CHARACTERIZING THE ROLE OF PUTATIVE VIRULENCE GENES ASSOCIATED WITH INFECTION, COLONIZATION AND PERSISTENCE OF SALMONELLA ENTERITIDIS IN CHICKEN USING A BIOLUMINESCENT REPORTER
Salmonella enterica subspecies enterica serovar Enteritidis (SEn) is a major bacterial cause of gastroenteritis in humans. It was first identified by Dr. Daniel Salmon and Dr. Theobald Smith (1980) during an outbreak of swine diarrhea. Initially, it was given the name Salmonella cholerasuis. Later it was reclassified and given the name S. Enteritidis according to the Kauffmann-White nomenclature scheme (1952). Since its discovery, consumption of contaminated chicken meat, eggs, and other poultry products has been driving sporadic outbreaks of salmonellosis around the globe. Chicken (Gallus gallus domesticus) is considered a hot spot for carrying SEn without overt clinical signs. We investigated the kinetics of early events after oral gavage in day-old chicken, using the bioluminescent imaging (BLI) technique. SEn was genetically engineered to continuously produce light (490nm) using the light-emitting module called lux operon. This technique has been widely used in various bacteria enabling live imaging in mouse models. We were able to accurately define the onset of primary colonization as early as 30 hours post-infection (p.i.) in the cecum with the aid of photonic signal strength detected by ex-vivo imaging and colony count values. Thus, colonization in the cecum is a rapid process in young birds. Other predilection sites were the crop and yolk sac, which were colonized at 24 hours p.i. (in crop) and 4 days p.i. (in yolk sac), respectively. Our findings regarding the temporal pattern of colonization were not possible without the BLI and establishing in-vivo imaging capabilities will be advantageous for the poultry disease field in the future. To further strengthen the concept, we have enhanced the signal strength of the lux operon by exciting a fluorophore-bound protein called lumazine which showed some improvement in the -limit of detection in-vitro and ex-vivo. Even though in-vivo imaging was not accomplished, ex-vivo imaging provided useful insights into the fitness advantages of various virulence genes which were hitherto not well characterized in chicken. For the first time, utilizing BLI, we identified candidate virulence that can significantly impact yolk sac colonization/infection. The virulence profile of SEn during yolk sac infection hinted that Salmonella iron homeostasis may be critical to pathogenesis in a chicken model. The contribution of various iron uptake systems has been investigated comprehensively in murine models with inconsistent data. Lack of information in a chicken model encouraged us to investigate the role of high-affinity iron uptake systems namely siderophore (enterobactin and salmochelin) mediated ferric iron uptake and FeoABC-mediated ferrous iron uptake. Our approach was to use mutant strains devoid of each of the uptake systems in a co-infection chicken model together with the wild-type strain. One strain in each mutant versus wildtype combination was chromosomally marked with the lux operon to enable differential detection and counting in the co-infection experiment. Our approach was superior because we avoided using two different antibiotic resistance markers to discriminate between the strains. A significant colonization defect in the cecum and extraintestinal sites was associated with a strain devoid of both siderophore synthesis genes and feoABC. The two uptake systems were functionally redundant by contributing to the virulence of SEn and the data suggested that chickens mount an iron starvation response towards SEn. To detect an iron starvation response during gastrointestinal colonization, a biosensor for iron limitation was introduced. The photonic signal strength increased towards the 48-hour post-infection during cecal colonization which pointed to iron limitation. But the total emitted signal did not reach maximum strength which would be equivalent to strict iron limitation. Our findings here indicated that the interaction of Salmonella with the avian host started in an environment close to the equilibrium regarding adequate iron supply to SEn but facing mounting iron starvation during early colonization. This also underlines the importance of having alternatives to import iron as an essential element and explains a functional redundancy between the two high-affinity iron-acquisition systems.
Salmonella Enteritidis, Bioluminescence, Lumazine, Chicken, Yolk sac, Iron uptake, Fur
Doctor of Philosophy (Ph.D.)