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Title: Physiological characterisation of the neonatal meningitic bacterium Cronobacter sakazakii
Author: Sonbol, H. S.
ISNI:       0000 0004 5920 1878
Awarding Body: Nottingham Trent University
Current Institution: Nottingham Trent University
Date of Award: 2015
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The Cronobacter genus is a member of the Enterobacteriaceae family that comprises 7 species. A multiple locus sequence typing (MLST) scheme was designed for Cronobacter and was used to identify over 400 sequence types (STs). Notably, C. sakazakii ST4 and its single/double loci variants, which together form clonal complex 4 (CC4), are predominantly associated with neonatal meningitis. The present PhD study aimed to investigate the diversity of C. sakazakii especially CC4 using MLST analysis, physiological characterisation of stress responses along with genomic analysis associated genes. In the first part of the study, MLST was used to analyse three collections of environmental bacterial isolates. The isolates were from powdered infant formula, milk powder factory environments, and milk powder processing equipment; they had not been profiled prior to this study and had previously been identified as E. sakazakii. These environmental strains were obtained from geographically diverse countries. A total of 39 STs were identified across the studied isolates. The analysis revealed that despite their geographical and temporal spread, only 4 Cronobacter species were isolated from these three collections of strains, namely C. sakazakii, C. malonaticus, C. turicensis, and C. muytjensii. Interestingly, this study demonstrated that C. sakazakii CC4 isolates represent a predominantly stable lineage within the Cronobacter genus in these environments. This result led to further investigation to study the physiological factors in the CC4 clone associated with its persistence in the environment. This included desiccation stress, heat tolerance, acid resistance and serum resistance assays. The desiccation and serum resistance assays indicated that all of the tested C. sakazakii CC4 and non-CC4 strains showed equal resistance against desiccation and serum. The experiments also indicated that C. sakazakii CC4 strains were more heat tolerant than non-CC4 strains to 100°C heat. The most interesting observation was that C. sakazakii CC4 strains were significantly more acid-resistant than non-CC4 strains to pH 3.5, which is the pH of the neonatal stomach. The results were supported by the outer membrane protein (OMP) profiling where C. sakazakii strains revealed more OMPs than non-CC4 at pH 3.5. Capsule production assays showed that most of the C. sakazakii CC4 and non-CC4 strains produced mucoid capsules when cultured on milk agar. The motility of the tested strains was associated with the presence of the fliRQPON flagellar genes. An exhaustive BLAST search of sequenced strains to identify genes associated with virulence or with environmental fitness found no significant difference between the virulence potential of C. sakazakii CC4 and C. sakazakii non-CC4 strains. The present study provides important insights into neonatal meningitis C. sakazakii CC4. Further studies are warranted to characterise the OMPs that are predominant in C. sakazakii CC4 and elucidate their significance towards virulence of this important neonatal health-associated pathogen.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available