NEW APPROACHES AND AUTOMATION IN FOOD MICROBIOLOGY

Advantages of rapid methods can include: Greater accuracy, better sensitivity, increased sample throughput, automated data capturing allowing easier data handling, and reduced cost for product release.There are three main categories; qualitative, quantitative and identification.

Qualitative Rapid Methods detect the presence of microorganisms in products. There are a number of tests currently available that measure changes in impedance, CO2 (via colour change in media) or pressure (headspace pressure) that signal microorganism growth. There are also methods such as polymerase chain reaction (PCR), flow cytometry and endotoxin tests (Limulus ameobocyte assay test) that can be used to more rapidly detect presence of microorganisms (or related bacterial endotoxins in the case of LAL). ATP measurements are also becoming more commonplace and have been used very successfully in hygiene monitoring to detect contamination.

Quantitative Rapid Methods are capable of providing a numerical value on the microorganisms present in a known sample unit. This is useful across the spectrum of microbiological tests. PCR is again useful as it is capable of quantifying as well as detecting the presence of microorganisms (commonly RT-PCR) in a matter of hours. There are also other methods that utilise direct detection of microorganisms using digital imaging of microcolonies (far too small to be seen with the naked eye) growing on solid media or filters as in direct laser scanning, light scattering, ATP bioluminescence and auto fluorescence methods. Methods such as flow cytometry and raman spectroscopy are also coming to the fore in quantitative RMMs, providing a much quicker time to result than the standard incubation and detection procedures used in most microbiology laboratories.

Identification methods - PCR or nucleic acid-based methods can be used to identify organisms based on the 16s rRNA gene (or 16sDNA) in bacteria or the 26s rRNA gene, ITS or D2 region in fungi. They can provide a result in a matter of hours, most likely an overnight run, but can identify to a very distinct level (type or sub-type) so are used commonly in determining the exact source of sterility failures or serious contaminant route cause analysis.

Immunological techniques

Immunological methods rely on binding of antibodies to specific antigens of target bacteria and serological assays. These methods improved the microbial diagnosis because of their high-throughput capacity, time- and cost-efficiency and ease-of-use .

-Enzyme-Linked Immunosorbent Assay (ELISA) - uses an enzyme-mediated colour change reaction to detect the presence of the specific pathogen/antigen. An advantage of this method is the possibility to quantify the pathogen/antigen. But like all other techniques, there are also some limitations: both specificity and sensitivity are reduced due to the difficulty to generate selective antibodies and the requirement of large amounts of antigens for quantification.

– The serological assay - another immunological approach is used to detect human serum antibodies that appear specifically in response to acute or past microbial infection. However, these assays are usually inadequate due to their long time the antibodies need to be produced within the human body.

Nucleic acid-based techniques – for highly specific detection

Nucleic acid-based technologies include several variations of hybridisations, polymerase chain reaction (PCR) and sequencing as well as DNA/RNA microarrays. All these methods base on the selection of genetic sequences through which pathogens can be specifically identified. For this purpose, generally ubiquitously conserved genes called housekeeping genes are used or random parts of the genome are screened. However, these methods don’t provide information about the cellular metabolic state because they just detect intact DNA.

-Hybridisation methods- The assays are based on a direct hybridisation of labelled (e. g. isotopes, fluorophores) oligonucleotide probes with species-specific DNA/RNA. A disadvantage of this method is the necessity of pre-cultivation of the microorganisms to produce significant amounts of cells to obtain a detectable signal. In addition, the number of probes that can be used in one experiment is limited and background noise might be a problem.

-PCR - The advent of PCR enabled the detection of even small amounts of genetic material encompassing DNA and RNA by amplification. PCR is very fast, sensitive and highly specific for bacteria whose sequence is already known. But they are limited in multiplexing and discovery of novel species or detection of variant strains of a known species.

In contrast, sequencing technologies provide the most detailed, unbiased information of all nucleic-acid based methods and are able to reveal unknown organisms. But depending on the application, it can be cost-intensive and time-consuming. Multiplex sequencing reduces the costs but decreases the sensitivity of the analysis, which might be an issue when pathogens have low abundance in the clinical sample (13).

-DNA/RNA-Microarrays – collection of various microscopic DNA/RNA-spots (probe) attached to a solid surface are found in the middle range regarding cost, processing time, sensitivity, specificity and ability to detect novel organisms. The currently available arrays are able to test for the presence of thousands of different microbes simultaneously (e.g. LLMDA), but generally, a previous labelling (e.g. fluorophore, silver, chemiluminescence) of the samples is necessary making it more labour- and cost-intensive. However, the usage of label-free devices might overcome this issue and turn microarrays to a more attractive technique.

-MALDI-TOF mass spectrometry

Matrix-assisted laser desorption ionisation time of flight mass spectrometry (MALDI-ToF)-  This is a different phenotypic identification method used to determine the identity of a microorganism on the basis of a protein profile or ‘fingerprint’. It is relatively inexpensive to run samples in the MALDIToF equipment and results are available in minutes, but there is a large outlay cost that is seen to be prohibitive for small throughput in smaller laboratories. This technique has fast, easy and high-throughput characteristics, does not need bacterial cultivation and generates simple and easily interpretable spectra. However, clinical samples are usually rich in host proteins and normal flora, which might result in overlapping mass spectra. Moreover, MALDI-TOF MS is limited in sensitivity since a sufficient number of cells is necessary to prevent their lost in background noise.

 

Automation in dilution and dispension

In most laboratories, much time is spent in routine manipulations involving media preparation, plate pouring, specimen dilution and inoculation and various staining procedures. There are several instruments now available which help with these mundane tasks.

-The "Stomacher"  - breaks and mixes raw samples such as meats, cheese, beans, peas, etc. The sample is placed in a sterile ethylene bag with a small amount of diluent. Paddles then massage the bag externally, creating violent shear forces which pulverize the specimens.

-The Colworth 2000 -  is a programmable instrument which takes a pre-determined volume of sample and makes aseptic dilutions (up to eight times) in as many as four media. It mixes these in molten agar, pours petri plates, prints the sample number on the plates, and stacks them for incubation.

-Auto-streaker - which streaks 1 to 10 plates of various media, selects the desired plates, streaks for isolation or count, labels with specimen number and sorts the finished plates for the correct incubator.

-Dynastainer - This instrument can efficiently perform gram, acid-fast, and other stains seven to eight times faster than manual operations.

-Automated pipettors and dilutors for rapid delivery and/or dilution of specimens, antibiotics, antigens, and various other fluids.

-Dynatiter or Autotiter - can automatically dilute antibiotics and add nutrient and test solutions for rapid MIC studies. Other immunological and serological studies can also be done.

Other biophysical and biochemical approaches

Bactec - is a completely automated system for radiometric determination of microbial growth in clinical and food samples. The degree of radioactive C02 production can be correlated with growth and is translated into a "growth index" on the instrument. The radiometric method is based on the premise that in a suitable medium containing radioactive glucose (or other compounds), microorganisms will liberate radioactive C02 as they metabolize the compound. The liberated gas is passed through an ionization chamber and can be determined. Usually [14C] compounds are employed. The Bactec 310 was used with labeled sodium formate to determine the microbial acceptability of cooked vegetables. This instrument has also been used to determine the microbiological burden of raw hamburger samples. The Bactec technique is basically designed for fermentative bacteria. To make use of this procedure for "non-fermentors," a special radiometric medium was developed which allowed rapid detection of Pseudomonas and Alcaligenes. Patulin, a toxin produced by Penicillium patulum in foods and feeds can be labeled with [14C] acetate so that its metabolism and mode of action can be studied.

-Chemiluminescence. These are based on the presence of iron-porphyrin compounds in the bacterial cell. These porphyrins appear to remain at a constant level in the cell regardless of environmental conditions or age of the cells. When luminol (or myeloperoxidase) and hydrogen peroxide are added to bacterial cells, light is generated. As with bioluminescent reactions, the amount of light generated is proportional to the amount of iron-porphyrin compounds present and can be equated with bacterial concentration

-Pyrolysis in combination with gas or gas-liquid chromatography provides another means for rapid iden-tification of bacteria or bacterial products and the method has been successfully automated. Of importance to food microbiologists, Emswiler and Kotula were able to correctly classify Salmonella serotypes by analyzing pyrochromatograms of flagella and DNA. Using this combination of pyrolysis and gas chromatography, an instrument named the Bacterial ID System 700 series is marketed by Chemical Data System Inc., Oxford, PA. Most major instrument companies market various types of chromatographs. Chromatape, the first fully automated system for thin-layer chromato-graphy, is marketed by J.T. Baker (Phillipsburg, N.J.). This has potential for food industry laboratories.

 

Agar based kits - The Enterotube system consists of 11 agar based tests housed in a compartmented plastic tube (Roche Diagnostics, Nutley, N.J.). Inoculation is done by touching an isolated bacterial colony with the tip of a special needle of the kit and pulling the needle through the kit, depositing the bacteria in the various chambers in the process. After an 18 to 24 h incubation, appropriate reagents are added to some of the chambers and reactions are recorded. To facilitate identification of unknowns, a convenient booklet (ENCISE) is available. This contains special codes and keys to identify the enteric microorganisms. A computer consultation service is available for helping the technician to identify the cultures. This system is easy to use and seems to have wide acceptance. The disadvantage of the system involves the fact that only 11 tests are available and the kit has a short shelf life. The original system was designed for Enterobacteriaceae.

Dehydrated media kits - A series of dehydrated systems called API are marketed by Analytab Products (Division of Ayerst Laboratories, Inc., Plainview, NY). The API 20 E (20 tests) and the API SOE (50 tests) are used for identification of Enterobacteriaceae. The API 20A is an anaerobic system, the API 20C identifies clinical yeasts, and the API 3S or lOS detects the possible presence of enteric pathogens in stool cultures (in 5 h). The dehydrated media are housed in small plastic chambers or "microtubes" on a convenient plastic card. Sus-pensions of the organisms are introduced into the individual chambers. After a 18 to 24-h incubation period, appropriate reagents are added and reactions are recorded. The data are matched with the API Coder and API Profile Register for identification. To facilitate accurate identification, the company also has a computer service to help with interpretation of the data, either by telephone or direct use of the computer bank.