Co-exposure to polyethylene fiber and Salmonella enterica serovar Typhimurium alters microbiome and metabolome of in vitro chicken cecal mesocosms.

Humans and animals encounter a summation of exposures during their lifetime (the exposome). In recent years, the scope of the exposome has begun to include microplastics. Microplastics (MPs) have increasingly been found in locations, including in animal gastrointestinal tracts, where there could be an interaction with Salmonella enterica serovar Typhimurium, one of the commonly isolated serovars from processed chicken. However, there is limited knowledge on how gut microbiomes are affected by microplastics and if an effect would be exacerbated by the presence of a pathogen. In this study, we aimed to determine if acute exposure to microplastics in vitro altered the gut microbiome membership and activity. The microbiota response to a 24 h co-exposure to Salmonella enterica serovar Typhimurium and/or low-density polyethylene (PE) microplastics in an in vitro broiler cecal model was determined using 16S rRNA amplicon sequencing (Illumina) and untargeted metabolomics. Community sequencing results indicated that PE fiber with and without S. Typhimurium yielded a lower Firmicutes/Bacteroides ratio compared with other treatment groups, which is associated with poor gut health, and overall had greater changes to the cecal microbial community composition. However, changes in the total metabolome were primarily driven by the presence of S. Typhimurium. Additionally, the co-exposure to PE fiber and S. Typhimurium caused greater cecal microbial community and metabolome changes than either exposure alone. Our results indicate that polymer shape is an important factor in effects resulting from exposure. It also demonstrates that microplastic-pathogen interactions cause metabolic alterations to the chicken cecal microbiome in an in vitro chicken cecal mesocosm. IMPORTANCE: Researching the exposome, a summation of exposure to one's lifespan, will aid in determining the environmental factors that contribute to disease states. There is an emerging concern that microplastic-pathogen interactions in the gastrointestinal tract of broiler chickens may lead to an increase in Salmonella infection across flocks and eventually increased incidence of human salmonellosis cases. In this research article, we elucidated the effects of acute co-exposure to polyethylene microplastics and Salmonella enterica serovar Typhimurium on the ceca microbial community in vitro. Salmonella presence caused strong shifts in the cecal metabolome but not the microbiome. The inverse was true for polyethylene fiber. Polyethylene powder had almost no effect. The co-exposure had worse effects than either alone. This demonstrates that exposure effects to the gut microbial community are contaminant-specific. When combined, the interactions between exposures exacerbate changes to the gut environment, necessitating future experiments studying low-dose chronic exposure effects with in vivo model systems.

General media over enrichment media supports growth of Campylobacter jejuni and maintains poultry cecal microbiota enabling translatable in vitro microbial interaction experiments.

AIM: This study aimed to assess the suitability of two media types, Bolton enrichment broth (BEB) and anaerobic dilution solution (ADS), in replicating the poultry cecal environment to investigate metabolic interactions and Campylobacter presence within poultry ceca. METHODS: Using an anaerobic in vitro poultry cecal model, cecal contents (free of culturable Campylobacter) were diluted in BEB and ADS, inoculated with 105 CFU of Campylobacter jejuni, and incubated for 48 h at 42°C under microaerophilic conditions. Samples were collected at 0, 24, and 48 h. Genomic DNA was extracted, amplified, and sequenced on Illumina MiSeq platform. Data underwent analysis within QIIME2-2021.11, including alpha and beta diversity assessments, ANOVA, ADONIS, ANCOM, and Bradford assay for protein concentration. RESULTS: ADS supported a more diverse microbial population than BEB, influencing C. jejuni presence. ANCOM highlighted dominant genera in BEB (Lactobacillus and Campylobacter) and affirmed C. jejuni growth in ADS. Core microbiota analysis revealed unique associations with each media type, while the Bradford assay indicated ADS consistently yielded more uniform microbial growth. CONCLUSIONS: ADS was identified as a preferred diluent for faithfully replicating cecal microbial changes in the presence of Campylobacter.

Comparison of optical density-based growth kinetics for pure culture Campylobacter jejuni, coli and lari grown in blood-free Bolton broth.

Campylobacter growth kinetic parameters can be used to refine the sensitivity and efficiency of microbial growth-based methods. Therefore, the aim of this study was to construct growth curves for C. jejuni, C. coli, and C. lari in pure culture and calculate growth kinetics for each Campylobacter species in the same environmental conditions. Campylobacter jejuni, C. coli and C. lari were grown over 48 h and inoculated into 15 mL Hungate tubes (N = 3 trials per species; 5 biological replicates per trial; 3 species; 1 strain per species). Absorbance measurements were taken in 45 min intervals over 24 h. Optical density readings were plotted versus time to calculate growth kinetic parameters. C. jejuni exhibited the longest lag phase (p < 0.001) at 15 h 20 min ± 30 min, versus C. coli at 11 h 15 min ± 17 min, and C. lari at 9 h 27 min ± 15 min. The exponential phase duration was no longer than 5 h for all species, and doubling times were all less than 1h 30 min. The variation in growth kinetics for the three species of Campylobacter illustrates the importance of determining individual Campylobacter spp. growth responses for optimizing detection based on low bacterial levels. This study provides kinetics and estimates to define enrichment times necessary for low concentration Campylobacter detection.

Salmonella in eggs and egg-laying chickens: pathways to effective control.

Eggs contaminated with Salmonella have been internationally significant sources of human illness for several decades. Most egg-associated illness has been attributed to Salmonella serovar Enteritidis, but a few other serovars (notably S. Heidelberg and S. Typhimurium) are also sometimes implicated. The edible interior contents of eggs typically become contaminated with S. Enteritidis because the pathogen's unique virulence attributes enable it to colonize reproductive tissues in systemically infected laying hens. Other serovars are more commonly associated with surface contamination of eggshells. Both research and field experience have demonstrated that the most effective overall Salmonella control strategy in commercial laying flocks is the application of multiple interventions throughout the egg production cycle. At the preharvest (egg production) level, intervention options of demonstrated efficacy include vaccination and gastrointestinal colonization control via treatments such as prebiotics, probiotics, and bacteriophages, Effective environmental management of housing systems used for commercial laying flocks is also essential for minimizing opportunities for the introduction, transmission, and persistence of Salmonella in laying flocks. At the postharvest (egg processing and handling) level, careful regulation of egg storage temperatures is critical for limiting Salmonella multiplication inside the interior contents.

Variations in bacterial community structure and antimicrobial resistance gene abundance in cattle manure and poultry litter.

Cattle manure and poultry litter are widely used as fertilizers as they are excellent sources of nutrients; however, potential adverse environmental effects exist during land applications, due to the release of zoonotic bacteria and antimicrobial resistance (AMR) genes. This study was conducted to understand linkages between physiochemical composition, bacterial diversity, and AMR gene presence of cattle manure and poultry litter using quantitative polymerase chain reaction to enumerate four AMR genes (ermB, sulI, intlI, and blactx-m-32), Illumina sequencing of the 16 S region, and analysis of physical and chemical properties. Principal coordinate analysis of Bray-Curtis distance revealed distinct bacterial community structures between the two manure sources. Greater alpha diversity occurred in cattle manure compared to poultry litter (P < 0.05). Redundancy analysis showed a strong relationship between manure physiochemical and composition and bacterial abundance, with positive relationships occurring among electrical conductivity and carbon/nitrogen, and negative associations for total solids and soluble fractions of heavy metals. Cattle manure exhibited greater abundance of macrolide (ermB) and sulfonamide (sulI) resistant genes. Consequently, fresh cattle manure applications may result in greater potential spread of AMR genes to the soil-water environment (relative to poultry litter) and novel best management strategies (such as composting) may reduce the release of AMR genes to the soil-water environment.

Identification of bacterial isolates from commercial poultry feed via 16S rDNA.

The study's objective was to identify typical aerobic isolates from commercial, corn-soybean meal poultry diets utilizing 16S rDNA, assign them their corresponding taxonomy, and compare the data with the previously published WGS analysis of these same isolates. Ten grams of a commercial corn-soybean meal poultry diet was homogenized in 100 mL of tryptic soy broth for 2 min, serially diluted, plated onto tryptic soy agar (TSA), and incubated aerobically for 24 h at 37 °C. Subsequently, 20 unique colonies were streaked for isolation on TSA and incubated aerobically for 24 h at 37 °C. This process was repeated three consecutive times for purification of isolates until only 11 morphologically distinct colonies were obtained. DNA was extracted using Qiagen's DNeasey® Blood and Tissue Kit. The 16S rRNA V4 region was targeted using an Illumina MiSeq and analyzed via QIIME2-2020.2. Alpha diversity and Beta diversity metrics were generated, and taxa were aligned using Silva in Qiime2-2020.2. Twenty-five distinct genera were identified within the 11 different colonies. Because 16S rDNA identification can provide an understanding of pathogen associations and microbial niches within an ecosystem, the information may present a potential method to establish and characterize the hygienic indicator microorganisms associated with poultry feed.

The biological effects of microencapsulated organic acids and botanicals induces tissue-specific and dose-dependent changes to the Gallus gallus microbiota.

BACKGROUND: Microencapsulated organic acids and botanicals have the potential to develop into important tools for the poultry industry. A blend of organic acids and botanicals (AviPlus®P) has previously shown to reduce Salmonella and Campylobacter in chickens; however, changes to the microbiota of the jejunum and ileum have not been evaluated. Microbiota diversity is linked to, but not correlated with, the efficacy of natural products; therefore, understanding the effects on the microbiota is necessary for evaluating their potential as an antibiotic alternative. RESULTS: Ileal and jejunal segments from control and supplement-fed chickens (300 and 500 g/metric ton [MT]) were subjected to alpha diversity analysis including Shannon's diversity and Pielou's Evenness. In both analytics, the diversity in the ileum was significantly decreased compared to the jejunum irrespective of treatment. Similarly, beta diversity metrics including Bray-Curtis dissimilarity index and Weighted Unifrac Distance Matrix, were significant (Q < 0.05) for both tissue and treatments comparisons. Alpha and beta diversity analytics indicated compartmentalization effects between the ileum and jejunum. Additionally, analysis of communities in the microbiota (ANCOM) analysis showed Lactobacilliaceae predominated the total operational taxonomic units (OTU), with a stepwise increase from 53% in the no treatment control (NTC) to 56% in the 300 g/MT and 67% in the 500 g/MT group. Staphylococcaceae were 2% in NTC and 2 and 0% in 300 and 500 g/MT groups. Enterobacteriaceae decreased in the 500 g/MT (31%) and increased in the 300 g/MT (37%) compared to the NTC (35%). Aerococcaceae was 0% for both doses and 7% in NTC. Ruminococcaceae were 0% in NTC and 2 and 1% in the 300 and 500 g/MT. These changes in the microbial consortia were statistically (Q < 0.05) associated with treatment groups in the jejunum that were not observed in the ileum. Least discriminant analysis effect size (LEfSE) indicated different changes directly corresponding to treatment. Enterobacteriaceae demonstrated a stepwise decrease (from NTC onward) while Clostridiaceae, were significantly increased in the 500 g/MT compared to NTC and 300 g/MT (P < 0.05). CONCLUSION: The bioactive site for the microencapsulated blend of organic acids and botanicals was the jejunum, and dietary inclusion enhanced the GIT microbiota and may be a viable antibiotic alternative for the poultry industry.

Swabbing the surface: critical factors in environmental monitoring and a path towards standardization and improvement.

Cross-contamination can be broadly defined as the transfer, direct or indirect, of microorganisms from a contaminated product to a non-contaminated product. Events that may result in cross-contamination include inadequate hygiene practices, contaminated equipment surfaces, contamination via food handling personnel, further product processing, or storage abuse All of these niches require consistent environmental surveillance systems to monitor microbial harborage sites to prevent foodborne illnesses via cross-contamination. Environmental surveillance is achieved through routine surface sampling of the food contact surfaces and surrounding areas. To better understand cross-contamination, the role of environmental surface transmission during outbreaks due to the presence and persistence of pathogenic microorganisms on various food contact surfaces must be investigated. However, studies on environmental sampling techniques are rarely performed in an actual food processing environment but rather under controlled variables within a laboratory-setting. Moreover, results and conclusions of studies differ because of the considerable variability across surface sampling tools due to individual operator dependency, low recovery rates, and low reproducibility. Information is also often lacking on environmental sampling tools used within a processing facility, the characterization of these tools, and the optimization of recovery of microorganisms for surface sampling. Thus, this review aims to: (1) discuss and compare factors impacting the recovery of microorganisms and the standardization of surface sampling methods for optimal recovery of microorganisms and (2) examine how research strategies could focus more towards the development of standard methodologies for surface sampling.

Aerobic plate count, Salmonella and Campylobacter loads of whole bird carcass rinses from pre-chillers with different water management strategies in a commercial poultry processing plant.

Salmonella and Campylobacter are significant issues for poultry processors because of increasing regulatory standards as well as public health concerns. The goal of this study is to report the effects of two different pre-chiller systems that utilize different temperatures and water recirculation systems on whole bird carcass rinsates. Both pre-chiller tanks were contained within a single poultry processing facility and operated at different temperatures and water systems. The incidence of Campylobacter spp. and Salmonella spp., as well as the aerobic plate counts on whole bird carcass rinses are reported in this study from each pre-chiller system. The results from this study reveal that there are significant differences in how microbial populations and pathogens change over time in each pre-chiller system. Furthermore, we identify that these patterns are different per system. Such data are impactful as it indicates that measuring carcasses within a plant must consider both temperature and water recirculation as it may prevent comparability of different lines within a single processing facility.

Non-molecular characterization of pellicle formation by poultry Salmonella Kentucky strains and other poultry-associated Salmonella serovars in Luria Bertani broth.

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.

Salmonella Cold Stress Response: Mechanisms and Occurrence in Foods.

Since bacteria in foods often encounter various cold environments during food processing, such as chilling, cold chain distribution, and cold storage, lower temperatures can become a major stress environment for foodborne pathogens. Bacterial responses in stressful environments have been considered in the past, but now the importance of stress responses at the molecular level is becoming recognized. Documenting how bacterial changes occur at the molecular level may help to achieve the in-depth understanding of stress responses, to predict microbial fate when they encounter cold temperatures, and to design and develop more effective strategies to control pathogens in food for ensuring food safety. Microorganisms differ in responding to a sudden downshift in temperature and this, in turn, impacts their metabolic processes and can cause various structural modifications. In this review, the fundamental aspects of bacterial cold stress responses focused on cell membrane modification, DNA supercoiling modification, transcriptional and translational responses, cold-induced protein synthesis including CspA, CsdA, NusA, DnaA, RecA, RbfA, PNPase, KsgA, SrmB, trigger factors, and initiation factors are discussed. In this context, specific Salmonella responses to cold temperature including growth, injury, and survival and their physiological and genetic responses to cold environments with a focus on cross-protection, different gene expression levels, and virulence factors will be discussed.

Molecular identification of common Salmonella serovars using multiplex DNA sensor-based suspension array.

Salmonella is one of major foodborne pathogens and the leading cause of foodborne illness-related hospitalizations and deaths. It is critical to develop a sensitive and rapid detection assay that can identify Salmonella to ensure food safety. In this study, a DNA sensor-based suspension array system of high multiplexing ability was developed to identify eight Salmonella serovars commonly associated with foodborne outbreaks to the serotype level. Each DNA sensor was prepared by activating pre-encoded microspheres with oligonucleotide probes that are targeting virulence genes and serovar-specific regions. The mixture of 12 different types of DNA sensors were loaded into a 96-well microplate and used as a 12-plex DNA sensor array platform. DNA isolated from Salmonella was amplified by multiplex polymerase chain reaction (mPCR), and the presence of Salmonella was determined by reading fluorescent signals from hybridization between probes on DNA sensors and fluorescently labeled target DNA using the Bio-Plex® system. The developed multiplex array was able to detect synthetic DNA at the concentration as low as 100 fM and various Salmonella serovars as low as 100 CFU/mL within 1 h post-PCR. Sensitivity of this assay was further improved to 1 CFU/mL with 6-h enrichment. The array system also correctly and specifically identified serotype of tested Salmonella strains without any cross-reactivity with other common foodborne pathogens. Our results indicate the developed DNA sensor suspension array can be a rapid and reliable high-throughput method for simultaneous detection and molecular identification of common Salmonella serotypes.

Evaluation of the Genetics and Functionality of Plasmids in Incompatibility Group I1-Positive Salmonella enterica.

Salmonella is a predominant foodborne pathogen in the United States and other countries. Mobile genetic elements such as plasmids allow Salmonella to adapt to external stress factors such as nutrient deprivation and host factors. Incompatibility group I1 (IncI1) plasmid-carrying Salmonella enterica strains were examined to determine the presence of plasmid-associated genes and their influence on phenotypic characteristics. The objective of this study was to understand the genetic determinants on IncI1 plasmids and their impact on antimicrobial susceptibility, competitive growth inhibition of Escherichia coli, and plasmid transfer. Primers were designed for genes that play a role in virulence, antimicrobial resistance, and plasmid transfer based on previously sequenced IncI1 plasmids. Polymerase chain reaction assays were conducted on 92 incompatibility group I1 (IncI1)-positive S. enterica strains. Phenotypic characterization included conjugation assays, antimicrobial susceptibility testing, and bacteriocin production based on the inhibition of growth of colicin-negative E. coli J53. The antimicrobial resistance genes aadA1, tetA, sul1, and blaCMY were detected in 88%, 87%, 80%, and 48% of the strains, respectively. Over half of the strains were resistant or intermediately resistant to streptomycin (85%), sulfonamides (76%), tetracycline (74%), and ampicillin (68%) and 57% of the strains inhibited growth of E. coli J53 strain. Among putative virulence genes, colicin-associated colI and cib were detected in 23% and 35% of strains and imm and ccdA were present in 58% and 54% of strains, respectively. Approximately 61% of strains contained plasmids that conjugally transferred antimicrobial resistance, including 83% where the recipient received IncI1 plasmids. Most of the strains carried an assortment of transfer associated (pil and tra) genes with between 63% and 99% of strains being positive for individual genes. Taken together the study affirms that IncI1 plasmids likely play roles in the dissemination of antimicrobial resistance and virulence-associated factors among enteric organisms.

Impact of Prebiotics on Poultry Production and Food Safety.

With the phasing out of routine use of antibiotics in animal agriculture, interest has grown for the need to develop feed supplements that augment commercial poultry performance and provide food safety benefits. From a food safety perspective, alternative feed supplements can be broadly categorized as either agents which reduce or eliminate already colonized foodborne pathogens or prevent colonization of incoming pathogens. Prebiotics are considered preventative agents since they select for gastrointestinal microbiota which not only benefits the host but can serve as a barrier to pathogen colonization. In poultry, prebiotics can elicit both indirect effects on the bird by shifting the composition and fermentation patterns of the gastrointestinal microbiota or directly by influencing host systems such as immune responses. Generation of short chain fatty acids is believed to be a primary inhibitory mechanism against pathogens when prebiotics are fermented by gastrointestinal bacteria, but other mechanisms such as interference with attachment can occur as well. While most of the impact of the prebiotic is believed to occur in the lower parts of the bird gastrointestinal tract, particularly the ceca, it is possible that some microbial hydrolysis could occur in upper sections such as the crop. Development of next generation sequencing has increased the resolution of identifying gastrointestinal organisms that are involved in metabolism of prebiotics either directly or indirectly. Novel sources of non-digestible oligosaccharides such as cereal grain brans are being explored for potential use in poultry to limit Salmonella establishment. This review will cover the current applications and prospects for use of prebiotics in poultry to improve performance and limit pathogens in the gastrointestinal tract.

Current aspects of Salmonella contamination in the US poultry production chain and the potential application of risk strategies in understanding emerging hazards.

One of the leading causes of foodborne illness in poultry products is Salmonella enterica. Salmonella hazards in poultry may be estimated and possible control methods modeled and evaluated through the use of quantitative microbiological risk assessment (QMRA) models and tools. From farm to table, there are many possible routes of Salmonella dissemination and contamination in poultry. From the time chicks are hatched through growth, transportation, processing, storage, preparation, and finally consumption, the product could be contaminated through exposure to different materials and sources. Examination of each step of the process is necessary as well as an examination of the overall picture to create effective countermeasures against contamination and prevent disease. QMRA simulation models can use either point estimates or probability distributions to examine variables such as Salmonella concentrations at retail or at any given point of processing to gain insight on the chance of illness due to Salmonella ingestion. For modeling Salmonella risk in poultry, it is important to look at variables such as Salmonella transfer and cross contamination during processing. QMRA results may be useful for the identification and control of critical sources of Salmonella contamination.

Heat Survival and Phenotype Microarray Profiling of Salmonella Typhimurium Mutants.

Contamination of food products by pathogenic microorganisms continues to be a major public health and food industry concern. Non-typhoidal Salmonella species have led to numerous outbreaks associated with various foods. A wide variety of methods have been applied and introduced for treatment of fresh foods to eliminate pathogenic as well as spoilage microorganisms. Salmonella can become exposed to elevated temperatures while associated with hosts such as poultry. In addition, heat treatment is also applied at various stages of processing to retain the shelf life of food products. Despite this, these microorganisms may overcome exposure to such treatments through the efficient expression of stress response mechanisms and result in illness following consumption. Thermal stress induces a range of destructive exposures to bacterial cells such as protein damage and DNA damage caused by reactive oxygen species. In this study, we chose three genes (∆recD, ∆STM14_5307, and ∆aroD) associated with conditionally essential genes required for different aspects of optimal growth at 42 °C and evaluated the responses of wild type and mutant Salmonella Typhimurium strains to uncover potential mechanisms that may enable survival and resistance under thermal stress. The RecBCD complex that initiates repair of double-stranded DNA breaks through homologous recombination. STM14_5307 is a transcriptional regulator involved in stationary phase growth and inositol metabolism. The gene aroD is involved in metabolism and stationary phase growth. These strains were characterized via high throughput phenotypic profiling in response to two different growth temperatures (37 °C (human host temperature) and 42 °C (poultry host temperature)). The ∆aroD strain exhibited the highest sensitivity to the various temperatures followed by the ∆recD and ∆STM14_5307 strains, respectively. Achieving more understanding of the molecular mechanisms of heat survival may lead to the development of more effective strategies to limit Salmonella in food products through thermal treatment by developing interventions that specifically target the pathways these genes are involved in.

Development of a rapid method to quantify Salmonella Typhimurium using a combination of MPN with qPCR and a shortened time incubation.

A novel method was developed for the specific quantification of S. Typhimurium using a most-probable-number (MPN) combined with qPCR and a shortened incubation time (MPN-qPCR-SIT). For S. Typhimurium enumeration, dilutions of samples were transferred into three wells on a microtiter plate and the plate was incubated for 4 h. The S. Typhimurium presence in the wells was identified using a qPCR and populations were determined based on an MPN calculation. The R2 between the MPN-qPCR-SIT and conventional MPN exhibited a high level of correlation (0.9335-0.9752), suggesting that the MPN-qPCR-SIT offers a reliable alternative method for S. Typhimurium quantification. Although plating and qPCR were limited in their ability to detect low levels of S. Typhimurium (e.g. 0.18 log MPN/ml), these levels could be successfully detected with the MPN-qPCR-SIT. Chicken breast samples inoculated with S. Typhimurium were incubated at 0, 4, and 24 h and incubated samples were subjected to microbiome analysis. Levels of Salmonella and Enterobacteriaceae increased significantly with incubation time. The obvious benefits of the MPN-qPCR-SIT are: 1) a further confirmation step is not required, 2) the detection limit is as low as conventional MPN, but 3) is more rapid, requiring approximately 7 h to simultaneously complete quantification.

Sodium bisulfate and a sodium bisulfate/tannin mixture decreases pH when added to an in vitro incubated poultry cecal or fecal contents while reducing Salmonella Typhimurium marker strain survival and altering the microbiome.

The objective of the present study was to investigate the ability of animal feed-grade sodium bisulfate (SBS) and a mixture of sodium bisulfate/tannin to inhibit the growth of Salmonella using an anerobic in vitro mixed cecal culture to mimic the conditions within the chicken cecum. An initial inoculum of Salmonella Typhimurium was introduced to an anerobic dilution solution containing 1/3000 diluted cecal bacteria and solids consisting of ground chicken feed and different percentages of solid SBS or SBS/tannin, and surviving organisms were enumerated. Two different experimental designs were employed. In the "unadapted" treatment, the S. Typhimurium was added at the beginning of the culture incubation along with cecal bacteria and chicken feed/SBS or chicken feed/SBS/tannin. In the "adapted" treatment, S. Typhimurium was added after a 24 hour pre-incubation of the cecal bacteria with the chicken feed/SBS or chicken feed/SBS/tannin. Adding SBS resulted in reduction of pH in the cultures which paralleled with the reduction of S. Typhimurium. The SBS alone was found to be inhibitory to S. Typhimurium in the adapted treatment at all concentrations tested (0.25, 0.5, and 0.75%), and the degree of inhibition was concentration-dependent. Salmonella Typhimurium was completely killed in the adapted culture with 0.5% SBS after 24 and 48 h. The SBS/tannin mixture was less inhibitory than SBS alone at the same concentrations in side-by-side comparisons. Testing at a 0.5% SBS concentration, chicken age had little or no effect on log reduction of S. Typhimurium relative to age-matched control cultures without SBS, but age did affect the absolute number of S. Typhimurium surviving, with the greatest decreases occurring at 2 and 4 weeks of age (approx. 103 S. Typhimurium surviving) compared to 6 weeks of age (approx. 105 Salmonella surviving). Microbiome analysis with an Illumina MiSeq platform was conducted to investigate bacterial compositional changes related to the addition of SBS. The relative abundance of Firmicutes (at the phylum level) was decreased, and genera Lactobacillus and Faecalibacterium were increased when SBS was added to the anaerobic mixed culture containing either fecal or cecal material. The antimicrobial action of feed-grade SBS may represent a potential pre-harvest control measure for Salmonella in poultry production.

Immuno-based detection of Shiga toxin-producing pathogenic Escherichia coli in food - A review on current approaches and potential strategies for optimization.

Certain pathogenic Escherichia coli known as Shiga toxin (Stx)-producing E. coli (STEC) are a public health threat to the consumer, and are problematic for the food industry. Food products containing STEC are deemed unfit for human consumption, and STEC illnesses can cause hemolytic uremic syndrome (HUS), a disease affecting the kidneys in susceptible individuals. Optimizing detection methods in foods have been focused on more prompt and accurate analysis. This review addresses the role and applications of immuno-based assays for STEC detection in food systems. Immunoassay antibody capture systems and flow cytometry platforms have been implemented into several food-based detection systems. By applying antibodies that will interact with target microorganisms, immunoassays can be used to directly detect and quantify pathogens. Immuno-based protocols could potentially be further implemented into the food industry, limit the duration of the detection process and increase accuracy.

Transposon sequencing: methods and expanding applications.

A comprehensive understanding of genotype-phenotype links in bacteria is the primary theme of bacterial functional genomics. Transposon sequencing (Tn-seq) or its equivalent methods that combine random transposon mutagenesis and next-generation sequencing (NGS) represent a powerful approach to understand gene functions in bacteria on a genome-wide scale. This approach has been utilized in a variety of bacterial species to provide comprehensive information on gene functions related to various phenotypes or biological processes of significance. With further improvements in the molecular protocol for specific amplification of transposon junction sequences and increasing capacity of next generation sequencing technologies, the applications of Tn-seq have been expanding to tackle questions that are important yet difficult to address in the past. In this review, we will discuss the technical aspects of different Tn-seq methods along with their pros and cons to provide a helpful guidance for those who want to implement or improve Tn-seq for their own research projects. In addition, we also provide a comprehensive summary of recent published studies based on Tn-seq methods to give an updated perspective on the current and emerging applications of Tn-seq.