Identification of Foodborne Bacterial Pathogens

Identification of Foodborne Bacterial Pathogens by Gene Probes
Source obtained from: http://www.cfsan.fda.gov/~ebam/bam-24.html

The above website contains information on:

• Some gene probes used to detect pathogenic bacteria in foods
• Description of individual probes to identify targets.

Example of Probes and their targets:

Campylobacter jejuni: Ribosomal RNA
A probe that is specific for C. jejuni ribosomal RNA genes has been developed (86,87) and is available commercially. A pool of randomly selected and tested chromosomal fragments is also specific for C. jejuni, but the target has not been reported (83).

Escherichia coli: Heat-labile enterotoxin genes
The heat-labile enterotoxins (LT) of E. coli are a closely related group of proteins; they are distinguished from heat-stable enterotoxins (ST) by being immunogenic and are inactivated by heating at 60°C for 10 Min (31). The toxins stimulate adenylate cyclase (30) and can be detected by tissue culture assays of Chinese hamster ovary cells (30) or mouse Y-l adrenal cells (13). Using these tests, So et al. (102) localized and cloned the structural gene for LT; Dallas et al. (8) recloned a smaller fragment into plasmid pEWD299. Although there are several different genes for LT, as evidenced by their nucleotide sequences (56,73,103,110,111), they all share a significant amount of genetic similarity. The region of the LT genes chosen as a gene probe target is identical in each of these genes, so that all strains with the genetic potential to produce LTs should be detected.
The LT probe, eltA11, is a 20 base synthetic oligonucleotide that encodes amino acids 45-51 of the A subunit of the E. coli LT (111).

Methods

Four different techniques can be used for colony hybridization tests:

1. Direct plating of samples for enumeration.
2. Direct plating of cultures after enrichment to determine presence/absence.
3. Spotting of individual colonies or pure cultures for an additional hybridization assay to confirm a positive result from colony hybridization with a mixed culture.
4. Returning to a "master" replica plate to make pure cultures of positive colonies for further study and analysis.

The first three techniques differ as to when solid media are inoculated. In the first, aliquots of the homogenized sample are plated immediately after blending. In the second, plates are inoculated after aliquots of the homogenized sample have been incubated under selective conditions. Samples from the first and second techniques are plated onto selective agar media whenever such appropriate media are available. For the third technique, individual positive colonies are re-streaked and an additional colony hybridization test is conducted to ensure that the initial positive or negative results can be repeated. With the last technique, a pure culture can be obtained without selective enrichment, and additional microbiological tests requiring a pure culture can then be performed.

Control cultures
Strains that are positive or negative for the various probe tests must be properly stored and periodically tested for the appropriate phenotypic characteristics. A test methodology other than a gene probe must be used to independently verify the genotype of the control microorganisms. Control cultures must also be stored appropriately to minimize the possibility of genetic change. Usually, freezing liquid cultures at -70°C in 10-25% glycerol will suffice, except for Vibrio species, which are particularly sensitive to cold. Appropriate control strains have been listed, but other strains can be used if they have been properly characterized.