and associated metadata deposited in public databases between 2017 and 2021 by federal agencies to determine serovar Kentucky incidence across different animal and human sources. Of 5151 genomes, 90.3 % were from isolates that came from broilers, while 5.9 % were from humans and 3.0 % were from cattle. Kentucky-I isolates were associated with broilers, while isolates belonging to Kentucky-II and a new lineage, Kentucky-III, were more commonly associated with cattle and humans. Very few serovar Kentucky isolates were associated with turkey and swine sources. Phylogenetic analyses showed that Kentucky-III genomes were more closely related to Kentucky-I, and this was confirmed by CRISPR-typing and multilocus sequence typing (MLST). In a macrophage assay, serovar Kentucky-II isolates were able to replicate over eight times better than Kentucky-I isolates. Analysis of virulence factors showed unique patterns across these three groups, and these differences may be linked to their association with different hosts.

Buffered Peptone Water Formulation Does Not Influence Growth of pESI-positive Salmonella enterica Serovar Infantis.

Salmonella enterica is a major cause of human foodborne illness and is often attributed to poultry food sources. S. enterica serovar Infantis, specifically those carrying the pESI plasmid, has become a frequently isolated serotype from poultry meat samples at processing and has caused numerous recent human infections. In 2016, the USDA-Food Safety and Inspection Service changed the official sampling method for raw poultry products from BPW to using neutralizing BPW (nBPW) as the rinsing agent in order to prevent residual antimicrobial effects from acidifying and oxidizing processing aids. This change was contemporaneous to the emergence of pESI-positive ser. Infantis as a prevalent serovar in poultry, prompting some to question if nBPW could be selecting for this prevalent serovar. We performed two experiments: a comparison of ser. Infantis growth in BPW versus nBPW, and a simulation of regulatory sampling methods. We found that when inoculated into both broths, ser. Infantis initially grows slightly slower in nBPW than in BPW but little difference was seen in abundance after 6 h of growth. Additionally, the use of nBPW to simulate poultry rinse sample and overnight cold shipping to a regulatory lab did not affect the survival or subsequent growth of ser. Infantis in BPW. We concluded that the change in USDA-FSIS methodology to include nBPW in sampling procedures has likely not affected the emergence of S. ser. Infantis as a prevalent serovar in chicken and turkey meat product samples.

Differences in biofilm formation of Salmonella serovars on two surfaces under two temperature conditions.

AIMS: Salmonella is extremely diverse, with >2500 serovars that are genetically and phenotypically diverse. The aim of this study was to build a collection of Salmonella isolates that are genetically diverse and to evaluate their ability to form biofilm under different conditions relevant to a processing environment. METHODS AND RESULTS: Twenty Salmonella isolates representative of 10 serovars were subtyped using Clustered regularly interspaced short palindromic repeats (CRISPR)-typing to assess the genetic diversity between isolates of each serovar. Biofilm formation of the isolates on both plastic and stainless-steel surfaces at 25 and 15°C was assessed. At 25°C, 8/20 isolates each produced strong and moderate biofilm on plastic surface compared to stainless-steel (3/20 and 13/20 respectively). At 15°C, 5/20 produced strong biofilm on plastic surface and none on stainless-steel. Several isolates produced weak biofilm on plastic (11/20) and stainless-steel (16/20) surfaces. Serovar Schwarzengrund consistently produced strong biofilm while serovars Heidelberg and Newport produced weak biofilm. CONCLUSION: These results suggest that Salmonellae differ in their attachment depending on the surface and temperature conditions encountered, which may influence persistence in the processing environment. SIGNIFICANCE AND IMPACT OF STUDY: These differences in biofilm formation could provide useful information for mitigation of Salmonella in processing environments.