RT info:eu-repo/semantics/doctoralThesis
T1 Viabilidad de células estresadas de Cronobacter sakazakii mediante citometría de flujo y espectrometría de masas
A1 Cal Sabater, Paloma de la
A2 Universidad de Valladolid. Escuela de Doctorado
K1 Microbiología
K1 Cronobacter sakazakii
K1 Cronobacter sakazakii
K1 Flow cytometry
K1 Citometría de flujo
K1 Mass spectrometry
K1 Espectrometría de masas
K1 3309.90 Microbiología de Alimentos
AB C. sakazakii is a thermotolerant pathogen associated with outbreaks in infants fed with reconstituted powdered infant formulae. These products are not stored under sterile conditions, so their contamination followed by improper preparation and storage can lead to bacterial growth. To limit the risk of C. sakazakii infection, reconstitution with water at 70°C is recommended. Although C. sakazakii infection has a moderate incidence, lethality and morbidity are high, with neurological sequelae occurring in many cases.The aim of the present study is to gain insight into the physiological states of C. sakazakii cells using flow cytometry to detect the compromised cells, which are viable but non-culturable using plate-based methods, and to evaluate the impact of milk heat treatments on those populations. Dead-cell suspensions as well as heat-treated and non-heat-treated cell suspensions were used. After 60 or 65 ◦C treatments, the number of compromised cells increased as a result of an increase in cells with damaged membranes, although they were not effective in inactivating the bacteria. Thus, mild heat treatments are not enough to guarantee the safety of reconstituted powdered infant formulae. Flow cytometry allowed the identification of C. sakazakii compromised cells that cannot be detected by classical plate count methods; hence, it could be used for screening studies in order to reduce the risk derived from the presence of pathogenic viable but non-culturable cells in food intended for newborn nutrition.In a second studio, dead cells were double-stained with SYTO 9- or SYBR Green I-PI. Cells were treated at different temperatures. Compromised cells were the most abundant at 60 and 65 ºC, but at 70 ºC onwards the most detected cells were dead cells. Over 80 ºC, all events were located in the dead cells area. Moreover, assays with different volumes of each dye solution were carried out at 70 ºC: from 0.125 to 1.5 µL. The populations showed statistically significant differences when comparing SYTO 9 and SYBR Green I staining. The maximum volume used is not necessary to detect live cells, but it is for detecting compromised or dead cells. Followed 2 h refrigeration at 4 ºC, sorted compromised cells showed no change in its detection. C. sakazakii cells from cross-contamination or poorly prepared infant formulae could recover and proliferate if there is a delay in consumption of more than 2 h at room temperature, constituting a public health risk.Finally, in a third work, the response of C. sakazakii to heat stress was studied by mass spectrometry. The expression of stress-related proteins and the peptide profile were analysed, with the aim of identifying biomarkers involved in the survival of the microorganism under sublethal environmental conditions. Four peptide markers were identified in the mass spectra corresponding to intact proteins. A potential stress biomarker of C. sakazakii was identified and validated at m/z 3336.6. The results showed that heat shock applied to the microorganism increased the expression of chaperones such as DnaK, ATP-independent proteins involved in proper folding and some virulence factors associated with the preservation of membrane integrity. These proteins seem to play a relevant role in the maintenance of protein synthesis as an adaptive mechanism to sublethal heat stress. The study of the resistance of C. sakazakii to sublethal temperature variations provides information on its viability and survival strategies, such as the generation of adaptive physiological states or the overexpression of peptide markers that may represent an additional pathogenicity pathway.
YR 2023
FD 2023
LK https://uvadoc.uva.es/handle/10324/67159
UL https://uvadoc.uva.es/handle/10324/67159
LA spa
NO Escuela de Doctorado
DS UVaDOC
RD 14-ene-2025