Roger J. Lewis

Rapid electrochemical detection and identification of catalase positive micro-organisms

The rapid detection and identification of bacteria has application in a number of fields, e.g. the food industry, environmental monitoring and biomedicine. While in biomedicine the number of organisms present during infection is multiples of millions in the other fields it is the detection of low numbers of organisms that is important, e.g. an infective dose of Escherichia coli O157:H7 from contaminated food is less than 100 organisms. A rapid and sensitive technique has been developed to detect low numbers of the model organism E. coli O55, combining Lateral Flow Immunoassay (LFI) for capture and amperometry for sensitive detection. Nitrocellulose membranes were used as the solid phase for selective capture of the bacteria using antibodies to E. coli O55. Different concentrations of E. coli O55 in Ringers solution were applied to LFI strips and allowed to flow through the membrane to an absorbent pad. The capture region of the LFI strip was placed in close contact with the electrodes of a Clarke cell poised at +0.7 V for the detection of hydrogen peroxide. Earlier research identified that the consumption of hydrogen peroxide by bacterial catalase provided a sensitive indicator of aerobic and facultative anaerobic microorganisms numbers. Modification and application of this technique to the LFI strips demonstrated that the consumption of 8 mM hydrogen peroxide was correlated with the number of microorganisms presented to the LFI strips in the range of 2 x 10(1)-2 x 10(7) colony forming units (cfu). Capture efficiency was dependent on the number of organisms applied and varied from 71% at 2 x 10(2) cfu to 25% at 2 x 10(7) cfu. The procedure was completed in less than 10 min and could detect less than 10 cfu captured from a 200 microl sample applied to the LFI strip. The approached adopted provides proof of principle for the basis of a new technological approach to the rapid, quantitative and sensitive detection of bacteria that express catalase activity.

Contributory factors to the spread of contamination in a model kitchen

Artificial contamination of chicken pieces with bioluminescent E. coli DH5a (pLITE 27) was used to examine the relationship between food hygiene interventions and the extent of contamination in a model kitchen. Analysis showed that, during the preparation of chicken casserole, bacteria were widely disseminated throughout the kitchen and equipment used. Food hygiene interventions were shown to reduce the extent of contamination. Demonstrates that effective cleaning and hand washing are important in preventing cross‐contamination in the domestic kitchen.

The rapid potentiometric detection of catalase positive microorganisms

The rate of fluoride ion release from the enzymatic cleavage of fluoride ion from 4-fluorophenol by horseradish peroxidase, in the presence of hydrogen peroxide, was measured using a fluoride ion selective electrode. Monitoring the utilisation of hydrogen peroxide by catalase (intracellularly present in almost all aerobic microorganisms) in the presence of 4-fluorophenol demonstrated the inhibition of the enzyme. Horseradish peroxidase appeared to impart a partial protective mechanism of this inhibition. The development of a sequential assay demonstrated the applicability of the proposed method in the assessment of aerobic microorganism numbers. The judicious variation of three parameters, the length of incubation, the concentration of the primary substrate (hydrogen peroxide) and the indicator enzyme activity (horseradish peroxidase), affected both the detection limit and the sensitivity of the assay. Typically with a 15 minute incubation, a detection limit for catalase activity of 1.5 x 10(-6) Uml-1 was obtained together with a sensitivity of 2.42 mumol l-1 s-1 per decade change in activity. Application of the developed catalase assay to the detection of Escherichia coli achieved a detection limit of 1 x 10(2) colony forming units (cfu) ml-1 with a sensitivity of 3.26 mumol l-1 s-1 per decade change in intact microorganisms. By lysis of the microorganisms the detection limit was further reduced to less than 10 cfu ml-1, indicating the future possibilities of the assay.

Bacterial Bioluminescent Biosensors: Applications in Food and Environmental Monitoring

Abstract Bioluminescence has emerged as an extremely useful and versatile reporter technology. It provides a sensitive, non‐destructive, and real‐time assay that allows for temporal and spatial measurement. The ability to emit light is dependent on the reducing power of the organism; hence, only metabolically active cells can produce light. The direct relationship between viability and light emission allows the use of bioluminescent bacteria to assess the effect of various chemical, biological, and physical signals. We have constructed a number of recombinant plasmids carrying the lux operon expressed constitutively in many Gram negative and Gram positive bacteria. These plasmids were used to transform a wide variety of bacteria, giving rise to a range of versatile and robust whole cell biosensors with varied applications. The clinical isolate Escherichia coli 16906 was transformed with the pUC19‐derived pLITE27 plasmid to study the antibacterial effect of xanthine oxidase found in bovine and human milk. The self‐bioluminescent strains Salmonella enterica serovar Typhimurium DT104 and E. coli O157 (tox‐) were used to monitor bacterial inactivation and recovery on food surfaces during and after heat treatment. Escherichia coli O157, transformed with the pLITE27 plasmid, was successfully employed to assess the uptake and survival of bacteria within the freshwater protozoa Tetrahymena pyriformis.