Turki M. Dawoud

Genome Scanning for Conditionally Essential Genes in Salmonella enterica Serotype Typhimurium

ABSTRACT As more whole-genome sequences become available, there is an increasing demand for high-throughput methods that link genes to phenotypes, facilitating discovery of new gene functions. In this study, we describe a new version of the Tn-seq method involving a modified EZ:Tn5 transposon for genome-wide and quantitative mapping of all insertions in a complex mutant library utilizing massively parallel Illumina sequencing. This Tn-seq method was applied to a genome-saturating Salmonella enterica serotype Typhimurium mutant library recovered from selection under 3 different in vitro growth conditions (diluted Luria-Bertani [LB] medium, LB medium plus bile acid, and LB medium at 42°C), mimicking some aspects of host stressors. We identified an overlapping set of 105 protein-coding genes in S. Typhimurium that are conditionally essential under at least one of the above selective conditions. Competition assays using 4 deletion mutants (pyrD, glnL, recD, and STM14_5307) confirmed the phenotypes predicted by Tn-seq data, validating the utility of this approach in discovering new gene functions. With continuously increasing sequencing capacity of next generation sequencing technologies, this robust Tn-seq method will aid in revealing unexplored genetic determinants and the underlying mechanisms of various biological processes in Salmonella and the other approximately 70 bacterial species for which EZ:Tn5 mutagenesis has been established.

Application of Molecular Methods for Traceability of Foodborne Pathogens in Food Safety Systems

Foodborne disease continues to be a considerable public health concern. It is becoming more important from a public accountability standpoint to be able to rapidly pinpoint the origin of a foodborne disease outbreak. However, this can be a challenge due to the complexity of food production systems and the biological variation that occurs in strains and isolates of a particular foodborne pathogen. Molecular methods have become more advanced and thus possess capabilities that can be utilized to more precisely identify a foodborne pathogen. In particular, whole genome sequencing offers the opportunity to detect subtle differences in organisms that not only will aid with identification of a particular strain but also will help to gauge the evolution of a particular pathogen variant prior to the outbreak. As genomic sequence data sets continue to expand it is anticipated that this information can be used to more rapidly assess pathogen identification and point of origin on a global basis.

Improving the Efficiency of Transposon Mutagenesis in Salmonella Enteritidis by Overcoming Host-Restriction Barriers

Transposon mutagenesis using transposome complex is a powerful method for functional genomics analysis in diverse bacteria by creating a large number of random mutants to prepare a genome-saturating mutant library. However, strong host restriction barriers can lead to limitations with species- or strain-specific restriction-modification systems. The purpose of this study was to enhance the transposon mutagenesis efficiency of Salmonella Enteritidis to generate a larger number of random insertion mutants. Host-adapted Tn5 DNA was used to form a transposome complex, and this simple approach significantly and consistently improved the efficiency of transposon mutagenesis, resulting in a 46-fold increase in the efficiency as compared to non-adapted transposon DNA fragments. Random nature of Tn5 insertions was confirmed by high-throughput sequencing of the Tn5-junction sequences. The result based on S. Enteritidis in this study should find broad applications in preparing a comprehensive mutant library of other species using transposome complex.

The Potential Link between Thermal Resistance and Virulence in Salmonella: A Review

In some animals, the typical body temperature can be higher than humans, for example, 42°C in poultry and 40°C in rabbits which can be a potential thermal stress challenge for pathogens. Even in animals with lower body temperatures, when infection occurs, the immune system may increase body temperature to reduce the chance of survival for pathogens. However, some pathogens can still easily overcome higher body temperatures and/or rise in body temperatures through expression of stress response mechanisms. Salmonella is the causative agent of one of the most prevalent foodborne illnesses, salmonellosis, and can readily survive over a wide range of temperatures due to the efficient expression of the heat (thermal) stress response. Therefore, thermal resistance mechanisms can provide cross protection against other stresses including the non-specific host defenses found within the human body thus increasing pathogenic potential. Understanding the molecular mechanisms associated with thermal responses in Salmonella is crucial in designing and developing more effective or new treatments for reducing and eliminating infection caused by Salmonella that have survived heat stress. In this review, Salmonella thermal resistance is assessed followed by an overview of the thermal stress responses with a focus on gene regulation by sigma factors, heat shock proteins, along with the corresponding thermosensors and their association with virulence expression including a focus on a potential link between heat resistance and potential for infection.

Overview of Salmonellosis and Food-borne Salmonella : Historical and Current Perspectives

Abstract Salmonella continues to be one of the more major food-borne pathogens from a public health standpoint. Historically pathogenic Salmonella have probably always been associated with human disease but the linkage between the pathogen and salmonellosis has not always been obvious and is more of a recent development. As epidemiology and identification methodologies became more sophisticated, the cause and effect between human clinical salmonellosis and water and food sources was established and appropriate control measures identified. However, food-borne salmonellosis continues to be problematic in part due to the biology of Salmonella and its occurrence in a wide array of environmental niches as well as the ability of some serovars to occupy the gastrointestinal tract of multiple host animals. In particular, food commodities including poultry and eggs continue to be prominent sources of food-borne salmonellosis. In this chapter, an overview of the historical milestones of Salmonellas initial identification as a pathogen causing human systemic disease, and later as a food-borne pathogen, will be discussed. Current patterns and trends of Salmonella occurrence worldwide will be described followed by an introduction to the presence of food-borne Salmonella in poultry and egg production.Salmonella continues to be one of the more major food-borne pathogens from a public health standpoint. Historically pathogenic Salmonella have probably always been associated with human disease but the linkage between the pathogen and salmonellosis has not always been obvious and is more of a recent development. As epidemiology and identification methodologies became more sophisticated, the cause and effect between human clinical salmonellosis and water and food sources was established and appropriate control measures identified. However, food-borne salmonellosis continues to be problematic in part due to the biology of Salmonella and its occurrence in a wide array of environmental niches as well as the ability of some serovars to occupy the gastrointestinal tract of multiple host animals. In particular, food commodities including poultry and eggs continue to be prominent sources of food-borne salmonellosis. In this chapter, an overview of the historical milestones of Salmonellas initial identification as a pathogen causing human systemic disease, and later as a food-borne pathogen, will be discussed. Current patterns and trends of Salmonella occurrence worldwide will be described followed by an introduction to the presence of food-borne Salmonella in poultry and egg production.

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.

Chapter 9 – Foodborne Salmonella in Laying Hens and Egg Production

Abstract Salmonella spp. remain an ongoing source of foodborne diseases. Numerous food products can serve as sources of this pathogen, including fresh produce, red meats, poultry, and egg products. With egg production, exposure to Salmonella can occur at any stage from the laying hen to egg processing. Salmonella Enteritidis has been the primary serovar of concern in egg contamination and considerable research has focused on understanding the relationship between this serovar and egg production. As more has become known, the interaction between the laying hen and infection by S. Enteritidis has been established. As a result, more effective control measures for limiting S. Enteritidis colonization of the laying hen and limiting egg contamination have been developed. However, more work needs to be conducted on the gastrointestinal ecology of S. Enteritidis and its relationship with the indigenous microbial population.

Salmonella and the Potential Role for Methods to Develop Microbial Process Indicators on Chicken Carcasses

Foodborne salmonellosis originating from poultry products is a major problem, and post harvest control is important to prevent Salmonella contamination. However, assessing when and where Salmonella contamination occurs, and, more importantly, which routes of contamination have the greatest impact on introduction and dissemination in the final poultry meat product is an extremely complex question with a tremendous number of variables. Estimating Salmonella prevalence in poultry processing and the significance/role of Salmonella prevalence in finished products is a critical aspect of food safety control, with USDA regulations updating regularly to improve food safety. Numerous cultural methodologies exist for the detection of Salmonella . However, it is also critical to establish non- Salmonella bacteria and indicator organism profiles that allow for a more continuous monitoring or oversight of microbial shifts that potentially could occur during processing. Development of quantitative risk assessment models, for assessing where the greatest risks for contamination occur, may also allow for prioritizing optimal intervention strategies to achieve the most effective reductions in Salmonella contamination of poultry products.