The evolution of bacterial resistance against high hydrostatic pressure

Exposure to high hydrostatic pressure (HHP) is becoming a valuable alternative to thermal pasteurization in food processing. While both heat and HHP (300 600 MPa) can inactivate vegetative cells of pathogenic and spoilage microorganisms, HHP treatment imposes less severe deteriorations of nutritional value, flavor, color, and texture on the food than does heat treatment. However, a sustained further exploitation of HHP technology in food processing nevertheless requires a more profound understanding of the potential impact of HHP stress on the physiology and evolution of food-borne bacteria. In this context, the focus of this doctoral work is to unravel the mechanisms underlying the acquisition of HHP resistance in bacteria. Extending the current knowledge regarding the piezophysiology of Escherichia coli as a food-borne model bacterium, a first set of experiments set out to probe for the extent of possible HHP resistance development in this bacterium by using specialized equipment to carry out HHP treatments in the GPa range (in collaboration with Prof. Paul McMillan, University College, London). In the course of these experiments, we were able to drastically extend the limits of HHP survival in vegetative cells by evolving mutants of E. coli that could withstand HHP treatments up to ≥ 2 GPa. In addition, our data further indicated that cellular heat and extreme HHP resistance seem independent features that are not necessarily correlated with each other.Subsequently the intrinsic potential for HHP resistance development in different Gram-positive and negative food-related pathogens (including Escherichia coli, Shigella flexneri, Salmonella Typhimurium, Salmonella Enteritidis, Yersinia enterocolitica, Aeromonas hydrophila, Pseudomonas aeruginosa and Listeria innocua) was examined and compared. This analysis revealed that E. coli strains in particular seem to have an outstanding propensity for HHP resistance development. In addition, we also observed that, once acquired, HHP resistance proved to be a very stable trait that does not affect cellular fitness. Mechanistic approaches also indicated that HHP resistance is not necessarily linked to derepression of the heat-shock genes or related to the phenomenon of persistence.Our focus then zoomed in on the peculiar progression of HHP resistance development in a pathogenic representative of E. coli, i.e. E. coli O157:H7 strain ATCC 43888, which revealed that HHP stress can rapidly select for strongly increased RpoS activity, thereby simultaneously increasing both HHP and heat resistance. Furthermore, extremely HHP-resistant mutants of ATCC 43888 clearly incurred a number of additional RpoS-dependent phenotypes as well, suggesting that the implementation of HHP processing in the food production chain can significantly alter the physiology of food-borne pathogens. Further extending on these latter findings, we embarked on the genetic dissection of HHP-resistance development in this E. coli O157:H7 ATCC 43888 strain. More specifically, a transposon mutagenesis screen was performed to identify loss-of-function mutations that could mediate the rapid evolution of HHP resistance in ATCC 43888. As such, individual disruptions in the rssB (Anti-RpoS factor), crp (catabolite response protein) or cyaA (adenylate cyclase) gene were picked up and shown to contribute to heat and HHP resistance. These mutations revealed that, aside RpoS activity, cAMP/CRP homeostasis can contribute to cellular HHP resistance as well. In conclusion, our research has highlighted the potential speed and extent of HHP resistance development in E. coli, and for the first time has shed light on the genetic strategies responsible for this adaptive evolution.nrpages: 150status: publishe

High hydrostatic pressure sensitivity of virulent avian pathogenic Escherichia coli

status: publishe

Novel cryptic prophage operon modulates stress resistance in Escherichia coli O157:H7

Prokaryotic genome plasticity has paved the way for an explosive diversification within bacterial species. Part of this plasticity originates from horizontal gene transfer that takes place within the bacterial kingdom with bacterial phage genes being amongst the most common exchanged fragments. These phage genetic elements can constitute up to one fifth of the complete prokaryotic genetic blueprint but its influence on the hosts physiology remains ill defined. Escherichia coli O157:H7 is a notorious foodborne pathogen potentiated by the large amount of horizontally transferred genomic material which differentiates it from harmless gut-inhabiting relatives such as E. coli K-12. Upon screening a transposon knock-out library of E. coli O157:H7 in search of high pressure (HP) resistant mutants, we came across a cryptic prophage encoded operon (hpsP-hpsQ) uniquely found in this strain that can modulate the stress resilience of the bacterial host. Disrupting the first part of this viral operon leads to a significant increase in resistance to HP as well as cross-resistance to heat with the underlying mechanism still being elusive. Upon overexpression HpsP not only proved to be a genotoxic protein with (in vitro and in vivo) DNA binding capacity, but was also able to quench the potent toxic effect of HpsQ, the second operon member. Moreover, HpsP functionality can be blocked by complementing with its own N-terminal ATPase domain thus abolishing its genotoxic DNA binding and starting host resilience modulation.status: publishe