Isolation of Salmonella spp. from artificially inoculated meat samples and detection by the PCR
The PCR assay and the microbiological method showed an equal level of sensitivity with a limit of detection for S. Typhimurium by both methods of 1 CFU/25 g of meat (initial inoculum level) (data not shown). The primer pair targeting the invA gene, designed by Rahn and others (1992), was tested using a collection of 630 Salmonella strains and showed 99.4% specificity and no amplification of DNA from non-Salmonella strains. Moreover, the adequacy of invA as a target gene in PCR assays was tested by Daum and others (2002) in a fluorogenic TaqMan PCR assay to confirm the presence of Salmonella directly from chicken in less than 3 h. Although in the current study a single serovar was tested using this assay, we have also amplified the PCR product of the invA gene in serovar S. Enteritidis using the same primer pair in assays for the detection of Salmonella in other types of food samples (unpublished data). Thus, the PCR assay targeting the invA gene can potentially be used to detect Salmonella in raw beef samples as an alternative to the cultural method. However, enrichment is a necessary step if fresh products are tested by either the microbiological method or the PCR, since it is possible that a low level of the pathogen of interest may be present. Growth of the target organism, however, could be inhibited somewhat by the accompanying flora, which might give a negative result by the cultural method, or the sensitivity of the PCR assay could be decreased. As such, it is important to select a suitable enrichment medium to inhibit the background flora and the appropriate enrichment time because PCR sensitivity tends to increase with increases in enrichment times (Guo and others 2000).
Microbiological assay and PCR of commercial meat samples
The data in Table 1 show results of 4 samples, which were positive by the PCR assay and the results of the same samples tested using the cultural method for the detection of Salmonella spp. The other 46 meat samples analyzed were negative using both methods. Three samples out of 50 (6%) were detected as positives by the microbiological method, while the PCR assay detected an additional sample (8%). Several reasons could explain why a sample was detected as positive by the PCR (sample nr 12) and not detected as positive by the microbiological method. It is possible that the cells in the meat sample were injured due to the use of sanitation products or due to stressful storage conditions, which can damage or even kill the Salmonella. This in turn would affect the ability to detect the pathogen using the cultural method, since this method is dependent on growth of the cells. Figure 1 shows the PCR products obtained following amplification of the positive meat samples. Sample nr 12 (lane 3) shows a weak band. Thus, it is possible that there was less PCR product from this sample because there were a lower number of cells after enrichment compared to the other samples. This was likely the reason why the pathogen was not detected by plating. Although, the sensitivity of both the microbiological and PCR methods was the same (1 CFU/25 g) when meat samples were inoculated with nonstressed cells, in naturally contaminated samples, the cells may be stressed affecting their ability to grow as rapidly as nonstressed cells in the enrichment medium.
Table 1—. Meat samples positive for Salmonella spp., by both the cultural and PCR methods.
|Sample nr||Cultural method||PCR assay|
Figure 1—. PCR products from the Salmonella invA gene in beef samples positive for Salmonella spp. Lane 1, molecular weight markers, 100-bp ladder (BIOLINE); lanes 2 to 5, samples 3, 12, 33, and 41, respectively; lane 6, negative control; and lane 7, positive control—S. Typhimurium.
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Cultural and PCR assays using cantaloupe rinse samples
The data in Table 2 show that in the 1st field, 4 positive samples were detected (quadrants I to III and V) by the microbiological method, and there were 4 positives from the same 4 quadrants by the PCR. However, for quadrants III and IV, the results of both methods differed. In the 2nd field, the results of both methods were the same, with positive results from samples in all 5 quadrants. In the 3rd and 4th fields, all results using the microbiological assay were negative, while using the PCR, results were positive from samples from the 3rd field from quadrants II and III and were negative from the 4th field from all 5 quadrants. With the exception of samples from quadrant III and IV from the 1st field where results differed, results of the PCR assay agreed with those of the cultural method, and in some cases the PCR assay was more sensitive for detection of Salmonella (quadrants II and III of the 3rd sampling). In summary, Salmonella spp. were detected by the microbiological method in 9 of 20 samples (45%), whereas the pathogen was detected by the PCR in 11 samples (55%). These results are partially in agreement with those of Espinoza-Medina and others (2006) who found that by the PCR method, 25.7% of samples from in-field cantaloupes were positive for Salmonella, whereas no positive samples were detected by the standard method.
Table 2—. Positive results for Salmonella spp. from cantaloupe surface washings using the cultural method and the PCR assay.
|First field||Second field||Third field||Fourth field|
The detection of this pathogen by the PCR was done from an enrichment culture; therefore, in addition to growth of Salmonella, the microflora from the melon samples also grew. Thus, the PCR was sensitive and specific, since Salmonella was detected in the presence of other microorganisms found in the melon production environment. Previous microbiological studies conducted in the Lagunera Region of Mexico (Froto and others 2004) found the presence of bacteria that belonged to the Enterobacteriaceae family and other microorganisms in the cuticle of melons, as well as plant pathogens such as Fusarium spp., Verticillium spp., and Rhizoctonia solani, and saprophytes such as Aspergillus spp., Rhizopus spp., and Penicillium spp. Also, human pathogens, including Clostridium botulinum, Listeria monocytogenes, Vibrio cholerae, Brucella melitensis, Salmonella Typhi, Salmonella Paratyphi, hepatitis A virus, Escherichia coli, and Shigella dysenteriae, were found. Most of these pathogens could be found in the soil coming from bovine and avian manure and from human feces (Froto and others 2004). The absence of the Salmonella invA sequence in other invasive bacteria such as Yersinia spp., Shigella spp., and enteroinvasive E. coli, which also have the capacity to invade epithelial cells, demonstrates the particular specificity and utility of this primer pair for detection of Salmonella spp. (Galán and Curtiss 1991). The invA gene has been used as the target in PCR assays mainly for detecting Salmonella in poultry, meats, and dairy products, and in vegetables and fruits (Guo and others 2000). The PCR results of the current study shown in Figure 2 indicate that the primers could be used for detection of Salmonella from cantaloupe surface washings and potentially in other types of fruits and vegetable samples, as well.
Figure 2—. Detection of Salmonella spp., by the PCR from cantaloupe surface washings. Lanes 1 to 4, quadrants I to IV (3rd field); lanes 5 to 6 and 8 to 10, quadrants I to V (4th field); lane 7, molecular weight marker, 100-bp ladder (BIOLINE); lane 11, positive control—S. Typhimurium; lane 12, negative control.
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The differences in results from field to field may have been due to differences in levels of Salmonella contamination. This may have been influenced in part by changes in the environment, including loss of specific nutrients and fluctuations in humidity, temperature, and ultraviolet light, all of which could damage bacterial cells and combined with the low probability that human pathogens develop stress resistance (Dickinson 1986; O'Brien and Lindow 1988). Furthermore, each field was sampled only once during the 1.5 mo period, and Good Agricultural Practices (GAP) were not applied in the first 3 fields. For the 4th field, in which no positive results were obtained, the cantaloupes were obtained from a field in which GAP were applied. These included the use of plastic mulch, fertirrigation, water without evidence of microbiological contamination, portable restrooms in the fields, training of field workers in GAP, use of authorized pesticides, designation of areas where workers may eat and take breaks, and availability of potable water for workers, which according to HACCP (Hazard Analysis and Critical Control Point) plans are basic tools for reducing physical, chemical, and microbiological hazards in agricultural production.