Sixteen FERN (Food Emergency Response Network) member laboratories collaborated in this study to verify extension of the real-time PCR Salmonella detection method originally designed for the single-tube Cepheid SmartCycler II and validated against the Salmonella method of the U. S. Food and Drug Administration Bacteriological Analytical Manual to the Applied Biosystems (ABI) 7500 FAST Real-Time PCR system multiwell plate platform. Four foods were selected for this study: chili powder, soft cheese, fish, and tomatoes; these foods represent products that are commonly analyzed for the presence of Salmonella for regulatory purposes. Each food consisted of six uninoculated control samples, six samples inoculated with low Salmonella levels (target 1 to 5 CFU/25 g), and six samples inoculated with high levels (target 10 to 50 CFU/25 g). All samples were tested for Salmonella using the 24-h quantitative PCR (qPCR) method for detecting Salmonella, which utilizes modified buffered peptone water as the sole enrichment medium and an internal control for the qPCR. Each of these 18 samples was individually analyzed for Salmonella by the collaborating laboratories using both the ABI 7500 FAST system (alternative method) and the SmartCycler II system (reference method). Statistical analysis of the data revealed no significant difference (P ≥ 0.05) between these two qPCR platforms except for the chili powder samples. The differences noted with chili powder (P = 0.0455) were attributed to the enhanced sensitivity of the ABI 7500 FAST system compared with the SmartCycler II system. The detection limit of both qPCR methods was 0.02 to 0.15 CFU/g. These results provide a solid basis for extending the 24-h qPCR Salmonella method to the ABI 7500 FAST system for high-throughput detection of Salmonella in foods.

An assay to identify the common food-borne pathogens Salmonella, Escherichia coli, Shigella, and Listeria monocytogenes was developed in collaboration with Ibis Biosciences (a division of Abbott Molecular) for the Plex-ID biosensor system, a platform that uses electrospray ionization mass spectroscopy (ESI-MS) to detect the base composition of short PCR amplicons. The new food-borne pathogen (FBP) plate has been experimentally designed using four gene segments for a total of eight amplicon targets. Initial work built a DNA base count database that contains more than 140 Salmonella enterica, 139 E. coli, 11 Shigella, and 36 Listeria patterns and 18 other Enterobacteriaceae organisms. This assay was tested to determine the scope of the assay's ability to detect and differentiate the enteric pathogens and to improve the reference database associated with the assay. More than 800 bacterial isolates of S. enterica, E. coli, and Shigella species were analyzed. Overall, 100% of S. enterica, 99% of E. coli, and 73% of Shigella spp. were detected using this assay. The assay was also able to identify 30% of the S. enterica serovars to the serovar level. To further characterize the assay, spiked food matrices and food samples collected during regulatory field work were also studied. While analysis of preenrichment media was inconsistent, identification of S. enterica from selective enrichment media resulted in serovar-level identifications for 8 of 10 regulatory samples. The results of this study suggest that this high-throughput method may be useful in clinical and regulatory laboratories testing for these pathogens. Mass spectrometry is an established analytical technique with growing applications within microbiology. With high sensitivity and high resolution, mass spectrometry can be used to differentiate microbial species based on subcellular variations. Recently, several articles concerning the application of either matrix-assisted laser desorption ionization (MALDI) (6, 14, 36) or electrospray ionization (ESI) mass spectrometry (MS) (13,21,30,37) to the detection and identification of microbes have been published. While some methods examine protein expression, this work centers on the use of nucleic acid information to identify bacteria.MS techniques involving the analysis of DNA take advantage of the difference in mass between strands with different base compositions. In order to utilize MS for DNA-based identification of bacteria, a region of DNA that varies between species or subspecies is amplified by PCR, and the mass of this amplicon is then determined. Since the exact masses of the individual bases in DNA are known, the quantity of each of these bases within the amplified sequence can be calculated based on the exact mass of the strand. While the exact sequence is not obtained through this method, the base compositions, or base counts, can provide enough information to discriminate between species, subspecies, and even serovars depending on the organism and the assay (15,19).This technique is comparable to oth...

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Recently, there have been several tattoo-related outbreaks of nontuberculous mycobacterial skin infections in the United States. In an eort to halt the outbreaks and to prevent similar events from occurring, FDA conducted an investigation to determine the source of the contamination. During the investigation, environmental and water samples were collected from tattoo parlors and manufacturers of tattoo ink. These samples were subjected to isolation of mycobacteria at Wadsworth Center of the New York State Department of Health and at FDA PRLSW followed by species identification of the isolates at PRLSW. In order to conduct the investigational studies in a time-sensitive manner, a two-step screening and classification procedure was devised. In this scheme, suspect mycobacterial colonies were screened using multiplex real- time PCR coupled with melting curve analyses specific for the genus Mycobacterium and for dierentiating the species within the M. chelonae-M. abscessus group. Mycobacterial isolates were subsequently identified via sequencing analysis within the coding regions of both 16s rRNA and RNA polymerase subunit beta. In total, 45 colonies of Mycobacterium were isolated and identified as M. chelonae, M. immunogenum, and M. mucogenicum. The isolates from each set of samples contained the corresponding species of Mycobacterium recovered from outbreak patients. Our results suggested that both unsanitary manufacturing processes during production of tattoo ink and the use of non-sterile water for dilution of tattoo ink were possible causes for outbreaks of skin infection in clients of the aected parlors. In addition, the two-step approach taken for screening and identifying mycobacterial colonies in the current study facilitated rapid investigation of tattoo-related outbreaks of nontuberculous mycobacterial infection, thereby enhancing FDAs ability to better protect public health.