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Title: Improved recovery and detection of Salmonella enterica from complex matrices
Author: Elmerhebi, E.
ISNI:       0000 0004 7428 7198
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2018
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Salmonella enterica is the pathogen responsible for salmonellosis, a disease characterised by onset of fever, diarrhoea and nausea. The disease is commonly foodborne and has a significant burden on both the industrialised world and low-income nations. The organism is classed as a major public health concern due to the level of incidence and potential severity of infections. The majority of testing carried out for the detection of Salmonella enterica contamination, still heavily relies on cultural detection methods, such as enrichment and diagnostic agar. Current methodologies employ workflows that are either protracted, rely on flawed diagnostic reactions or are unable to deal with a low-level of target organism in the presence of high background and interfering matrices. The main aims for the studies presented in this thesis were to develop and evaluate a novel chromogenic plating medium, paired with a selective single stage enrichment workflow for detection of Salmonella enterica from complex matrices. The study also examined the development of immunomagnetic separation (IMS) beads for the detection of Salmonella enterica, and specifically their use for the capture and concentration of S. Typhi. The development of a novel chromogenic agar was achieved by pairing two chromogens in a highly engineered selective agar base. 5-bromo-4-chloro-3-indolyl nonanoate (X-nonanoate) was used as the target chromogen and 3,4-cyclohexenoesculetin ß-D-glucoside (CHE-glc) was used as the masking chromogen. Salmonella possess esterase activity capable of cleaving the X-nonanoate resulting in blue green colonies on the agar surface. Several Enterobacteriaceae can also utilise the X-nonanoate but their activity is masked by the expression of ß-D-glucosidase, which cleaves the CHE-glc resulting in black colonies. The agar was designated chromogenic agar for Salmonella esterase (CASE) and was shown to be superior to commercially available chromogenic media, regarding sensitivity and specificity. The development of a single stage enrichment for Salmonella enterica was achieved by pairing an antibiotic cocktail with buffered peptone water (BPW). The workflow was used in conjunction with CASE to replace the standard methodology (ISO 6579) for the detection of Salmonella enterica from a wide range of foodstuffs. The novel workflow was compared against the standard by screening retail meat samples for Salmonella contamination, specifically concerned with pork mince. The prevalence of contamination for pork food products was similar to that reported previously. The alternative method reported similar results to the standard method but achieved them 24 hours earlier. A low cost, effective IMS bead was designed and manufactured for Salmonella enterica. This allowed for the capture and concentration of Salmonella from complex matrices such as faeces, after only a six-hour resuscitation step. The produced bead was then used in a feasibility experiment, for recovering wild type S. Typhi isolates alongside a modified version of CASE. All isolates tested showed good reactivity with the produced IMS bead and good, typical growth on modified CASE. The improved diagnostic agar and methodology described in this thesis allows for faster, more sensitive recovery and detection of Salmonella enterica from complex matrices. This provides improved tools for surveillance for this globally important pathogen.
Supervisor: Williams, Nicola ; Wigley, Paul Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral