Methodology for detection and typing of foodborne microorganisms

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Abstract

Over the past decade many improvements have been seen in both conventional and modern methods for the detection of pathogenic bacteria in foods. Modifications and automation of conventional methods in food microbiology include sample preparation, plating techniques, counting and identification test kits. ATP bioluminescence techniques are increasingly used for measuring the efficiency of cleaning surfaces and utensils. Cell counting methods, including flow cytometry and the direct epifluorescent filter technique are suitable techniques for rapid detection of microorganisms, especially in fluids. Automated systems based on impedimetry are able to screen high numbers of samples based on total bacterial counts within 1 day. Immunoassays in a wide range of formats make rapid detection of many pathogens possible. Recently, there have been important developments in the use of nucleic acid-based assays for the detection and subtyping of foodborne pathogens. The sensitivity of these methods has been significantly increased by the use of the polymerase chain reaction and other amplification techniques. Alternative and rapid methods must meet several requirements concerning accuracy, validation, speed, automation, sample matrix, etc. Both conventional and rapid methods are used within hazard analysis critical control point programs. Further improvements especially in immunoassays and genetic methods can be expected, including the use of biosensors and DNA chip technology.

Introduction

The analysis of foods for the presence of both pathogenic and spoilage bacteria is a standard practice for ensuring food safety and quality. Conventional bacterial testing methods rely on specific microbiological media to isolate and enumerate viable bacterial cells in foods. These methods are very sensitive, inexpensive and can give both qualitative and quantitative information on the number and the nature of the microorganisms present in a food sample. However, conventional methods require several days to give results because they rely on the ability of microorganisms to multiply to visible colonies. Moreover, culture medium preparation, inoculation of plates, colony counting and biochemical characterization make these methods labour intensive. Especially in the food industry there is a need for more rapid methods to provide adequate information on the possible presence of pathogens in raw materials and finished food products, for manufacturing process control and for the monitoring of cleaning and hygiene practices. These rapid methods deal with the early detection and enumeration of microorganisms, but also with the characterization of isolates, by use of microbiological, chemical, biochemical, biophysical, molecular biological, immunological, and serological methods (Fung, 1995). Alternative and rapid methods can be divided into the following categories:

  • modified and automated conventional methods;

  • bioluminescence;

  • cell counting methods;

  • impedimetry;

  • immunological methods;

  • nucleic acid-based assays.

Section snippets

Modified and automated conventional methods

Many attempts have been made to improve laboratory efficiency by making the procedures for the traditional agar medium-based methods more convenient and user-friendly and to reduce the costs of material and labour (Table 1)

Bioluminescence

Microbial cells as well as cells from food ingredients contain adenosine-5′-triphosphate (ATP) which can be measured using the luciferase enzyme complex found in fireflies. The total light output of a sample is directly proportional to the amount of ATP present and can be quantitated by luminometers. At least 104 cells are required to produce a signal. Measurement of ATP from bacterial and nonbacterial cells can, in this way, be used for measuring the efficiency of cleaning surfaces and

Flow cytometry

This is an optically-based method for analyzing individual cells in complex matrixes. Microorganisms suspended in a liquid pass a beam of laser light. As this occurs, the light is both scattered and absorbed by the microorganisms. The extent and the nature of the scattering, which is an intrinsic property of the microorganisms, may be analyzed by collecting the scattered light with a system of lenses and photocells and can be used to estimate the number, size, and shape of microorganisms. The

Impedimetry

Impedimetry is based on changes in conductance in a medium where microbial growth and metabolism takes place. The time necessary for these changes to reach a threshold value, the detection time, is inversely proportional to the initial inoculum. Several automated systems based on impedimetry are commercially available. These systems are able to examine hundreds of samples at the same time. Instruments are fully automated and computer-driven to enable continuous monitoring of impedance changes

Immunological methods

Immunological methods rely on the specific binding of an antibody to an antigen. For the detection of specific microorganisms and microbial toxins a variety of antibodies which are employed in different assay types have been described (Märtlbauer and Becker, 1995). The suitability of these antibodies depends mainly on their specificity. Polyclonal antisera contain an assortment of antibodies having different cellular origins and, therefore, somewhat different specificities. Most polyclonal

Nucleic acid-based assays

The past decade has seen a significant increase in the development of genetically-based methods for the detection and characterization of pathogens in foods. Genetic detection methods are based on the hybridization of target DNA with a specific DNA probe. Depending on the desired specificity of the detection (genus-, species-, strain-specificity), different regions of the genome can be used as targets (Scheu et al., 1998).

Requirements for alternative and rapid methods

There are several factors which must be considered before adapting a new alternative or rapid method (Swaminathan and Feng, 1994, Fung, 1995, van der Zee and Huis in ’t Veld, 1997, Notermans et al., 1997):

  • 1.

    Accuracy — false-positive and false-negative results must be minimal or preferably zero. The method must be as sensitive as possible and the detection limit as low as possible. In many cases, the demand is less than one cell per 25 g of food, as small numbers of some pathogens may cause

Microbiological methods in HACCP programs

In the past decade, the control of the safety of foods has been mainly carried out by product testing rather than process control. The main problem with doing end-product testing is the high number of samples to be examined before one can decide on the safety of the product batch, especially when pathogens are expected to be heterogeneously distributed in the batch. Moreover, end-product testing detects only failures and does not identify causes (van Schothorst and Jongeneel, 1994). HACCP is

Future development

Improvements in the field of immunology, molecular biology, automation and computer technology continue to have a positive effect on the development of faster, more sensitive and more convenient methods in food microbiology. Further development of “on-line” microbiology, including ATP bioluminescence and cell counting methods, is important for rapid monitoring of cleanliness in HACCP programs.

One of the most challenging problems is sample preparation. More research is needed on techniques for

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