1 National Food Institute, Technical University of Denmark
Salmonella are zoonotic pathogens that cause food-borne illness and that are widely disseminated in nature. An estimated number of 93.8 million human cases of gastroenteritis caused by Salmonella occur annually throughout the world. The epidemiology of Salmonella is characterized by a temporal dominance of certain clones. These epidemically successful clones are often resistant to antibiotics and associated with severe human illness. They pose a major threat to public health and lead to heavy economic losses. So far, little is known about the environmental and bacterial factors leading to the emergence of successful clones. However, resistance to multiple antimicrobial drugs and quinolones seems to contribute to the epidemic success of Salmonella as it is associated with an increased severity of illness and epidemicity. In order to predict and prevent future outbreaks and epidemics, research should focus on the evolutionary mechanisms of emerging success clones. The ability to spread in different food production sectors and to cause human disease seems critical for a clone to become successful. The aim of this PhD study was to identify common phenotypic and genetic traits of success clones that may have an epidemic importance with respect to the survival in the food chain and to human infection. Non-typhoid Salmonella isolates from food, production animals and humans were carefully selected and characterized as successful or non successful based on epidemiological data. In addition, the isolates were categorized based on their antibiotic resistance pattern into quinolone resistant (Q; at least low level resistance to one fluoroquinolone), multidrug resistant (MR; resistant to four or more antibiotic agents), resistant (R; resistant to less than four antibiotic agents) and sensitive (S; fully susceptible to the tested antibiotics). The ability to survive and grow after physical stress experienced in the food chain is likely to contribute to a successful spread of Salmonella. Thus, the response of Salmonella isolates in exponential and stationary growth state to freezing in minced meat and to drying at two combinations of temperature and humidity was investigated. The results of this study are presented in Manuscript I. Stationary phase cells survived freezing for up to one year without major decline and initiated growth with a short lag time of 1.7 hours on average. Exponential phase cells showed reduced survival and a tendency to exhibit shorter lag times. Dehydration at 82 % relative humidity (RH) and 10 °C was less harmful for Salmonella than 49 % RH and 25 °C. Dehydration, in contrast to freezing, was differently tolerated by the Salmonella isolates but neither the tolerance to freezing nor to dehydration appears to contribute to the epidemic success of Salmonella. MR and Q Salmonella seem to have a virulence or fitness advantage apart from the effect posed by the antibiotic resistance phenotype. Manuscript II describes a study aiming to identify genes and genetic structures in Q and MR isolates that might confer fitness or virulence attributes by the use of array-based comparative genomic hybridizations and PCR screenings. The largest fraction of the genes dominating in Q and MR Salmonella were single genes and gene clusters belonging to prophages, among these the virulence genes sopE (STY4609) and gipA (STM2599) and the genes recT (SG1183), sugR (STM3753) and trhH (AF261825_S012). The latter three genes were knocked out and the mutants were used in functional analyses. The mutants were not significantly different from the wildtype but RecT knockout mutants seemed to be more susceptible to the early defense of macrophages and exhibited increased spontaneous mutation rates. The largest group of genes dominating in sensitive isolates were related to metabolism indicating that many of these genes are typically absent or diverged in Q and MR Salmonella. The acquisition and maintenance of bacteriophages conferring fitness and virulence attributes may be part of the evolution of successful MR and Q Salmonella. The bacterial and host factors leading to an increased severity of illness caused by quinolone resistant Salmonella were investigated as they are largely unknown. Quinolone resistance is mostly caused by point mutations and can develop in Salmonella during infection. As described in Manuscript III, the spontaneous mutations rates and virulence potential of ciprofloxacin (CIP) resistant Salmonella in presence and absence of CIP were assessed using cell cultures. The rate at which CIP sensitive isolates can experience a reduction in their CIP susceptibility after macrophage infection and in presence and absence of CIP was investigated. Strain dependent differences in the mutation rate, the ability to invade intestinal epithelial cells and to replicate within macrophages were found, but there was no correlation between virulence, mutation rate and resistance pattern. The presence of clinically relevant CIP concentrations decreased but failed to eliminate the number of CIP resistant intraphagocytic Salmonella. During macrophage infection, Salmonella adapted to higher CIP concentrations (to at least 2 – 8 fold the initial MIC). When CIP was added to the extracellular medium, the frequency of intraphagocytic bacteria exhibiting reduced CIP susceptibilities increased markedly. Thus virulent Salmonella strains that are likely to persist in macrophages for prolonged times might become resistant to quinolones and the administration of quinolones may increase the rate of resistant mutants. In conclusion, the emergence of successful clones is likely to be the result of a combination of factors including the acquisition and maintenance of bacteriophages encoding genes that increase the fitness or virulence of their bacterial host. Marker genes related to bacteriophages might be useful in the future for the early detection of these clones. Macrophages might play a role in the evolution of quinolone resistance in invasive Salmonella strains. Both in absence of antimicrobial drugs and when quinolones are administered, intraphagocytic Salmonella may become quinolone resistant and simultaneously may acquire other advantageous mutations. This highlights the importance of an antibiotic therapy that is effective in eliminating intracellular pathogens.
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Agersø, Yvonne, Aabo, Søren, Aarestrup, Frank Møller