Through the Looking Glass: Genome, Phenome, and Interactome of Salmonella enterica.

This review revisits previous concepts on biological phenomenon contributing to the success of the Salmonella enterica subspecies I as a pathogen and expands upon them to include progress in epidemiology based on whole genome sequencing (WGS). Discussion goes beyond epidemiological uses of WGS to consider how phenotype, which is the biological character of an organism, can be correlated with its genotype to develop a knowledge of the interactome. Deciphering genome interactions with proteins, the impact of metabolic flux, epigenetic modifications, and other complex biochemical processes will lead to new therapeutics, control measures, environmental remediations, and improved design of vaccines.

Tissue Colonization and Egg and Environmental Contamination Associated with the Experimental Infection of Cage-Free Laying Hens with Salmonella Braenderup.

In 2018, a national recall of shell eggs in the United States occurred due to human illness caused by Salmonella Braenderup. Although previous studies have identified Salmonella Braenderup in laying hens and the production environment, little is known about the ability of this Salmonella serovar to infect laying hens and contaminate eggs. The objective of this study was to examine the invasiveness of Salmonella Braenderup in laying hens as well as its ability to persist in the production environment. Specific-pathogen-free laying hens (four trials; 72 hens/trial) were orally challenged with 107 colony-forming units of Salmonella Braenderup. On day 6 postinoculation, half of the challenged hens were euthanatized, and samples of ileocecal junction (sections above and below it, and portions of both ceca), liver, spleen, ovary, and oviduct tissues were collected and cultured for Salmonella Braenderup. Egg and environmental (nest box swaps and substrate (litter)) samples were collected days 7-20 postinoculation (Trials 1 and 2; excluding weekends) and days 7-27 postinoculation (Trials 3 and 4; excluding weekends) to detect Salmonella Braenderup. Recovery of Salmonella Braenderup was highest in ileocecal tissue samples (11.1%-33.3%; P < 0.05), with little to no recovery in other collected tissue samples. Salmonella Braenderup was detected in a small number of shell emulsions (0%-2.9%; P < 0.01) and recovered in Trial 1 at a high rate (92.5%; P < 0.0001) in the substrate composite samples; however, recovery of Salmonella Braenderup was low in the other egg and environmental samples. These trials indicate that Salmonella Braenderup is not an invasive Salmonella serovar for cage-free laying hens, especially when compared to serovars of concern to the egg industry. However, it may persist in the environment at low levels.

Colonización de tejidos y contaminación ambiental y de huevo asociados con la infección experimental de gallinas de postura libres de jaulas por Salmonella Braenderup. En 2018, se retiraron del mercado a nivel nacional en los Estados Unidos huevos con cascarón debido a una enfermedad en humanos causada por Salmonella Braenderup. Aunque estudios anteriores han identificado Salmonella Braenderup en gallinas de postura y en ambientes de producción, se conoce poco sobre la capacidad de esta serovariedad de Salmonella para infectar a las gallinas ponedoras y contaminar el huevo. El objetivo de este estudio fue examinar la capacidad de invasión de Salmonella Braenderup en gallinas ponedoras, así como su capacidad para persistir en el ambiente de producción. Se desafiaron oralmente a gallinas de postura libres de patógenos específicos (cuatro ensayos; 72 gallinas/ensayo) con 107 unidades formadoras de colonias de Salmonella Braenderup. El día seis después de la inoculación, la mitad de las gallinas desafiadas se sacrificaron y se recolectaron y cultivaron muestras de la unión ileocecal (secciones anteriores y posteriores de la misma y porciones de ambos ciegos), hígado, bazo, ovario y oviducto y se cultivaron para Salmonella Braenderup. Se recolectaron muestras de huevos y ambientales (hisopos de las cajas de nido y sustrato [cama] en los días 7 a 20 después de la inoculación (Pruebas 1 y 2; excluyendo los fines de semana) y en los días 7 a 27 después de la inoculación (Pruebas 3 y 4; excluyendo los fines de semana) para detectar Salmonella Braenderup. La recuperación de Salmonella Braenderup fue mayor en las muestras de tejido ileocecal (11.1%­33.3%; P < 0.05), con poca o ninguna recuperación en otras muestras de tejido recolectadas. Se detectó Salmonella Braenderup en un pequeño número de emulsiones de cascarones (0%­2.9%; P < 0.01) y se recuperó en el Ensayo 1 a una tasa alta (92.5%; P < 0.0001) en las muestras compuestas de sustrato; sin embargo, la recuperación de Salmonella Braenderup fue baja en las otras muestras de huevos y ambientales. Estos ensayos indican que Salmonella Braenderup no es un serovar de Salmonella invasivo para gallinas de postura sin jaulas, especialmente cuando se compara con los serovares de interés para la industria del huevo. Sin embargo, puede persistir en el medio ambiente en niveles bajos.

Serotype Screening of Salmonella enterica Subspecies I by Intergenic Sequence Ribotyping (ISR): Critical Updates.

(1) Background: Foodborne illness from Salmonella enterica subspecies I is most associated with approximately 32 out of 1600 serotypes. While whole genome sequencing and other nucleic acid-based methods are preferred for serotyping, they require expertise in bioinformatics and often submission to an external agency. Intergenic Sequence Ribotyping (ISR) assigns serotype to Salmonella in coordination with information freely available at the National Center for Biotechnology Information. ISR requires updating because it was developed from 26 genomes while there are now currently 1804 genomes and 1685 plasmids. (2) Methods: Serotypes available for sequencing were analyzed by ISR to confirm primer efficacy and to identify any issues in application. Differences between the 2012 and 2022 ISR database were tabulated, nomenclature edited, and instances of multiple serotypes aligning to a single ISR were examined. (3) Results: The 2022 ISR database has 268 sequences and 40 of these were assigned new NCBI accession numbers that were not previously available. Extending boundaries of sequences resolved hdfR cross-alignment and reduced multiplicity of alignment for 37 ISRs. Comparison of gene cyaA sequences and some cell surface epitopes provided evidence that homologous recombination was potentially impacting results for this subset. There were 99 sequences that still had no match with an NCBI submission. (4) The 2022 ISR database is available for use as a serotype screening method for Salmonella enterica subspecies I. Finding that 36.9% of the sequences in the ISR database still have no match within the NCBI Salmonella enterica database suggests that there is more genomic heterogeneity yet to characterize.

AT Homopolymer Strings in Salmonella enterica Subspecies I Contribute to Speciation and Serovar Diversity.

Adenine and thymine homopolymer strings of at least 8 nucleotides (AT 8+mers) were characterized in Salmonella enterica subspecies I. The motif differed between other taxonomic classes but not between Salmonella enterica serovars. The motif in plasmids was possibly associated with serovar. Approximately 12.3% of the S. enterica motif loci had mutations. Mutability of AT 8+mers suggests that genomes undergo frequent repair to maintain optimal gene content, and that the motif facilitates self-recognition; in addition, serovar diversity is associated with plasmid content. A theory that genome regeneration accounts for both persistence of predominant Salmonella serovars and serovar diversity provides a new framework for investigating root causes of foodborne illness.

Horizontal Gene Transfer Is the Main Driver of Antimicrobial Resistance in Broiler Chicks Infected with Salmonella enterica Serovar Heidelberg.

The overuse and misuse of antibiotics in clinical settings and in food production have been linked to the increased prevalence and spread of antimicrobial resistance (AR). Consequently, public health and consumer concerns have resulted in a remarkable reduction in antibiotics used for food animal production. However, there are no data on the effectiveness of antibiotic removal in reducing AR shared through horizontal gene transfer (HGT). In this study, we used neonatal broiler chicks and Salmonella enterica serovar Heidelberg, a model food pathogen, to test if chicks raised antibiotic free harbor transferable AR. We challenged chicks with an antibiotic-susceptible S. Heidelberg strain using various routes of inoculation and determined if S. Heidelberg isolates recovered carried plasmids conferring AR. We used antimicrobial susceptibility testing and whole-genome sequencing (WGS) to show that chicks grown without antibiotics harbored an antimicrobial resistant S. Heidelberg population at 14 days after challenge and chicks challenged orally acquired AR at a higher rate than chicks inoculated via the cloaca. Using 16S rRNA gene sequencing, we found that S. Heidelberg infection perturbed the microbiota of broiler chicks, and we used metagenomics and WGS to confirm that a commensal Escherichia coli population was the main reservoir of an IncI1 plasmid acquired by S. Heidelberg. The carriage of this IncI1 plasmid posed no fitness cost to S. Heidelberg but increased its fitness when exposed to acidic pH in vitro. These results suggest that HGT of plasmids carrying AR shaped the evolution of S. Heidelberg and that antibiotic use reduction alone is insufficient to limit antibiotic resistance transfer from commensal bacteria to Salmonella enterica. IMPORTANCE The reported increase in antibiotic-resistant bacteria in humans has resulted in a major shift away from antibiotic use in food animal production. This shift has been driven by the assumption that removing antibiotics will select for antibiotic susceptible bacterial taxa, which in turn will allow the currently available antibiotic arsenal to be more effective. This change in practice has highlighted new questions that need to be answered to assess the effectiveness of antibiotic removal in reducing the spread of antibiotic resistance bacteria. This research demonstrates that antibiotic-susceptible Salmonella enterica serovar Heidelberg strains can acquire multidrug resistance from commensal bacteria present in the gut of neonatal broiler chicks, even in the absence of antibiotic selection. We demonstrate that exposure to acidic pH drove the horizontal transfer of antimicrobial resistance plasmids and suggest that simply removing antibiotics from food animal production might not be sufficient to limit the spread of antimicrobial resistance.

The occurrence of Salmonella, extended-spectrum ß-lactamase producing Escherichia coli and carbapenem resistant non-fermenting Gram-negative bacteria in a backyard poultry flock environment.

Increase in the number of small-scale backyard poultry flocks in the USA has substantially increased human-to-live poultry contact, leading to increased public health risks of the transmission of multi-drug resistant (MDR) zoonotic and food-borne bacteria. The objective of this study was to detect the occurrence of Salmonella and MDR Gram-negative bacteria (GNB) in the backyard poultry flock environment. A total of 34 backyard poultry flocks in Washington State (WA) were sampled. From each flock, one composite coop sample and three drag swabs from nest floor, waterer-feeder, and a random site with visible faecal smearing, respectively, were collected. The samples were processed for isolation of Salmonella and other fermenting and non-fermenting GNB under ceftiofur selection. Each isolate was identified to species level using MALDI-TOFF and tested for resistance against 16 antibiotics belonging to eight antibiotic classes. Salmonella serovar 1,4,[5],12:i:- was isolated from one (3%) out of 34 flocks. Additionally, a total of 133 ceftiofur resistant (CefR ) GNB including Escherichia coli (53), Acinetobacter spp. (45), Pseudomonas spp. (22), Achromobacter spp. (8), Bordetella trematum (1), Hafnia alvei (1), Ochrobactrum intermedium (1), Raoultella ornithinolytica (1), and Stenotrophomonas maltophilia (1) were isolated. Of these, 110 (82%) isolates displayed MDR. Each flock was found positive for the presence of one or more CefR GNB. Several MDR E. coli (n = 15) were identified as extended-spectrum ß-lactamase (ESBL) positive. Carbapenem resistance was detected in non-fermenting GNB including Acinetobacter spp. (n = 20), Pseudomonas spp. (n = 11) and Stenotrophomonas maltophila (n = 1). ESBL positive E. coli and carbapenem resistant non-fermenting GNB are widespread in the backyard poultry flock environment in WA State. These GNB are known to cause opportunistic infections, especially in immunocompromised hosts. Better understanding of the ecology and epidemiology of these GNB in the backyard poultry flock settings is needed to identify potential risks of transmission to people in proximity.

Low Dose Infection of Hens in Lay with Salmonella enterica Serovar Enteritidis from Different Genomic Clades.

Salmonella enterica serovar Enteritidis is the leading cause of salmonellosis in people, and modeling of infections in chickens is used to identify intervention strategies. A review of 80 manuscripts encompassing 119 experiments indicated that the mean dose of infection was 108 CFU per bird. Experiments of less than 106 CFU were primarily conducted in immature birds. To address a lack of information on the impact of low dosages on the hen at lay, two experiments were conducted in triplicate. Experiment A addressed issues associated with vaccination; thus, hens were infected intramuscularly at 103, 105, and 107 CFU. For Experiment B, which was focused more on colonization and invasion, hens were infected orally with 5 × 103 CFU with 4 strains from different genomic clades. Samples from liver, spleen, ovarian pedicle, and paired ceca in both experiments were cultured 5, 6, 7, and 8 days postinfection. Eggshell microbiome taxa were assessed in Experiment B. Results indicated that dosages of 103 CFU in both experiments produced enough positive samples to be used within models. The intramuscular route resulted in approximately twice as many positive samples as the oral route. The kinetics of infection appeared to differ between low and high dosages suggestive of a J-curve response. These results could impact risk assessments if the hen at lay has a nonlinear response to infectious dose.

Infección por dosis baja en gallinas de postura con Salmonella enterica Serovar Enteritidis de diferentes clados genómicos. Salmonella enterica serovar Enteritidis es la principal causa de salmonelosis en las personas y el modelo de infecciones en pollos se utiliza para identificar estrategias de intervención. Una revisión de 80 manuscritos que abarca 119 experimentos indicó que la dosis media de infección fue de 108 unidades formadoras de colonias (UFC) por ave. Los experimentos de menos de 106 UFC se realizaron principalmente en aves inmaduras. Para abordar la falta de información sobre el impacto de las dosis bajas en gallinas de postura, se realizaron dos experimentos por triplicado. El experimento A abordó los problemas asociados con la vacunación; así, las gallinas se infectaron por vía intramuscular con 103, 105 y 107 UFC. Para el Experimento B, que se enfocó más en la colonización y la invasión, las gallinas se infectaron por vía oral con 5×103 UFC con 4 cepas de diferentes clados genómicos. Las muestras de hígado, bazo, pedículo ovárico y pares de sacos ciegos se cultivaron en ambos experimentos a los cinco, seis, siete y ocho días después de la infección. Los taxones del microbioma del cascarón de huevo se evaluaron en el Experimento B. Los resultados indicaron que las dosis de 103 UFC en ambos experimentos produjeron suficientes muestras positivas para ser utilizadas dentro de los modelos. La ruta intramuscular dio como resultado aproximadamente el doble de muestras positivas que la ruta oral. La cinética de la infección parece diferir entre las dosis bajas y altas que sugieren una respuesta tipo curva J. Estos resultados podrían afectar las evaluaciones de riesgo si las gallinas de postura tienen una respuesta no lineal a la dosis infecciosa.

Genome sequence analysis of 91 Salmonella Enteritidis isolates from mice caught on poultry farms in the mid 1990s.

A total of 91 draft genome sequences were used to analyze isolates of Salmonella enterica serovar Enteritidis obtained from feral mice caught on poultry farms in Pennsylvania. One objective was to find mutations disrupting open reading frames (ORFs) and another was to determine if ORF-disruptive mutations were present in isolates obtained from other sources. A total of 83 mice were obtained between 1995-1998. Isolates separated into two genomic clades and 12 subgroups due to 742 mutations. Nineteen ORF-disruptive mutations were found, and in addition, bigA had exceptional heterogeneity requiring additional evaluation. The TRAMS algorithm detected only 6 ORF disruptions. The sefD mutation was the most frequently encountered mutation and it was prevalent in human, poultry, environmental and mouse isolates. These results confirm previous assessments of the mouse as a rich source of Salmonella enterica serovar Enteritidis that varies in genotype and phenotype.

A Microbiomic Analysis of a Pasture-Raised Broiler Flock Elucidates Foodborne Pathogen Ecology Along the Farm-To-Fork Continuum.

While conventionally grown poultry continues to dominate the U. S. poultry industry, there is an increasing demand for locally-grown, "all natural" alternatives. The use of next generation sequencing allows for not only the gross (e.g., community structure) but also fine-scale (e.g., taxa abundances) examination of these complex microbial communities. This data provides a better understanding of how a pasture flock's microbiome changes throughout the production life cycle and how that change in microbial ecology changes foodborne pathogens in alternative poultry production systems. In order to understand this ecology better, pooled broiler samples were taken during the entire flock life cycle, from pre-hatch gastrointestinal samples (N = 12) to fecal samples from the brood (N = 5), and pasture (N = 10) periods. Additional samples were taken during processing, including skin and feather rinsates (N = 12), ceca (N = 12), and whole carcass rinses (N = 12), and finally whole carcasss rinsates of final products (N = 3). Genomic DNA was extracted, 16S rDNA microbiome sequencing was conducted (Illumina MiSeq), and microbiomes were analyzed and compared using QIIME 1.9.1 to determine how microbiomes shifted throughout production continuum, as well as what environmental factors may be influencing these shifts. Significant microbiome shifts occurred during the life cycle of the pasture broiler flock, with the brood and pasture fecal samples and cecal samples being very distinct from the other pre-hatch, processing, and final product samples. Throughout these varied microbiomes, there was a stable core microbiome containing 13 taxa. Within this core microbiome, five taxa represented known foodborne pathogens (Salmonella, Campylobacter) or potential/emerging pathogens (Pseudomonas, Enterococcus, Acinetobacter) whose relative abundances varied throughout the farm-to-fork continuum, although all were more prevalent in the fecal samples. Additionally, of the 25 physiochemical and nutrient variables measured from the fecal samples, the carbon to nitrogen ratio was one of the most significant variables to warrant further investigations because it impacted both general fecal microbial ecology and Campylobacter and Enterococcus taxa within the core fecal microbiomes. These findings demonstrate the need for further longitudinal, farm-to-fork studies to understand the ecology of the microbial ecology of pasture production flocks to improve animal, environmental, and public health.

Genomic organization and role of SPI-13 in nutritional fitness of Salmonella.

Salmonella pathogenicity island 13 (SPI-13) contributes to the virulence of Salmonella. The majority of the SPI-13 genes encode proteins putatively involved in bacterial metabolism, however, their functions largely remain uncharacterized. It is currently unknown if SPI-13 contributes to metabolic fitness of Salmonella and, if so, what are the metabolic substrates for the protein encoded by genes within SPI-13. We employed Phenotype Microarray (Biolog, USA) to compare the metabolic properties of SPI-13 deficient mutant (ΔSPI-13) and the WT parent strain of non-typhoidal Salmonella enterica sub sp. enterica serovar Enteritidis (S. Enteritidis). The results of Phenotype Microarray revealed that SPI-13 is required for efficient utilization of two micronutrients, namely, d-glucuronic acid (DGA) and tyramine (TYR), as sole sources of carbon and/or nitrogen. By systematic deletion of the individual gene(s), we identified specific genes within SPI-13 that are required for efficient utilization of DGA (SEN2977-80) and TYR (SEN2967 and SEN2971-72) as sole nutrient sources. The results show that SPI-13 mediated DGA and TYR metabolic pathways afford nutritional fitness to S. Enteritidis. Comparative genomics analysis of the SPI-13 locus from 247 Salmonella strains belonging to 57 different serovars revealed that SPI-13 genes specifically involved in the metabolism of DGA and TYR are highly conserved in Salmonella enterica. Because DGA and TYR are naturally present as metabolic byproducts in the gastrointestinal tract and other host tissues, we propose a metabolic model that shows that the role of SPI-13 mediated DGA and TYR metabolism in the nutritional fitness of Salmonella is likely linked to nutritional virulence of this pathogen.

Multiplication in Egg Yolk and Survival in Egg Albumen of Genetically and Phenotypically Characterized Salmonella Enteritidis Strains.

Prompt refrigeration of eggs to prevent the multiplication of Salmonella Enteritidis to high levels during storage is an important practice for reducing the risk of egg-transmitted human illness. The efficacy of egg refrigeration for achieving this goal depends on the interaction among the location of contamination, the ability of contaminant strains to survive or multiply, and the rate at which growth-restricting temperatures are attained. The present study assessed the significance of several characterized genetic and phenotypic properties for the capabilities of 10 Salmonella Enteritidis isolates to multiply rapidly in egg yolk and survive for several days in egg albumen during unrefrigerated (25°C) storage. The growth of small numbers of each Salmonella Enteritidis strain (approximately 101 CFU/mL) inoculated into egg yolk samples was determined after 6 and 24 h of incubation. The survival of larger numbers of Salmonella Enteritidis (approximately 105 CFU/mL) inoculated into albumen samples was determined at 24 and 96 h of incubation. In yolk, the inoculated Salmonella Enteritidis strains multiplied to mean levels of approximately 102.6 CFU/mL after 6 h of incubation and 108.3 CFU/mL after 24 h. In albumen, mean levels of approximately 104.6 CFU/mL Salmonella Enteritidis were maintained through 96 h. The concentrations of the various Salmonella strains after incubation in either yolk or albumen were distributed over relatively narrow ranges of values. Significant ( P < 0.01) differences observed among individual strains suggested that maintenance of the fimbrial gene sefD may have positive genetic selection value by improving fitness to grow inside egg yolk, whereas the antibiotic resistance gene blaTEM-1 tet(A) appeared to have negative genetic selection value by decreasing fitness to survive in egg albumen.

Complete Genome Sequence of a Ciprofloxacin-Resistant Salmonella enterica subsp. enterica Serovar Kentucky Sequence Type 198 Strain, PU131, Isolated from a Human Patient in Washington State.

Strains of the ciprofloxacin-resistant (Cipr) Salmonella enterica subsp. enterica serovar Kentucky sequence type 198 (ST198) have rapidly and extensively disseminated globally to become a major food safety and public health concern. Here, we report the complete genome sequence of a CiprS. Kentucky ST198 strain, PU131, isolated from a human patient in Washington State (USA).

Serotyping of Salmonella Enterica Isolated from Mice Caught on US Poultry Farms 1995 through 1998.

In this study, a collection of Salmonella enterica subspecies obtained from live mice caught on 32 poultry farms in the Northeast US between 1995 to 1998 was evaluated to provide a historical reference for serotype distribution during a time when egg contamination by serotype Enteritidis was at its peak. Of 821 mice cultured, 157 were positive (19.1%). Seven mice harbored two serotypes of Salmonella. Nine serotypes were detected, eight of which are often associated with foodborne illness. The three most prevalent serotypes were Enteritidis, Heidelberg, and Typhimurium. Enteritidis and Typhimurium were obtained from both spleens and intestines without preference according to type of sample. In contrast, Heidelberg was isolated most often from intestines and Schwarzengrund was most often obtained from spleens. These results support that the house mouse Mus musculus was a risk factor for introduction of multiple pathogenic Salmonella serotypes in poultry raised in the Northeast US during the mid-1990s. Isolates were submitted to the Food and Drug Administration and draft genomes for 64 isolates of Salmonella enterica serovar Enteritidis data have been released through the National Center for Biotechnology Information via the GenomeTrakr network.

Simultaneous Detection of Multiple Salmonella Serovars from Milk and Chicken Meat by Real-Time PCR Using Unique Genomic Target Regions.

A novel genomic and plasmid target-based PCR platform was developed for the detection of Salmonella serovars Heidelberg, Dublin, Hadar, Kentucky, and Enteritidis. Unique genome loci were obtained through extensive genome mining of protein databases and comparative genomic analysis of these serovars. Assays targeting Salmonella serovars Hadar, Heidelberg, Kentucky, and Dublin had 100% specificity and sensitivity, whereas those for Salmonella Enteritidis had 97% specificity and 88% sensitivity. The limits of detection for Salmonella serovars Heidelberg, Kentucky, Hadar, Enteritidis, and Dublin were 12, 9, 40, 13, and 5,280 CFU, respectively. A sensitivity assay was also performed by using milk artificially inoculated with pooled Salmonella serovars, yielding a detection limit of 1 to10 CFU/25 mL of milk samples after enrichment. The minimum DNA detected using the multiplexed TaqMan assay was 75.8 fg (1.53 × 101 genomic equivalents [GE]) for Salmonella Heidelberg, 140.8 fg (2.8 × 101 GE) for Salmonella Enteritidis, and 3.48 pg (6.96 × 102 GE) for Salmonella Dublin. PCR efficiencies were 89.8% for Salmonella Heidelberg, 94.5% for Salmonella Enteritidis, and 75.5% for Salmonella Dublin. Four types of 30 pasteurized milk samples were tested negative by culture techniques and with a genus-specific Salmonella invA gene PCR assay. Among 30 chicken samples similarly tested, 12 (40%) were positive by both culture and the invA PCR. Testing of these 12 samples with the serovar-specific PCR assay detected single and mixed contamination with Salmonella Kentucky, Salmonella Enteritidis, and Salmonella Heidelberg. Five unique primers were designed and tested by multiplex conventional PCR in conjunction with the use of the multiplex TaqMan assay with three of the primers. The diagnostic assays developed in this study could be used as tools for routine detection of these five Salmonella serovars and for epidemiological investigations of foodborne disease outbreaks.

Frequency and Duration of Fecal Shedding of Salmonella Serovars Heidelberg and Typhimurium by Experimentally Infected Laying Hens Housed in Enriched Colony Cages at Different Stocking Densities.

Eggs contaminated with Salmonella Enteritidis are leading sources of human salmonellosis, but Salmonella Heidelberg and Salmonella Typhimurium are also egg-associated pathogens. The management practices and housing facilities characterizing different systems for housing commercial egg flocks can influence Salmonella persistence and transmission. Animal welfare aspects of poultry housing have been widely debated, but their food safety ramifications are not thoroughly understood. The present study assessed the effects of two different bird stocking densities on the frequency and duration of fecal shedding of strains of Salmonella Heidelberg and Salmonella Typhimurium in groups of experimentally infected laying hens housed in colony cages enriched with perching and nesting areas. In separate trials, laying hens were distributed into two groups housed in enriched colony cages at stocking densities of 648 and 973 cm2/bird, and a third group was housed in conventional cages at 648 cm2/bird. All hens were orally inoculated with doses of 108 colony-forming units (CFU) of either Salmonella Heidelberg or Salmonella Typhimurium. At eight weekly postinoculation intervals, samples of voided feces were collected from beneath each cage and cultured to detect Salmonella. Fecal shedding of Salmonella Heidelberg continued for 8 wk in all housing groups, but Salmonella Typhimurium shedding ceased after as little as 5 wk in enriched colony cages at low stocking density. After Salmonella Heidelberg infection, the overall frequency of positive fecal cultures for all sampling dates combined was significantly (P < 0.05) greater from either conventional cages (51.0%) or enriched colony cages (46.5%) at high stocking density than from enriched colony cages at low stocking density (33.3%). No significant differences in Salmonella Typhimurium fecal isolation were identified between housing groups. These results demonstrate that stocking density can affect intestinal colonization and fecal shedding in laying hens for some (but not necessarily all) Salmonella serovars or strains.

Draft Genome Sequences of 64 Salmonella enterica Serotype Enteritidis Isolates Obtained from Wild Mice.

Salmonella enterica serotype Enteritidis is a foodborne pathogen of global concern, because it is frequently isolated from foods and patients. Draft genome sequences are reported here for 64 S Enteritidis strains isolated from the intestines and spleens of mice caught live on chicken farms in the U.S. Northeast. The availability of these genomes provides baseline information on the genomic diversity of S Enteritidis during the 1990s, when foodborne outbreaks traced to internal contamination of eggs were prevalent.

Population dynamics and antimicrobial resistance of the most prevalent poultry-associated Salmonella serotypes.

Salmonella spp. is the most predominant bacterial cause of foodborne gastroenteritis in humans. Due to the risk of human infection associated with poultry products and the prevalence of antimicrobial resistance, Salmonella also poses a significant challenge to commercial poultry production. During the last decade (2002 to 2012), the 12 most prevalent poultry-associated Salmonella serotypes (MPPSTs) were frequently and consistently isolated from poultry products in the United States. These MPPSTs and their percent prevalence in poultry products include Kentucky (4%), Enteritidis (2%) Heidelberg (2%), Typhimurium (2%), S. I 4,[5],12:i:- (0.31%), Montevideo (0.20%), Infantis (0.16%) Schwarzengrund (0.15%), Hadar (0.15%), Mbandaka (0.13%), Thompson (0.12%), and Senftenberg (0.04%). All MPPSTs except Kentucky are among the top 30 clinically significant serotypes that cause human illnesses in the United States. However with the exception of a few widely studied serotypes such as S. Enteritidis and Typhimurium, the ecology and epidemiology of the majority of MPPSTs still remain poorly investigated. Published data from the United States suggests that MPPSTs such as Heidelberg, Typhimurium, Kentucky, and Sentfenberg are more likely to be multi-drug resistant (MDR, ≥3 antimicobial classes) whereas Enteritidis, Montevideo, Schwarzengrund, Hadar, Infantis, Thompson, and Mbandaka are generally pan-susceptible or display resistance to fewer antimicobials. In contrast, the majority of MPPSTs isolated globally have been reported to display MDR phenotype. There also appears to be an international spread of a few MDR serotypes including Kentucky, Schwarzengrund, Hadar, Thomson, Sentfenberg, and Enteritidis, which may pose significant challenges to the public health. The current knowledge gaps on the ecology, epidemiology, and antimicrobial resistance of MPPSTs are discussed.

Colonization of internal organs by Salmonella serovars Heidelberg and Typhimurium in experimentally infected laying hens housed in enriched colony cages at different stocking densities.

Contaminated eggs produced by infected commercial laying flocks are often implicated as sources of human infections with Salmonella Enteritidis, but Salmonella serovars Heidelberg and Typhimurium have also been associated with egg-transmitted illness. Contamination of the edible contents of eggs is a consequence of the colonization of reproductive tissues in systemically infected hens. In recent years, the animal welfare implications of diverse poultry housing and management systems have been vigorously debated, but the food safety significance of laying hen housing remains uncertain. The present study evaluated the effects of 2 different bird stocking densities on the invasion of internal organs by Salmonella serovars Heidelberg and Typhimurium in groups of experimentally infected laying hens housed in colony cages enriched with perching and nesting areas. Laying hens were distributed at 2 different stocking densities (648 and 973 cm2/bird) into colony cages and (along with a group housed in conventional cages at 648 cm2/bird) orally inoculated with doses of 107 cfu of 2-strain cocktails of either Salmonella Heidelberg or Salmonella Typhimurium. At 5 to 6 d post-inoculation, hens were euthanized and samples of internal organs (cecum, liver, spleen, ovary, and oviduct) were removed for bacteriologic culturing. The overall frequency of Salmonella isolation from ceca after inoculation with strains of serovar Heidelberg (83.3%) was significantly (P < 0.001) greater than the corresponding value for strains of serovar Typhimurium (53.8%), whereas Salmonella was recovered significantly more often from both livers (85.2% vs. 53.7%; P < 0.0001) and spleens (78.7% vs. 56.5%; P = 0.0008) after inoculation with strains of serovar Typhimurium than strains of serovar Heidelberg. However, there were no significant differences (P > 0.05) between stocking densities or cage systems in the frequencies of isolation of either Salmonella serovar from any of the five sampled tissues. These results contrast with prior studies, which reported increased susceptibility to internal organ invasion by Salmonella Enteritidis among hens in conventional cages at higher stocking densities.

Genome Sequences of Two Salmonella enterica Serovar Kentucky Isolates Recovered from Poultry Carcasses in the United States.

We report here the draft genome sequences of two Salmonella enterica serovar Kentucky eBurstGroup 15 isolates collected from poultry carcasses in Georgia (USA).