Submitted to: Journal of Rapid Methods and Automation in Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/1/1999
Publication Date: N/A
Citation: N/A Interpretive Summary: In order to obtain the high numbers of bacteria needed for rapid detection tests, food samples are usually incubated overnight to allow the bacteria to grow to high numbers before conducting the tests. Rapid methods for concentrating the bacteria from the food samples to detectable levels could eliminate this added incubation time, thus shortening the time needed for detection. In previous work, it was shown that high numbers of bacteria adhere to the mineral hydroxyapatite (HA), and are rapidly concentrated from suspension. This work examined the use of HA to speed rapid detection of bacteria, by combining HA concentration and polymerase chain reaction (PCR) detection of the bacterium Salmonella. PCR detection of bacteria is a very sensitive detection method, because it involves the detection of genes that are unique to the target bacteria. High numbers of 14 different strains of Salmonella were found to adhere to HA, and nine strains adhered at percentages greater than 98.0%. HA was used to concentrate Salmonella from ground beef and beef carcass sponge samples, then PCR was used to detect this organism in the concentrated samples. HA typically removed and concentrated more than 95% both high and low levels of Salmonella from these beef samples. HA concentration improved the sensitivity of PCR detection of Salmonella in ground beef 1000 to 10,000-fold and in carcass sponge samples 100 to 1000-fold, as compared to detection in the unconcentrated samples. Detection was improved further upon short incubations (2-4 hours) of the concentrated samples. HA concentration is a both simple and fast way to concentrate bacteria from ground beef and carcass samples for PCR detection.
Technical Abstract: Hydroxyapatite (HA) concentration of bacteria from ground beef and bovine carcass sponge samples was examined as a method to enhance the PCR detection of low levels of Salmonella typhimurium in these samples. Ground beef and sponge samples taken from 24 h chilled carcasses were inoculated with progressively lower levels of S. typhimurium, such that prepared sample stomachates contained levels of S. typhimurium from 10**5 to 10**0 cells/ml. Concentrated (10% HA) and unconcentrated samples were prepared for PCR after 0, 2, 3, or 4 h of enrichment. Percent adherence values of Salmonella from the ground beef and sponge stomachates ranged from 81.3 to 99.4%, and typically were greater than 95% regardless of the sample type or the initial cell level. Without HA concentration and enrichment, Salmonella in ground beef was not detected by seminested PCR, even when present at levels up to 105 cells/ml in the 1:10 stomached samples of the ground beef. However, when ground beef samples were extracted with HA, limits of detection in non-enriched samples were 10**2-10**3 CFU/ml and in enriched samples were 10**1 CFU/ml (after 2 and 3 h enrichment) and 10**0 CFU/ml (after 4 h enrichment). Without concentration or enrichment, the limit of detection of Salmonella in bovine carcass sponge samples was 103 cells/ml. HA concentration of these non-enriched sponge samples lowered this limit to 10**0-10**1 CFU/ml. Percent HA adherence characteristics of 14 different Salmonella serotypes were determined, and ranged from 39.5% to 99.6%, with nine serotypes adhering at proportions of 98.0% or more.