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In mammals, enteric salmonellas can use tetrathionate (ttr), formed as a by-product from the inflammatory process in the intestine, as electron acceptor in anaerobic respiration, and it can fuel its energy metabolism by degrading the microbial fermentation product 1,2-propanediol. However, recent studies have shown that this mechanism is not important for Salmonella infection in the intestine of poultry, while it prolongs the persistence of Salmonella at systemic sites in this species. In the current study, we show that ΔttrApduA strains of Salmonella enterica have lower net survival within chicken-derived HD-11 macrophages, as CFU was only 2.3% (S. Enteritidis ΔttrApduA), 2.3% (S. Heidelberg ΔttrApduA), and 3.0% (S. Typhimurium ΔttrApduA) compared to wild-type strains after 24 h inside HD-11 macrophage cells. The difference was not related to increased lysis of macrophages, and deletion of ttrA and pduA did not impair the ability of the strains to grow anaerobically. Further studies are indicated to determine the reason why Salmonella ΔttrApduA strains survive less well inside macrophage cell lines. [ABSTRACT FROM AUTHOR]

The Salmonella enterica serovars Typhimurium (S. Typhimurium), Heidelberg (S. Heidelberg), and their monophasic variants (S. 1,4,[5],12:i:-, S. 1,4,[5],12:r:- and S. 1,4,[5],12:-:1,2) are highly disseminated in poultry farming and can contaminate chicken meat, eggs, and other foods of avian origin. A time-consuming bacteriological and serological analysis is usually required to identify serovars by traditional methods. Incomplete and inconclusive serological results are frequent in routine analysis, mainly due to the occurrence of bacterial isolates presenting similar antigenic profiles. Molecular biology assays have been developed to improve the detection of specific Salmonella serovars and strains. This study aimed to develop a multiplex real-time PCR (SHTAmp) for the rapid DNA detection of S. Typhimurium, S. Heidelberg, and their monophasic variants from poultry samples. The methodology was used in the analysis of 147 field isolates from Brazilian poultry flocks previously evaluated with serological analysis. The results demonstrated that it was able to specifically and rapidly detect 21 S. Typhimurium and 57 S. Heidelberg isolates with complete antigenic formulae. Furthermore, SHTAmp was able to differentiate nine S. Typhimurium and 44 S. Heidelberg isolates with incomplete serological formulae (monophasic and aphasic variants). The complete methodology was also successfully used to detect these bacteria directly from 34 poultry samples after pre-enrichment in buffered peptone water (BPW). In conclusion, SHTAmp is a fast and accurate method to detect the two frequent and concerning serovars S. Typhimurium and S. Heidelberg directly from poultry samples.

The Salmonella enterica serovars Typhimurium (S. Typhimurium), Heidelberg (S. Heidelberg), and their monophasic variants (S. 1,4,[5],12:i:-, S. 1,4,[5],12:r:- and S. 1,4,[5],12:-:1,2) are highly disseminated in poultry farming and can contaminate chicken meat, eggs, and other foods of avian origin. A time-consuming bacteriological and serological analysis is usually required to identify serovars by traditional methods. Incomplete and inconclusive serological results are frequent in routine analysis, mainly due to the occurrence of bacterial isolates presenting similar antigenic profiles. Molecular biology assays have been developed to improve the detection of specific Salmonella serovars and strains. This study aimed to develop a multiplex real-time PCR (SHTAmp) for the rapid DNA detection of S. Typhimurium, S. Heidelberg, and their monophasic variants from poultry samples. The methodology was used in the analysis of 147 field isolates from Brazilian poultry flocks previously evaluated with serological analysis. The results demonstrated that it was able to specifically and rapidly detect 21 S. Typhimurium and 57 S. Heidelberg isolates with complete antigenic formulae. Furthermore, SHTAmp was able to differentiate nine S. Typhimurium and 44 S. Heidelberg isolates with incomplete serological formulae (monophasic and aphasic variants). The complete methodology was also successfully used to detect these bacteria directly from 34 poultry samples after pre-enrichment in buffered peptone water (BPW). In conclusion, SHTAmp is a fast and accurate method to detect the two frequent and concerning serovars S. Typhimurium and S. Heidelberg directly from poultry samples. [ABSTRACT FROM AUTHOR]

ABSTRACT Brazilian chicken meat is exported to more than 150 countries and consumed by consumer markets that demand high quality and food safety, thus, requiring very strict control of pathogens present in food to guarantee these rigorous safety standards. This study evaluates the reports from the Salmonella spp. Control and Monitoring Program of the Brazilian Ministry of Agriculture, Livestock and Food Supply of seven slaughterhouses inspected by the Federal Inspection Service from the western region of Paraná state, Brazil, from March 2017 to February 2019. The broiler litter swab and carcass analyses revealed a Salmonella spp. positivity ratio of 5.9% (19/319) and 23.5% (75/319), respectively. The concomitant presence of Salmonella spp. in the broiler litter swab and chicken carcasses occurred in 58% of the positive samples. The most frequently isolated serovar in the carcasses was Salmonella Heidelberg (85.3%) followed by Salmonella spp. (10.6%). During slaughter, carcass positivity to Salmonella spp. was significantly different (p=0.047) between the first (19.6%) and the second (29.4%) shifts. The results alert for the possibility of carcass contamination during slaughtering and, therefore, more stringent hygiene measures between shifts must be implemented to mitigate carcass contamination.

The incidence of salmonellosis, and other enteric zoonotic diseases, has been associated with various food and environmental exposures and socioeconomic factors. Increasingly, there is interest in exploring differences among serotypes of Salmonella to better inform public health prevention efforts. Consequently, we investigated whether rates of Salmonella Heidelberg and Typhimurium in Ontario communities in 2015 were influenced by household characteristics, agricultural factors, and the presence of meat plants. Data for each reported case of S. Heidelberg and S. Typhimurium in Ontario in 2015 were collected. Expected cases of each serotype were calculated, stratified by age group and sex, according to the underlying population distribution from the Canadian census. Socioeconomic, agricultural census data, and data concerning provincial and federally inspected meat plants were combined with observed and expected case counts. The association between community‐level agricultural, meat processing, and socioeconomic variables, serotype, and the rate of salmonellosis in each census subdivision (CSD) was explored using multilevel Poisson models, with random intercepts for CSD and census division (CD). Rates of S. Heidelberg and S. Typhimurium were associated with the proportion of married individuals in a CSD, and were higher in CSDs with the highest quantile of labour participation compared to those in the lowest quantile. There was an interaction effect between cattle, poultry and swine farm density in a CD and serotype, with rates of either serotype decreasing as cattle, poultry, or swine farm density in the encompassing CD increased. The rate of the decrease varied by serotype. Our findings concerning community‐level household characteristics may be explained by the influence of family structure and occupation on food consumption patterns and environmental exposures. Rates of S. Heidelberg and S. Typhimurium may be lower in areas with increased animal farm density due to naturally acquired immunity from routine exposure to Salmonella via livestock. [ABSTRACT FROM AUTHOR]

Salmonella enterica serovar Heidelberg (S. Heidelberg) is a zoonotic, ubiquitous, and worldwide-distributed pathogen, responsible for gastroenteritis in humans caused by the consumption of contaminated food. In this study, 11 S. Heidelberg strains isolated from chicken and bovine meat, drag swab, and animal feed between 2013 and 2017 in states of the southern region of Brazil were characterized by whole-genome sequencing (WGS) analyses. Antimicrobial resistance against 18 antimicrobials was determined by disk-diffusion and ciprofloxacin's minimum inhibitory concentration by Etest®. The search for resistance and virulence genes, plasmids, Salmonella Pathogenicity Islands (SPIs) plus multi-locus sequence typing (MLST), and single-nucleotide polymorphisms (SNPs) analyses was conducted using WGS data. All strains harbored resistance genes fosA7, aac(6')-Iaa, sul2, tet(A), blaCMY-2, mdsA, and mdsB, and point mutations in gyrA and parC. All strains showed a phenotypic multidrug-resistant profile, with resistant or intermediate resistant profiles against 14 antimicrobials tested. Plasmids ColpVC, IncC, IncX1, and IncI1-I(Alpha) were detected. Virulence genes related to adherence, macrophage induction, magnesium uptake, regulation, and type III secretion systems plus 10 SPIs were detected. All strains were assigned to ST15 and belonged to two SNP clusters showing high similarity to isolates from the United Kingdom, Chile, Germany, the Netherlands, China, South Africa, and South Korea. In conclusion, the presence of multidrug-resistant S. Heidelberg strains in Brazil showing a global genomic relationship may alert for the necessity of stronger surveillance measures by food safety and public health authorities to limit its spread to humans and animals through foods. [ABSTRACT FROM AUTHOR]

ABSTRACT Salmonellosis is an important gastrointestinal infection in humans and cause of foodborne outbreaks in the world. In this context, molecular characterization is essential to understand how the strains circulate. The aim of this study was to evaluate the genotypic distribution of S. Heidelberg according to the source of isolation. The genetic relatedness of the S. Heidelberg isolates was determined by pulsed-field gel electrophoresis (PFGE). The most prevalent pulsotypes of cluster A were BRJF6X01.006 (27/95 = 28,42%) related between 1995 and 2011 in broilers, poultry meat and poultry farms, meat product and human, and BRJF6X01.001 (21/95 = 22,10%) related between 2011 and 2017 in wild animals, broilers, poultry meat, poultry farms, meat product, animal feed, and pork meat. The pulsotype BRJF6X01.001 shows a high distribution in the environmental and productive chain. The degree of similarity between pulsotypes BRJF6X01.006 and BRJF6X01.001 is 88%. To ensure the safety of human and animal health, holistic approaches, including surveillance of Salmonella throughout the environment and in the production chain, together with control measures, are critical. As transmission of Salmonella from food producing animals to wildlife and to the environment is considered potential public health problem, information on the survival and persistence of Salmonella in the environment and in potential reservoirs is of considerable importance.

Salmonella infections have become increasingly resistant to antibiotics, including fluoroquinolones, third-generation cephalosporins (C3G), and even carbapenems. This report describes the emergence of a strain of Salmonella enterica serovar Heidelberg that produces the carbapenemase OXA 48. The strain was isolated from a stool sample taken from a newborn. Antimicrobial susceptibility testing was carried out following the recommendations of the Clinical and Laboratory Standard Institute. Whole genome sequencing was performed on MiSeq Illumina™. The strain was resistant to ertapenem (minimal inhibitory concentration [MIC] = 12 µg/mL), intermediate to imipenem (MIC = 1.5 µg/mL), resistant to nalidixic acid, and intermediate to fluoroquinolones but was susceptible to C3G, cotrimoxazole, chloramphenicol, and colistin (MIC = 0.064 µg/mL). The strain was identified as ST-15. The strain of Salmonella Heidelberg ST-15 was found to have antimicrobial resistance genes, specifically blaOXA-48 , aac(6')-Iaa and fosA7 , which mediate resistance to carbapenems, aminoglycosides and fosfomycin, respectively. Additionally, mutations were detected in the gyrA , parC. Three plasmid replicon type IncL, IncX1, and Col156 have been identified. The strain has the potential to cause an epidemic. The genomic analysis of the strain allowed us to understand the mechanisms of resistance. Preventing the spread of Salmonella carbapenemase-producing strains is crucial, particularly in hospital settings. Epidemiological measures are necessary to achieve this goal.

SUMMARY: Salmonella remains the second most common cause of gastrointestinal illness in the United States, European Union and other regions of the globe. In the food chain, poultry, especially turkeys and chickens, are considered significant contributors to salmonellosis in humans, as their intestinal tracts are often colonized with Salmonella. Due to changes in the prevalence of serovars, Salmonella Heidelberg (SH) has gained prominence, especially in the United States and Brazil, where outbreaks have occurred in recent years. The transmission of salmonella can occur by different routes, whether by infected breeding flocks, previously infected flocks, contaminated food or water. Thus, eliminating the bacteria from the installations can be a difficult task. In this sense, several countries have implemented salmonella control programs on poultry farms, in order to reduce its presence. Our study demonstrated that, despite the similarity between bird species and their rearing systems, turkeys are more sensitive to the Heidelberg serovar, especially in relation to microbiological contamination of internal organs and elimination of the agent into the environment. This information is extremely relevant for the poultry industry, enabling it to improve biosecurity programs in this species.

Salmonella enterica subsp. enterica serovar Heidelberg has been associated with a broad host range, such as poultry, dairy calves, swine, wild birds, environment, and humans. The continuous evolution of S. Heidelberg raises a public health concern since there is a global dispersal of lineages harboring a wide resistome and virulome on a global scale. Here, we characterized the resistome, phylogenetic structure and clustered regularly interspaced short palindromic repeats (CRISPR) array composition of 81 S. Heidelberg strains isolated from broiler farms (n = 16), transport and lairage (n = 5), slaughterhouse (n = 22), and retail market (n = 38) of the poultry production chain in Brazil, between 2015 and 2016 using high-resolution approaches including whole-genome sequencing (WGS) and WGS-derived CRISPR genotyping. More than 91% of the S. Heidelberg strains were multidrug-resistant. The total antimicrobial resistance (AMR) gene abundances did not vary significantly across regions and sources suggesting the widespread distribution of antibiotic-resistant strains from farm to market. The highest AMR gene abundance was observed for fosA7 , aac(6′)-Iaa , sul2 , tet(A) , gyrA , and parC for 100% of the isolates, followed by 88.8% for bla CMY –2. The β-lactam resistance was essentially driven by the presence of the plasmid-mediated AmpC (pAmpC) bla CMY –2 gene, given the isolates which did not carry this gene were susceptible to cefoxitin (FOX). Most S. Heidelberg strains were classified within international lineages, which were phylogenetically nested with Salmonella strains from European countries; while CRISPR genotyping analysis revealed that the spacer content was overall highly conserved, but distributed into 13 distinct groups. In summary, our findings underscore the potential role of S. Heidelberg as a key pathogen disseminated from farm to fork in Brazil and reinforce the importance of CRISPR-based genotyping for salmonellae. Hence, we emphasized the need for continuous mitigation programs to monitor the dissemination of this high-priority pathogen. [ABSTRACT FROM AUTHOR]

This study compares the plate count (PC) and the Propidium Monoazide-quantitative Polymerase Chain Reaction (PMA-qPCR) methods to assess the growth of a cocktail of three serotypes of Salmonella enterica (Heidelberg, Typhimurium, and Enteritidis) in cooked, sliced, and vacuum-packaged turkey breast (STB) under isothermal storage temperatures (8 °C–20 °C), using predictive models. Standard curves were developed for PMA-qPCR, demonstrating high efficiency (101%) and sensitivity, with quantification limits ranging from 1 to 2 log 10 CFU/g for all temperatures studied. Comparative analysis revealed a significant correlation (R 2 = 0.99; 95% CI) between the PC and PMA-qPCR methods; however, the agreement analysis indicated a mean difference (Bias) of −0.11 log 10 CFU/g (p < 0.05), suggesting underestimation by the PC method. This indicates the presence of stressed or viable but nonculturable (VBNC) cells, detectable by PMA-qPCR but not by PC. The Baranyi and Roberts model showed a good ability to describe the behavior of S. enterica cocktail in STB for PC and PMA-qPCR data under all isothermal conditions. The exponential secondary model more accurately represented the temperature dependence of the maximum specific growth rate compared to the Ratkowsky square root model, with R 2 values ≥ 0.984 and RMSE values ≤ 0.011 for both methods. These results suggest that combining PMA-qPCR with predictive modeling allows for a more accurate prediction of S. enterica growth, compared to PC method. In the event of cold chain disruptions of meat products, the use of PMA-qPCR method allow the quantification of VBNC cells, that can still pose a health risk to consumers, especially in ready-to-eat products. • PMA-qPCR monitor the growth of S. enterica in turkey breast ham better than PC. • The PC method underestimated S. enterica growth more at stressful temperatures. • LoQ values were ranging from 1 to 2 log 10 CFU/g for all temperatures studied. • PMA-qPCR detects VBNC cells showing higher sensitivity than the PC method. • Predictive models described the S. enterica behavior in turkey ham for both methods. [ABSTRACT FROM AUTHOR]

Among the animal derived food products, contamination of poultry eggs, and egg shell surface is one of the major causes for foodborne salmonellosis in the United States. As a means of reducing the pathogen transfer to the internal egg contents, polysaccharide-based coatings containing antimicrobial phytochemicals could potentially serve as a biocontrol strategy for shelled egg products. The current study investigated the efficacy of four GRAS (Generally Recognized as Safe)-status plant-derived compounds, namely, caproic acid (CAO), caprylic acid (CAY), linalool (LIN) and cuminaldehyde (CUM), as pectin-based coating treatments, individually or in combination, for reducing Salmonella Heidelberg (SH) on shell eggs. A three-strain mixture of SH (~8.0 log CFU in 50 μL inoculum) was spot-inoculated on surface sterilized white-shelled eggs. Eggs were evenly coated with either pectin-based treatments of CAO (1%), CAY (1%), LIN (1%) and CUM (1%), individually, or a combination of 4 phytochemicals (COMB- each phytochemical at 0.5% v/v level of inclusion). The treated eggs were stored at 4°C and SH counts were enumerated on days 0, 1, 3, 5, 7, 14, and 21 of storage. The study was replicated thrice, 3 eggs/treatment/day time point, and the data were analyzed using two-way ANOVA with significance tested at p < 0.05. On day 0, pectin-coated control eggs had ~7.6 log CFU of SH/egg. At the end of refrigerated storage (day 21), pectin-based coating of CAO and CAY at 1% level reduced SH by 2.0–2.5 log CFU/egg (P < 0.05) when compared to controls. In addition, the CUM and LIN based coatings produced 3.0 log and 3.9 log reduction, respectively, in SH counts on eggs by day 21 of storage. Among the treatments with phytochemical combinations, COMB1 [pectin (2%) + Caprylic acid, caproic acid and cuminaldehyde (each at 0.5% level)] was found to be most effective, reducing SH counts to 2.5–3.3 log CFU/egg from day 0 through day 14, and by the end of storage period (day 21), a 3.5 log CFU reduction/egg (p < 0.05) compared to untreated controls. Morphological studies of treated eggs using atomic force microscopy (AFM) have shown that the roughness of eggs can be influenced by a combination of various compounds. Results indicate the potential efficacy of the aforesaid phytochemicals in reducing SH on shell eggs; however, further studies investigating their industrial feasibility and effects on sensory attributes of eggs are warranted.

Salmonella enterica subsp. enterica serovar Heidelberg has been associated with a broad host range, such as poultry, dairy calves, swine, wild birds, environment, and humans. The continuous evolution of S. Heidelberg raises a public health concern since there is a global dispersal of lineages harboring a wide resistome and virulome on a global scale. Here, we characterized the resistome, phylogenetic structure and clustered regularly interspaced short palindromic repeats (CRISPR) array composition of 81 S. Heidelberg strains isolated from broiler farms (n = 16), transport and lairage (n = 5), slaughterhouse (n = 22), and retail market (n = 38) of the poultry production chain in Brazil, between 2015 and 2016 using high-resolution approaches including whole-genome sequencing (WGS) and WGS-derived CRISPR genotyping. More than 91% of the S. Heidelberg strains were multidrug-resistant. The total antimicrobial resistance (AMR) gene abundances did not vary significantly across regions and sources suggesting the widespread distribution of antibiotic-resistant strains from farm to market. The highest AMR gene abundance was observed for fosA7, aac(6′)-Iaa, sul2, tet(A), gyrA, and parC for 100% of the isolates, followed by 88.8% for blaCMY–2. The β-lactam resistance was essentially driven by the presence of the plasmid-mediated AmpC (pAmpC) blaCMY–2 gene, given the isolates which did not carry this gene were susceptible to cefoxitin (FOX). Most S. Heidelberg strains were classified within international lineages, which were phylogenetically nested with Salmonella strains from European countries; while CRISPR genotyping analysis revealed that the spacer content was overall highly conserved, but distributed into 13 distinct groups. In summary, our findings underscore the potential role of S. Heidelberg as a key pathogen disseminated from farm to fork in Brazil and reinforce the importance of CRISPR-based genotyping for salmonellae. Hence, we emphasized the need for continuous mitigation programs to monitor the dissemination of this high-priority pathogen.

The current public health worry revolves around infections stemming from the consumption of meat contaminated with Salmonella spp., which has been linked to the growing issue of antimicrobial resistance. We investigated the presence of non-typhoidal Salmonella (NTS) in the retail of spicy chicken meat and its antimicrobial susceptibility profile. Forty establishments (supermarkets and butcheries) in Mid-West Brazil were visited in February and July 2017, totaling 80 samples. Salmonella isolation, molecular serotyping and antimicrobial analyses were performed. Of the total establishments, 18 sold meat NTS-positive, 6 of which were contaminated in the two visits, resulting in 24 contaminated samples (30%). Of these, the prevalent serovars were Heidelberg (58%–14/24), Minnesota (21%–05/24), Muenchen (17%–04/24) and Mbandaka (4%–01/24), but none was positive for Typhimurium and Enteritidis. Still, 55.6% (10/18) of the establishments did not have a veterinary inspection seal, and 50% (09/18) sold products beyond their expiration date. Worse still, 87.5% (21/24) of NTS isolates were resistant to at least one antimicrobial agent, being 66.7% (16/24) multidrug-resistant. The bla gene was detected in 41.6% (10/24), confirming resistance by β-lactamase enzymes. These findings highlighted the great risk of foodborne illness due to the possibility of contaminated seasoned chicken meat and transmission of bla resistant genes through the food chain. • Non-typhoidal Salmonella (NTS) in spicy chicken meats were investigated. • Heidelberg (58%) was the prevalent serovar; none were Typhimurium or Enteritidis. • 55.6% of the samples lacked vet inspection seal, and 50% sold expired products. • 87.5% of NTS isolates were antimicrobial resistant, and 66.7% of these were MDR. • 41.6% NTS isolates presented bla gene, indicating resistance β-lactamase enzymes. [ABSTRACT FROM AUTHOR]

A dairy-originated probiotic bacterium, Propionibacterium freudenreichii subsp. freudenreichii B3523 (PF) was found to be effective in reducing multidrug-resistant Salmonella Heidelberg (MDR SH) colonization in turkey poults (2-week-old) and growing (7-week-old) and finishing (12-week-old) turkeys. In this study, we explored the potential for microbiome modulation in the cecum of turkeys of different age groups due to PF supplementation in conjunction with MDR SH challenge. One-day-old commercial turkey poults were allocated to 3 treatment groups: negative control (N; turkeys without PF supplementation or SH challenge), SH control (S; turkeys challenged with SH without PF supplementation), and test group (P; turkeys supplemented with PF and challenged with SH). Turkeys were supplemented with 1010 CFU PF in 5-gallon (18.9 L) water until 7 or 12 week of age. At the 6th or 11th wk, turkeys were challenged with SH at 106 and 108 CFU/bird by crop gavage, respectively. After 2 and 7 d of challenge (2-d postinoculation [PI] and 7-d PI, respectively), cecal samples were collected and microbiome analysis was conducted using Illumina MiSeq. The experiments were repeated twice with 8 and 10 turkeys/group for 7- and 12-wk studies, respectively. Results indicated that the species richness and abundance (Shannon diversity index) was similar among the treatment groups. However, treatments caused apparent clustering of the samples among each other (P < 0.05). Firmicutes was the predominant phylum in the growing and finishing turkey cecum which was evenly distributed among the treatments except on wk 12 where the relative abundance of Firmicutes was significantly higher in P than in N (P = 0.02). The MDR SH challenge resulted in modulation of microflora such as Streptococcus, Gordonibacter, and Turicibacter (P < 0.05) in the S groups compared with the P and N groups, known to be associated with inflammatory responses in birds and mammals. The supplementation of PF increased the relative abundance of carbohydrate-fermenting and short-chain fatty acid–producing genera in the P group compared with the S group (P < 0.05). Moreover, the results revealed that PF supplementation potentially modulated the beneficial microbiota in the P group, which could mitigate SH carriage in turkeys.

SUMMARY: Drinking water contaminated with Salmonella could serve as a source for cecal colonization of the pathogen in birds. In this study, we investigated the efficacy of a generally recognized as safe—status essential oil (lemongrass essential oil, LGEO) against multidrug-resistant Salmonella enterica serovar Heidelberg (S. Heidelberg) in poultry drinking water. Farm water with and without added droppings, litter, or feed [0.5% (w/v)] inoculated with 6.0 log10 cfu/mL S. Heidelberg was treated with 0, 0.03, 0.06, 0.125, 0.25, or 0.5% (v/v) LGEO and incubated at 12.5°C or 22°C for up to 7 d. The number of viable S. Heidelberg populations was determined on d 0, 1, 3, 5, and 7. At 12.5°C, all concentrations of LGEO inactivatedS. Heidelberg to non-detectable levels in water alone and water contaminated with droppings or litter by d 7 (>6 log10 cfu/mL; P < 0.05). The highest LGEO concentration tested (0.5%) resulted in >5.0 log10 cfu/mL reduction of S. Heidelberg on d 7 in water contaminated with feed (P < 0.05). At 22°C, all concentrations of LGEO resulted in the reduction of S. Heidelberg to non-detectable levels in water alone by d 5 (>6 log10 cfu/mL; P < 0.05). Concentrations of LGEO ≥0.125% resulted in inhibition of S. Heidelberg to non-detectable levels in water contaminated with droppings from d 5 onwards (P < 0.05). The LGEO at the highest tested concentration (0.5%) resulted in >5.0 log10 cfu/mL reduction of S. Heidelberg in water contaminated with litter on d 7 (P < 0.05) but did not result in reduction of the pathogen in water contaminated with feed at 22°C. Results indicate the potential of LGEO against S. Heidelberg in water alone or water containing droppings or litter, indicating its efficacy as a natural disinfectant in poultry drinking water.

Salmonellosis is one of several zoonotic diseases for which individuals with occupational animal contact, including abattoir workers, are at an increased risk. If meat is contaminated during slaughter, this can increase the risk of enteric illness for consumers. In this study, we investigated whether reported cases of Salmonella Heidelberg and Typhimurium were clustered around abattoirs in Ontario in 2015 and whether there was any evidence (laboratory/exposure) to suggest an abattoir at the centre of a cluster might be the source of exposure. Data for each reported case of S. Heidelberg and S. Typhimurium in Ontario in 2015 were collected. Multi‐focused and non‐focused spatial and space‐time cluster detection tests were performed for each serotype, with and without cases linked to known outbreaks, using Poisson and space‐time permutation models. Focused tests included the location of abattoirs operational in all or part of 2015. Laboratory data and exposure information were used to explore the relatedness of cases within identified clusters. Focused spatial tests identified clusters of S. Heidelberg and S. Typhimurium around abattoirs. Focused space‐time permutation tests identified 2 significant space‐time clusters of S. Heidelberg; one cluster (n = 11 cases) included 8 of 9 cases associated with a known outbreak and the other cluster (n = 18 cases) was not part of a previously identified outbreak. Review of laboratory and risk factor information suggested that cases within each cluster shared a common exposure. Cases were not asked about goat or sheep meat consumption. The focused cluster test, particularly with the space‐time permutation model, could assist in identifying outbreaks associated with a particular physical location, such as an abattoir. Improvements to the current case investigation process, such as consistent collection and reporting of high‐risk occupation information and more detailed food consumption history, could assist in outbreak identification when coupled with this statistic. [ABSTRACT FROM AUTHOR]

ABSTRACT: The antimicrobial efficacy of caprylic acid (CA), a medium-chain fatty acid, against multidrug-resistant Salmonella Heidelberg (MDR SH) on chicken drumsticks in a soft-scalding temperature-time setup was investigated. Based on the standardization experiments in nutrient media and on chicken breast fillet portions, intact chicken drumsticks were spot inoculated with MDR SH and immersed in water with or without antimicrobial treatments at 54°C for 2 min. The treatments included 0.5% CA, 1% CA, 0.05% peracetic acid (PAA), 0.5% CA + 0.05% PAA, and 1.0% CA + 0.05% PAA. Additionally, the efficacy of the potential scald treatments against MDR SH survival on drumsticks for a storage period of 48 h at 4°C was determined. Furthermore, the effect of these treatments on the surface color of the drumsticks was also evaluated. Appropriate controls were included for statistical comparisons. The antimicrobial treatments resulted in a significant reduction of MDR SH on drumsticks. For the lower inoculum (∼2.5 log10 CFU/g) experiments, 0.5% CA, 1% CA, 0.05% PAA, 0.5% CA + 0.05% PAA, and 1.0% CA + 0.05% PAA resulted in 0.7-, 1.0-, 2.5-, 1.4-, and 1.5- log10 CFU/g reduction of MDR SH on drumsticks, respectively (P < 0.05). The same treatments resulted in 0.9-, 1.3-, 2.5-, 2.2-, and 2.6- log10 CFU/g reduction of MDR SH when the drumsticks were contaminated with a higher inoculum (∼4.5 log10 CFU/g) level (P < 0.05). Moreover, the antimicrobial treatments inactivated MDR SH in the treatment water to undetectable levels, whereas 2.0- to 4.0- log10 CFU/mL MDR SH survived in the positive controls (P < 0.05). Also, the treatments were effective in inhibiting MDR SH on the drumsticks compared to the respective controls during a storage period of 48 h at 4°C; however, the magnitude of reduction remained the same as observed during the treatment (P < 0.05). Additionally, none of the treatments affected the color of the drumsticks (P > 0.05). Results indicate that CA could be an effective natural processing aid against MDR SH on chicken products.

ABSTRACT: Salmonella is a major cause of foodborne illness worldwide, and poultry and its derived products are the most common food products associated with salmonellosis outbreaks. Some countries, including Brazil, have experienced an increased prevalence of Salmonella Heidelberg among their poultry flocks. Some isolates have also presented high resistance to antimicrobial agents and persist in the poultry farm environment. This study aimed to compare the susceptibility of S. Heidelberg strains isolated in 2006 with those isolated in 2016 against disinfectants and antimicrobial agents. The results showed that all the strains were highly susceptible to sodium hypochlorite, regardless of the conditions and year of isolation. Resistance to benzalkonium chloride varied according to the conditions applied, but not to the year of isolation. Increased antimicrobial resistance from 2006-2016 was observed only for tetracycline. The results suggest that the antimicrobial and disinfectant resistance of S. Heidelberg did not increase for ten years (2006-2016). However, further analysis should include a larger number of S. Heidelberg isolates from poultry origin and additional antimicrobial agents for more precise conclusions about the increasing in antimicrobial resistance in the last years.

ABSTRACT: Salmonella Heidelberg (S. Heidelberg) is a major pathogen implicated in foodborne outbreaks for which poultry products can serve as an epidemiological source. This study determined the efficacy of GRAS-status lemongrass essential oil (LGEO) against S. Heidelberg in vitro and on the pathogen's attachment to skin and meat. At first, employing in vitro assays, the effect of LGEO on multidrug-resistant S. Heidelberg multiplication and motility was examined. Biofilm inhibition and inactivation assays were also performed. The quorum-sensing modulating effect of LGEO was determined. In follow-up experiments, chicken skin or meat samples inoculated with S. Heidelberg were treated with various concentrations of LGEO at different time points at simulated scalding (54°C) and chilling (4°C) temperatures. The samples were incubated, and the surviving populations of S. Heidelberg were enumerated to determine if LGEO could be a potential processing aid in poultry operations. Duplicate samples were included in each treatment, and the experiments were repeated at least 3 times. Significant reductions of S. Heidelberg of at least 4.0 log10 CFU/mL after 24 h in nutrient broth and poultry cecal contents was observed with 0.5% LGEO. Complete inhibition of motility, biofilm formation, and inactivation of pre-formed biofilms was observed with 0.15% LGEO (P ≤ 0.05). Concentrations of LGEO at 0.5% and 1% affected violacein production (P ≤ 0.05). On skin samples, all concentrations significantly reduced S. Heidelberg by 1.2 to 3.9 log10 CFU/sample after 2 min at 54°C. We obtained a significant reduction of the pathogen in meat samples at 54°C and skin samples at 4°C with 2% LGEO. All concentrations significantly reduced S. Heidelberg from the treatment water kept at 4°C and 54°C (P ≤ 0.05). In conclusion, LGEO could potentially serve as a natural antimicrobial strategy in scalding and chilling waters to reduce S. Heidelberg during processing. However, additional studies are warranted before recommending its commercial use.

In the last 10 years, Salmonella Heidelberg has been extensively isolated from poultry in several countries. In this context, molecular characterization is essential to understand whether the strains have entered the farms from a single or several sources. Thus, the aim of this study was to determine the genetic relationship and antimicrobial susceptibility of S. Heidelberg strains isolated between 2011 and 2012 from broiler farms belonging to three integrated poultry companies located in Argentina. The genetic relatedness of the S. Heidelberg isolates was determined by pulsed‐field gel electrophoresis (PFGE), and resistance to 21 antimicrobials was determined by the disc diffusion method. The isolates were assigned to four PFGE patterns. Most of the strains showed 100% similarity and belonged to the same integrated poultry company. This PFGE pattern was also prevalent in S. Heidelberg strains isolated from humans in several provinces of Argentina, which suggests an epidemiological association between human and poultry strains. All the isolates were classified as multidrug‐resistant (MDR), and no clear relationship was observed between PFGE and resistance patterns. S. Heidelberg strains may circulate among farms from the same integrated company due to common sources of contamination. To guarantee the safety of the poultry product for the consumers, holistic approaches including surveillance of Salmonella throughout the production chain together with control measures are crucial. [ABSTRACT FROM AUTHOR]

Before starting a study with many birds, it helps to know the method of chick inoculation. The objective was to compare 3 methods of Salmonella challenge (oral gavage [OR], intracloacal inoculation [IC], and seeder bird [SB]). Day-old broiler chicks (n = 100) were inoculated with 106 colony forming units (CFU) per chick of a marker strain of Salmonella Heidelberg (SH) with each route of inoculation. Chicks (n = 25) inoculated by each route were placed in floor pens on fresh pine shavings litter. For the seeder batch, 5 colonized chicks, each orally gavaged with 106 CFUs, were placed with 20 pen mates. Two weeks after inoculation, 10 birds from each pen and the 5 inoculated seeder birds were euthanized, the ceca were aseptically removed and macerated with a rubber mallet and weighed, and 3 times (w/v) buffered peptone was added and stomached for 60 s. Serial dilutions were made and plated onto Brilliant Green Sulfa plates containing 200 ppm nalidixic acid. Plates were incubated along with the stomached ceca for 24 h at 37°C. If no colonies appeared on the plates, an additional plate was streaked from the preenriched bag and incubated for 24 h at 37°C. In addition to all seeder birds being positive, the number of SH-positive birds out of 20 sampled in each group was 13, 17, and 7 for OR, IC, and SB, respectively. The level of SH per g of ceca and cecal contents was log (SE) 3.0 (0.7), 2.0 (0.4), and 2.6 (0.4) for OR, IC, and SB, respectively. After enrichment, the number of colonized birds out of 20 was 18, 20, and 10 for OR, IC, and SB, respectively. In conclusion, this study suggests that IC is the method to use to ensure most of the challenged birds are colonized. However, if you prefer to have a smaller percentage of the birds colonized with higher levels, then OR might be better.

Abstract Salmonella enterica serotype Heidelberg is primarily a poultry adapted serotype of Salmonella that can also colonize other hosts and cause human disease. In this study, we compared the genomes of outbreak associated non-outbreak causing Salmonella ser. Heidelberg strains from diverse hosts and geographical regions. Human outbreak associated strains in this study were from a 2015 multistate outbreak of Salmonella ser. Heidelberg involving 15 states in the United States which originated from bull calves. Our clinicopathologic examination revealed that cases involving Salmonella ser. Heidelberg strains were predominantly young, less than weeks-old, dairy calves. Pre-existing or concurrent disease was found in the majority of the calves. Detection of Salmonella ser. Heidelberg correlated with markedly increased death losses clinically comparable to those seen in herds infected with S. Dublin, a known serious pathogen of cattle. Whole genome based single nucleotide polymorphism based analysis revealed that these calf isolates formed a distinct cluster along with outbreak associated human isolates. The defining feature of the outbreak associated strains, when compared to older isolates of S. Heidelberg, is that all isolates in this cluster contained Saf fimbrial genes which are generally absent in S. Heidelberg. The acquisition of several single nucleotide polymorphisms and the gain of Saf fimbrial genes may have contributed to the increased disease severity of these Salmonella ser. Heidelberg strains.

Summary: Previously, we reported that a dairy-originated probiotic bacterium, Propionibacterium freudenreichii subsp. freudenreichii B3523 (PF) reduced cecal colonization of multidrug-resistant Salmonella enterica serovar Heidelberg (SH) in poults (2-wk-old) and growing (7-wk-old) turkeys. In the present study, we validated the antibacterial potential of PF in finisher (12-wk-old) turkeys using 2 separate experiments. In each experiment, day-old poults were randomly allocated to 3 treatment groups, with 10 turkeys in each group (N = 30 turkeys/experiment). The treatment groups were a negative control (turkeys without PF supplementation or SH challenge), an SH control (SC; turkeys challenged with SH without PF supplementation), and a test group (PFS; turkeys supplemented with PF and challenged with SH). Supplementation of PF at 1012 cfu/5 gallons water was provided to the turkeys in the PFS groups for 12 wk. The turkeys in the SC and PFS groups were challenged with SH at 108 cfu/turkey at 11 wk of age. Five turkeys from each group were euthanized 2 and 7 d after SH challenge. The cecum, liver, and spleen samples were collected to determine colonization of SH in the cecum and SH dissemination to the targeted internal organs. Propionibacterium freudenreichii subsp. freudenreichii B3523 was recovered at 4.0 log10 cfu/g in the turkey cecum. Supplementation of PF reduced the cecal colonization of SH by >2.0 log10 cfu/g in finishing turkeys (P < 0.05). Similarly, PF supplementation reduced SH dissemination to the spleen (P < 0.05). Our investigations revealed that PF could be a biocontrol strategy to reduce Salmonella colonization and its spread to internal organs in turkeys.

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Purpose: Salmonellosis is one of several reportable diseases in Ontario (ON). Two or more cases of the same serotype that are linked to a common exposure or related to one another in time and/or space are considered a potential outbreak. While laboratory data can help to determine the molecular relatedness of cases, results may take up to several weeks. This study aimed to assess the utility of the retrospective spatial scan statistic in detecting clusters of Salmonella Heidelberg and Salmonella Typhimurium cases using data from ON in 2015. Identified clusters were validated by laboratory data (where available) to determine whether identified clusters were likely outbreaks. Methods: Data representing the location of each reported S. Heidelberg or S. Typhimurium case in 2015, responsible serotype and symptom onset date were exported to SaTScan for retrospective spatial, temporal, and space‐time analyses using the spatial scan statistic with Bernoulli models and a space‐time permutation model. Analyses were performed with and without those cases linked to known outbreaks. Laboratory subtyping data (i.e. pulsed field gel electrophoresis (PFGE) and/or phage type) and food and environmental exposure information (e.g. travel, animal contact, poultry and other food item consumption) were used to explore the relatedness of cases within identified clusters. Results: Spatial, temporal and space‐time analyses identified a known outbreak of S. Heidelberg in 2015 (n = 9 cases) and a previously unidentified cluster of S. Heidelberg cases. Most cases (94%) within a cluster detected via a space‐time permutation model of S. Heidelberg cases shared an identical PFGE pattern and appeared to represent a true outbreak. Conclusions: The spatial scan statistic, and particularly the space‐time permutation model, could assist in outbreak identification before laboratory data are available, allowing for faster cluster identification and implementation of control measures. [ABSTRACT FROM AUTHOR]

Salmonella Heidelberg and Salmonella Typhimurium are among the most common serotypes responsible for human salmonellosis in Ontario. Introduction of the Ontario Investigation Tools (OIT) in 2014 allowed for standardized case investigation and reporting. This study compared the risk factors and symptomatology for sporadic S. Heidelberg and S. Typhimurium cases reported in Ontario in 2015, following implementation of the OIT. Multilevel logistic regression models were applied to assess associations between serotype and individual‐level demographic characteristics, exposures and symptoms for sporadic confirmed cases of S. Heidelberg and S. Typhimurium in Ontario in 2015. There were 476 sporadic cases of S. Typhimurium (n = 278) and S. Heidelberg (n = 198) reported in Ontario in 2015. There were significant associations between the odds of the isolate from a case being one of these serotypes, and travel, consumption of sprouts (any type), contact with reptiles and development of malaise, fever or bloody diarrhoea. The S. Typhimurium and S. Heidelberg cases differed in both symptom presentation and risk factors for illness. Case–case comparisons of Salmonella serotypes have some advantages over case–control studies in that these are less susceptible to selection and recall bias while allowing for rapid comparison of cases to identify potential high‐risk exposures that are unique to one of the serotypes when compared to the other. Comparing cases of two different Salmonella serotypes can help to highlight risk factors that may be uniquely associated with one serotype, or more strongly associated with one serotype compared to another. This information may be useful for understanding relative source attribution between common serotypes of Salmonella. [ABSTRACT FROM AUTHOR]

There is limited research concerning the biofilm-forming capabilities of Salmonella Kentucky, a common poultry isolate. The objective was to quantitate pellicle formation of S. Kentucky versus better-characterized Salmonella strains of Enteritidis and Heidelberg. In separate experiments, Salmonella strains and serovars were tested for their biofilm-forming abilities in different Luria-Bertani (LB) broths (1); pellicle formation in different volumes of LB without salt (2); and the potential priming effects on formation after pellicles were transferred three consecutive times (3). Data were analyzed using One-Way ANOVA with means separated using Tukey's HSD (P ≤ 0.05). In the first experiment, there was no significant effect between strain and serovars (P > 0.05), but media type affected pellicle formation significantly with LB Miller and LB minus NaCl plus 2% glucose resulting in no pellicle formation (P < 0.001). When grown in 50 mL, Kentucky 38-0085 produced larger pellicles than Kentucky 38-0055, and Heidelberg strain 38-0127 (P < 0.0001). Serial transfers of pellicles did not significantly affect pellicle formation (P > 0.05); however, Kentucky 38-0084, 38-0085 and 38-0086 produced larger pellicles than Kentucky 38-0055 and 38-0056 and Heidelberg 38-0126, 38-0127 and 38-0152. The current study demonstrates the consistent biofilm forming capabilities of Kentucky and may explain why Kentucky is frequently isolated in poultry processing facilities. [ABSTRACT FROM AUTHOR]

Drinking water contaminated with Salmonella could serve as a source for cecal colonization of the pathogen in birds. In this study, we investigated the efficacy of a generally recognized as safe—status essential oil (lemongrass essential oil, LGEO) against multidrug-resistant Salmonella enterica serovar Heidelberg (S. Heidelberg) in poultry drinking water. Farm water with and without added droppings, litter, or feed [0.5% (w/v)] inoculated with 6.0 log10 cfu/mL S. Heidelberg was treated with 0, 0.03, 0.06, 0.125, 0.25, or 0.5% (v/v) LGEO and incubated at 12.5°C or 22°C for up to 7 d. The number of viable S. Heidelberg populations was determined on d 0, 1, 3, 5, and 7. At 12.5°C, all concentrations of LGEO inactivated S. Heidelberg to non-detectable levels in water alone and water contaminated with droppings or litter by d 7 (>6 log10 cfu/mL; P < 0.05). The highest LGEO concentration tested (0.5%) resulted in >5.0 log10 cfu/mL reduction of S. Heidelberg on d 7 in water contaminated with feed (P < 0.05). At 22°C, all concentrations of LGEO resulted in the reduction of S. Heidelberg to non-detectable levels in water alone by d 5 (>6 log10 cfu/mL; P < 0.05). Concentrations of LGEO ≥0.125% resulted in inhibition of S. Heidelberg to non-detectable levels in water contaminated with droppings from d 5 onwards (P < 0.05). The LGEO at the highest tested concentration (0.5%) resulted in >5.0 log10 cfu/mL reduction of S. Heidelberg in water contaminated with litter on d 7 (P < 0.05) but did not result in reduction of the pathogen in water contaminated with feed at 22°C. Results indicate the potential of LGEO against S. Heidelberg in water alone or water containing droppings or litter, indicating its efficacy as a natural disinfectant in poultry drinking water. [ABSTRACT FROM AUTHOR]

The vast capacity for maintenance and dissemination in the environment are major challenges for the control of Salmonella spp. in poultry farms. The aim of this study was to assess environmental contamination by non-typhoidal Salmonella in successive broiler flocks in nine commercial broiler farms integrated with three companies in the south of Brazil, for a twelve-month production period. Recycled broiler litter, feed and swabs from the evaporative cooling system pads were analyzed, and the total enterobacteria count in the litter samples was ascertained. Positive broiler houses were identified in two of the three broiler companies studied, in which non-typhoidal Salmonella were detected for the first time in the first or second flock, and recurred in the recycled litter of subsequent flocks. Feed and evaporative cooling pad swab samples were also positive in at least one of the assessed flocks. The majority of the isolates (87.5%) originating from different flocks, broiler houses and companies that were sampled were identified as S. Heidelberg, with the prevalence of one single genotype. The total enterobacteria levels in the litter diminished as the flocks progressed, but the presence of Salmonella spp. was constant over the course of time, indicating that the litter management procedures were not capable of interrupting the cycle of residual contamination. The predominance of S. Heidelberg highlights its emergence and dissemination in this region, as well as its resistance and maintenance in the environment, and reinforces the need to improve prevention and recycled litter management measures. [ABSTRACT FROM AUTHOR]

We investigated the antibacterial efficacy of a dairy-origin probiotic bacterium, Propionibacterium freudenreichii freudenreichii B3523 (PF), on a multidrug-resistant strain of Salmonella enterica serovar Heidelberg (SH) in growing turkeys. In 2 independent experiments, day-old poults (N = 24/experiment) were randomly assigned to 3 treatment groups (8 turkeys/group). The groups were: a negative control (NC; turkeys without PF supplementation or SH challenge), an SH control (SC; turkeys challenged with SH without PF supplementation), and a test group (PFS; turkeys supplemented with PF and challenged with SH) raised until 7 wk of age. Turkeys in the PFS group received PF once in 2 d at 1012 cfu in 5 gallon drinking water from day 0 until 6 wk of age. Turkeys in SC and PFS groups were challenged with SH as crop gavage at 6 wk of age. After SH inoculation, the PFS group received PF daily for 7 d until the end of the study. The colonization of SH and PF in turkey ceca was determined 2 and 7 d post-inoculation of the pathogen. In addition, the effect of PF supplementation on the dissemination of SH to liver and spleen was determined. Results indicated that PF was recoverable at 4 to 4.5 log10 cfu/g at the end of the study. Additionally, PF supplementation significantly reduced SH colonization in the PFS group by 1 to 1.3 log10 cfu/g in the turkey ceca 2 and 7 d after SH inoculation (P ≤ 0.05), compared to the SC group. In addition, PF supplementation reduced dissemination of SH to liver and spleen (P ≤ 0.05), compared to the SC group. Results indicate that PF could be a potential probiotic strategy in turkeys to control SH colonization at a later stage of turkey production. [ABSTRACT FROM AUTHOR]

Effects of turkey-derived beneficial bacteria Lactobacillus ingluviei UMNPBX19 and Lactobacillus salivarius UMNPBX2 on Salmonella Heidelberg (SH) in turkey poults was investigated. Using in vitro studies, we determined each strain's resistance to pH 2.5 and 0.3% bile salts and their β-hemolysis activity. We also tested each strain's adherence to avian epithelial cells and exhibition of antimicrobial activity against major poultry-associated Salmonella. Moreover, using three in vivo experiments, we determined the effect of the strains in combination (LBIS) against SH in turkey poults. The treatment groups were negative control (−SH, −LBIS), SH control (+SH, −LBIS), and LBIS group (+SH, +LBIS). Supplementation of LBIS was done in drinking water throughout the study at a dose of 8 log CFU/gal. On day 7, poults were challenged with a 2011 ground turkey outbreak strain of SH at 5 × 105 CFU/mL, and the surviving pathogens were determined on day 7 postinoculation from the cecum, spleen, and liver. Both Lactobacillus strains exerted resistance to low pH and bile salts (P < 0.05), showed adhesion to epithelial cells (P < 0.05), but did not exhibit β-hemolysis. Cell-free culture supernatants of strains showed antimicrobial activity against Salmonella (P < 0.05). Results from the in vivo studies revealed that LBIS significantly reduced dissemination of SH to the liver and spleen in all experiments, and colonization in the cecum in two of the three experiments (1.9- and 3.9-log CFU/g reductions), compared with the control. The results indicate that turkey-derived L. ingluviei UMNPBX19 and L. salivarius UMNPBX2 have potential beneficial effects against SH in turkeys. However, more studies to this effect are warranted. [ABSTRACT FROM AUTHOR]

In this study we report the characterization of plasmid-mediated CMY-2-producing Salmonella Heidelberg recovered from food, poultry, and poultry environment in Brazil, between 2014 and 2016. The blaCMY-2 resistance gene was allocated in large (90–148 kb) IncI1 type transferable plasmids. Salmonella Heidelberg isolates were genetically related, indicating the dissemination of closely related isolates among food, poultry, and its environment. This is the first report of IncI1 replicon-types of plasmids encoding the blaCMY-2 resistance gene in Salmonella Heidelberg isolates in Brazil, the world's biggest exporter of chickens. [ABSTRACT FROM AUTHOR]

This study was conducted to evaluate the effectiveness of different levels of Saccharomyces cerevisiae yeast cell wall or a yeast culture in reducing the direct colonization and horizontal transmission of Salmonella Heidelberg in broiler chickens. At d of hatch, 2,000 male broiler chicks were randomly assigned into 5 treatment groups with 8 replicate pens per treatment. Birds were challenged with nalidixic acid-resistant S. Heidelberg either by direct inoculation of 107 cfu orally (seeders) or by horizontal transmission through inoculated penmates (contacts). Yeast cell wall (YCW) at 500 ppm decreased salmonella prevalence of the total infected birds (seeders and contacts) to 41.7% as compared to 54.2% in the untreated birds. The effect of YCW in reducing prevalence of positive birds was even greater when we considered the contact birds only; 32.5% in contact birds treated with 500 ppm as compared to 57.5% in the untreated group (P = 0.09). Furthermore, enumeration of S. Heidelberg colonization level in the cecum using the most probable number (MPN) method showed that YCW at 500 ppm reduced the bacterial load in the cecum of positive birds to 1.7 MPN/g as compared to 2.7 MPN/g in the control group. The reduction was statistically significant (P = 0. 04) in the contact birds. The same effect was not seen in the yeast culture treatment. [ABSTRACT FROM AUTHOR]

Abstract We report a novel RNase H2-dependent PCR (rhPCR) genotyping assay for a small number of discriminatory single-nucleotide polymorphisms (SNPs) that identify lineages and sub-lineages of the highly clonal pathogen Salmonella Heidelberg (SH). Standard PCR primers targeting numerous SNP locations were initially designed in silico , modified to be RNase H2-compatible, and then optimized by laboratory testing. Optimization often required repeated cycling through variations in primer design, assay conditions, reagent concentrations and selection of alternative SNP targets. The final rhPCR assay uses 28 independent rhPCR reactions to target 14 DNA bases that can distinguish 15 possible lineages and sub-lineages of SH. On evaluation, the assay correctly identified the 12 lineages and sub-lineages represented in a panel of 75 diverse SH strains. Non-specific amplicons were observed in 160 (15.2%) of the 1050 reactions, but due to their low intensity did not compromise assay performance. Furthermore, in silico analysis of 500 closed genomes from 103 Salmonella serovars and laboratory rhPCR testing of five prevalent Salmonella serovars including SH indicated the assay can identify Salmonella isolates as SH, since only SH isolates generated amplicons from all 14 target SNPs. The genotyping results can be fully correlated with whole genome sequencing (WGS) data in silico. This fast and economical assay, which can identify SH isolates and classify them into related or unrelated lineages and sub-lineages, has potential applications in outbreak identification, source attribution and microbial source tracking. Highlights • Highly clonal Salmonella Heidelberg requires discriminatory subtyping methods. • Current subtyping by PFGE and phage typing lacks discriminatory power. • A novel RNase H2-dependent PCR genotyping assay meets this need. • Genotyping results correlate fully with WGS data in silico. • Potentially useful in outbreak identification, source attribution and tracking. [ABSTRACT FROM AUTHOR]

During the last years, Brazilian government control programs have detected an increase of Salmonella Heidelberg in poultry slaughterhouses a condition that poses a threat to human health However, the reasons remain unclear. Differences in genetic virulence profiles may be a possible justification. In addition, effective control of Salmonella is related to an efficient epidemiological surveillance system through genotyping techniques. In this context, the aim of this study was the detection of 24 virulence-associated genes in 126 S. Heidelberg isolates. We classified the isolates into 56 different genetic profiles. None of the isolates presented all the virulence genes. The prevalence of these genes was high in all tested samples as the lowest number of genes detected in one isolate was 10/24. The lpfA and csgA (fimbriae), invA and sivH (TTSS), and msgA and tolC (intracellular survival) genes were present in 100% of the isolates analyzed. Genes encoding effector proteins were detected in the majority of SH isolates. No single isolate had the sefA gene. The pefA gene was found in only four isolates. We have also performed a screening of genes associated with iron metabolism: 88.9% of isolates had the iroN geneand 79.4% the sitC gene. Although all the isolates belong to the same serotype, several genotypic profiles were observed. These findings suggest that there is a diversity of S. Heidelberg isolates in poultry products. The fact that a single predominant profile was not found in this study indicates the presence of variable sources of contamination caused by SH. The detection of genetic profiles of Salmonella strains can be used to determine the virulence patterns of SH isolates. [ABSTRACT FROM AUTHOR]

Salmonella is a burden to the poultry, health, and food safety industries, resulting in illnesses, food contamination, and recalls. Salmonella enterica subspecies enterica Enteritidis (S. Enteritidis) is one of the most prevalent serotypes isolated from poultry. Salmonella enterica subspecies enterica Heidelberg (S. Heidelberg), which is becoming as prevalent as S. Enteritidis, is one of the five most isolated serotypes. Although S. Enteritidis and S. Heidelberg are almost genetically identical, they both are capable of inducing different immune and metabolic responses in host cells to successfully establish an infection. Therefore, using the kinome peptide array, we demonstrated that S. Enteritidis and S. Heidelberg infections induced differential phosphorylation of peptides on Rho proteins, caspases, toll-like receptors, and other proteins involved in metabolic- and immune-related signaling of HD11 chicken macrophages. Metabolic flux assays measuring extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) demonstrated that S. Enteritidis at 30 min postinfection (p.i.) increased glucose metabolism, while S. Heidelberg at 30 min p.i. decreased glucose metabolism. S. Enteritidis is more invasive than S. Heidelberg. These results show different immunometabolic responses of HD11 macrophages to S. Enteritidis and S. Heidelberg infections.

Salmonella is one of the leading causes of human foodborne gastroenteritis in the United States. In addition, Salmonella contributes to morbidity and mortality in livestock. The control of Salmonella is an increasing problematic issue in livestock production due to lack of effective control methods and the constant adaptation of Salmonella to new management practices, which is often related to horizontal acquisition of virulence or antibiotic resistance genes. Salmonella enterica serotype Heidelberg is one of the most commonly isolated serotypes in all poultry production systems in North America. Emergence and persistence of multi-drug resistant Salmonella Heidelberg isolates further impact the poultry production and public health. We hypothesized that distinct poultry production environments affect Salmonella genomic content, and by consequence its survival and virulence abilities. This study compared the genomic composition of S. Heidelberg isolated from environmental samples (19 chicken and 12 turkey isolates) of different breeder farms (16 chicken and 8 turkey farms) in the Midwest, United States. Whole genome comparison of 31 genomes using RAST and SEED identified differences in specific sub-systems in isolates between the chicken- and turkey-associated farm environmental samples. Genes associated with the type IV secretion system (n = 12) and conjugative transfer (n = 3) were absent in turkey farm isolates compared to the chicken ones (p-value < 0.01); Further, turkey farm isolates were enriched in prophage proteins (n = 53; p-value < 0.01). Complementary studies using PHASTER showed that prophages were all Caudovirales phages and were more represented in turkey environmental isolates than the chicken isolates. This study corroborates that isolates from distinct farm environment show differences in S. Heidelberg genome content related to horizontal transfer between bacteria or through viral infections. Complementary microbiome studies of these samples would provide critical insights on sources of these variations. Overall, our findings enhance the understanding of Salmonella genome plasticity and may aid in the development of future effective management practices to control Salmonella.

Antimicrobial potential of a dairy-origin probiotic bacteria, Propionibacterium freudenreichii, against multidrug-resistant Salmonella Heidelberg (SH) in turkey poults was determined in the current study. Employing in vitro experiments, two strains (subsp.) of P. freudenreichii: P. freudenreichii freudenreichii B3523 (PF) and P. freudenreichii shermanii B4327 (PS) were tested for their ability to resist low pH (2.5) and bile salts (0.3%). In addition, the ability of the strains to adhere to and invade avian epithelial cells was determined after exposure to Propionibacterium strains followed by SH challenge. Moreover, the antibacterial activity of the strains’ cell-free culture supernatants (CFCSs) were tested against three major foodborne pathogens, including SH. Furthermore, the susceptibility of the strains to common antibiotics used for human therapy was determined. The hemolytic properties of the strains were determined in comparison to Streptococcus pyogenes, a known hemolysis-causing pathogen. Appropriate controls were kept in all studies. Using two in vivo experiments, PF was tested against SH colonization of poult ceca and dissemination to liver and spleen. The four treatment groups were: negative control, PF control (PFC), SH control (SC), and a test group (PFS; PF + SH). The poults in the PFC and PFS groups were inoculated with 1010 CFU ml−1 PF on day 1 through crop gavage and subsequently supplemented through drinking water. On day 7, SC and PFS groups were challenged with SH at 106 CFU ml−1, and after 7 days, cecum, liver, and spleen were collected for determining surviving SH populations. Results indicated that both PF and PS resisted pH = 2.5 and 0.3% bile salts with surviving populations comparable to the control and adhered well onto the avian epithelial cell lines. The strains were susceptible to antibiotics and did not invade the epithelial cells or exhibit hemolytic properties. The CFCSs were highly bactericidal against all tested pathogens. In turkey poults, PF significantly reduced cecal colonization of SH and the dissemination of the pathogen to the liver, compared to the SH challenge controls (P < 0.05). Results revealed that PF, a non-host gastrointestinal tract-derived probiotic, could be an antibiotic alternative to prevent the early colonization of SH in poults, improving the preharvest safety of turkeys.

Salmonella enterica serovar Heidelberg (S. Heidelberg) is a major foodborne pathogen colonizing poultry. The pathogen is associated with a significant number of foodborne outbreaks through contaminated poultry meat, including turkeys. Recently, multidrug-resistant (MDR) strains of S. Heidelberg have emerged as a threat to human public health in the United States. The objective of this study was to determine the cecal colonization, dissemination to internal organs, and the potential for skeletal muscle deposition of an MDR S. Heidelberg isolate from the 2011 ground turkey outbreak in the United States after the experimental oral challenge of poults (young turkeys) and adult turkey hens. In the poult study, two separate experiments using day-old, straight-run, commercial hybrid converter poults were randomly assigned to five challenge groups (0, 10∧2, 10∧4, 10∧6, 10∧8 CFU groups; 12 poults/group; N = 60/experiment) and a week after, treatment groups were challenged separately with 0-, 2-, 4-, 6-, and 8- log10 CFU of S. Heidelberg orally. After 14 days post-challenge, the poults were euthanized, and samples were collected to determine MDR S. Heidelberg colonization in the cecum, dissemination to liver and spleen, and deposition in the thigh, drumstick, and breast muscles. A similar experimental design was followed for the adult turkey hens. In two separate experiments, 11-week-old commercial Hybrid Converter turkey hens (4 hens/group; N = 20/experiment) were challenged with MDR S. Heidelberg and on day 16 post-challenge, birds were euthanized and samples were collected to determine Salmonella populations in the samples. The results indicated that, in turkey poults, the recovery of MDR S. Heidelberg was highest in the cecum followed by spleen, liver, thigh, drumstick, and breast. All tested inoculum levels resulted in more than 3.5 log10 CFU/g colonization in the poult cecum. The cecal colonization, dissemination to internal organs, and tissue deposition of MDR S. Heidelberg were high in poults. The pathogen recovery from the cecum of adult turkey hens ranged from 37.5 to 62.5% in the challenge groups. The results signify the importance of controlling MDR S. Heidelberg in turkeys at the farm level to improve the safety of turkey products.

Before starting a study with many birds, it helps to know the method of chick inoculation. The objective was to compare 3 methods of Salmonella challenge (oral gavage [OR], intracloacal inoculation [IC], and seeder bird [SB]). Day-old broiler chicks (n = 100) were inoculated with 106 colony forming units (CFU) per chick of a marker strain of Salmonella Heidelberg (SH) with each route of inoculation. Chicks (n = 25) inoculated by each route were placed in floor pens on fresh pine shavings litter. For the seeder batch, 5 colonized chicks, each orally gavaged with 106 CFUs, were placed with 20 pen mates. Two weeks after inoculation, 10 birds from each pen and the 5 inoculated seeder birds were euthanized, the ceca were aseptically removed and macerated with a rubber mallet and weighed, and 3 times (w/v) buffered peptone was added and stomached for 60 s. Serial dilutions were made and plated onto Brilliant Green Sulfa plates containing 200 ppm nalidixic acid. Plates were incubated along with the stomached ceca for 24 h at 37°C. If no colonies appeared on the plates, an additional plate was streaked from the preen-riched bag and incubated for 24 h at 37°C. In addition to all seeder birds being positive, the number of SH-positive birds out of 20 sampled in each group was 13, 17, and 7 for OR, IC, and SB, respectively. The level of SH per g of ceca and cecal contents was log (SE) 3.0 (0.7), 2.0 (0.4), and 2.6 (0.4) for OR, IC, and SB, respectively. After enrichment, the number of colonized birds out of 20 was 18, 20, and 10 for OR, IC, and SB, respectively. In conclusion, this study suggests that IC is the method to use to ensure most of the challenged birds are colonized. However, if you prefer to have a smaller percentage of the birds colonized with higher levels, then OR might be better. [ABSTRACT FROM AUTHOR]

Salmonella is one of the leading causes of human foodborne gastroenteritis in the United States. In addition, Salmonella contributes to morbidity and mortality in livestock. The control of Salmonella is an increasing problematic issue in livestock production due to lack of effective control methods and the constant adaptation of Salmonella to new management practices, which is often related to horizontal acquisition of virulence or antibiotic resistance genes. Salmonella enterica serotype Heidelberg is one of the most commonly isolated serotypes in all poultry production systems in North America. Emergence and persistence of multi-drug resistant Salmonella Heidelberg isolates further impact the poultry production and public health. We hypothesized that distinct poultry production environments affect Salmonella genomic content, and by consequence its survival and virulence abilities. This study compared the genomic composition of S. Heidelberg isolated from environmental samples (19 chicken and 12 turkey isolates) of different breeder farms (16 chicken and 8 turkey farms) in the Midwest, United States. Whole genome comparison of 31 genomes using RAST and SEED identified differences in specific sub-systems in isolates between the chicken- and turkey-associated farm environmental samples. Genes associated with the type IV secretion system (n = 12) and conjugative transfer (n = 3) were absent in turkey farm isolates compared to the chicken ones (p -value < 0.01); Further, turkey farm isolates were enriched in prophage proteins (n = 53; p -value < 0.01). Complementary studies using PHASTER showed that prophages were all Caudovirales phages and were more represented in turkey environmental isolates than the chicken isolates. This study corroborates that isolates from distinct farm environment show differences in S. Heidelberg genome content related to horizontal transfer between bacteria or through viral infections. Complementary microbiome studies of these samples would provide critical insights on sources of these variations. Overall, our findings enhance the understanding of Salmonella genome plasticity and may aid in the development of future effective management practices to control Salmonella. [ABSTRACT FROM AUTHOR]

Salmonella enterica serovar Heidelberg ( S. Heidelberg) is one of the top serovars causing human salmonellosis. This serovar ranks second and third among serovars that cause human infections in Québec and Canada, respectively, and has been associated with severe infections. Traditional typing methods such as PFGE do not display adequate discrimination required to resolve outbreak investigations due to the low level of genetic diversity of isolates belonging to this serovar. This study evaluates the ability of four whole genome sequence (WGS)-based typing methods to differentiate among 145  S. Heidelberg strains involved in four distinct outbreak events and sporadic cases of salmonellosis that occurred in Québec between 2007 and 2016. Isolates from all outbreaks were indistinguishable by PFGE. The core genome single nucleotide variant (SNV), core genome multilocus sequence typing (MLST) and whole genome MLST approaches were highly discriminatory and separated outbreak strains into four distinct phylogenetic clusters that were concordant with the epidemiological data. The clustered regularly interspaced short palindromic repeats (CRISPR) typing method was less discriminatory. However, CRISPR typing may be used as a secondary method to differentiate isolates of S. Heidelberg that are genetically similar but epidemiologically unrelated to outbreak events. WGS-based typing methods provide a highly discriminatory alternative to PFGE for the laboratory investigation of foodborne outbreaks. [ABSTRACT FROM AUTHOR]

Antimicrobial potential of a dairy-origin probiotic bacteria, Propionibacterium freudenreichii, against multidrug-resistant Salmonella Heidelberg (SH) in turkey poults was determined in the current study. Employing in vitro experiments, two strains (subsp.) of P. freudenreichii: P. freudenreichii freudenreichii B3523 (PF) and P. freudenreichii shermanii B4327 (PS) were tested for their ability to resist low pH (2.5) and bile salts (0.3%). In addition, the ability of the strains to adhere to and invade avian epithelial cells was determined after exposure to Propionibacterium strains followed by SH challenge. Moreover, the antibacterial activity of the strains’ cell-free culture supernatants (CFCSs) were tested against three major foodborne pathogens, including SH. Furthermore, the susceptibility of the strains to common antibiotics used for human therapy was determined. The hemolytic properties of the strains were determined in comparison to Streptococcus pyogenes, a known hemolysis-causing pathogen. Appropriate controls were kept in all studies. Using two in vivo experiments, PF was tested against SH colonization of poult ceca and dissemination to liver and spleen. The four treatment groups were: negative control, PF control (PFC), SH control (SC), and a test group (PFS; PF + SH). The poults in the PFC and PFS groups were inoculated with 1010 CFU ml−1 PF on day 1 through crop gavage and subsequently supplemented through drinking water. On day 7, SC and PFS groups were challenged with SH at 106 CFU ml−1, and after 7 days, cecum, liver, and spleen were collected for determining surviving SH populations. Results indicated that both PF and PS resisted pH = 2.5 and 0.3% bile salts with surviving populations comparable to the control and adhered well onto the avian epithelial cell lines. The strains were susceptible to antibiotics and did not invade the epithelial cells or exhibit hemolytic properties. The CFCSs were highly bactericidal against all tested pathogens. In turkey poults, PF significantly reduced cecal colonization of SH and the dissemination of the pathogen to the liver, compared to the SH challenge controls (P < 0.05). Results revealed that PF, a non-host gastrointestinal tract-derived probiotic, could be an antibiotic alternative to prevent the early colonization of SH in poults, improving the preharvest safety of turkeys. [ABSTRACT FROM AUTHOR]

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