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PDF Use of isolation and antibody detection for salmonella assessment

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The IC method was optimized for incubation time, bacterial concentration, and capture efficiency.

The method was shown to be highly specific for the pathogens concerned. The IC method also showed better pathogen detection ability at different concentrations of cells from artificially contaminated food samples in comparison with culture based methods. The IC method poses to be the potential to be used as a method of choice for detection of foodborne pathogens in routine laboratory practice after proper validation.

Foodborne pathogens are a growing concern for human illness, death, and food safety and security [ 1 ]. The analysis of foods for pathogen presence is a standard practice for ensuring the safety of food, identifying agents of foodborne illness and determining sources of foodborne outbreaks.

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Conventionally, the microbiological analysis of food involves culture enrichment followed by isolation on selective media [ 2 ]. From there, the pathogen, if present, is isolated on selective agar, and purification and confirmation occur using morphological, biochemical, or physiological tests [ 3 ]. Conventional culture methods, however, are often problematic, in that many are time-consuming and require several days to complete, appropriate selective media are not currently available for all bacterial foodborne pathogens, some bacterial pathogens require specific atmospheric or other growth conditions which may be difficult to simulate in the laboratory and some bacterial pathogens may not be culturable by currently available methods [ 4 ].

The presence of high background indigenous microflora and complex matrix in food also limits detection of pathogen from food samples. Again, pathogen often exist in viable but non-culturable states in food which cannot be detected by the conventional culture based method [ 5 ]. There is a great need for improved methods for foodborne pathogen detection in food matrices. Concentration and separation of pathogens from the food matrix has been the focus of many studies investigating ways to improve sample assay detection limits and speed time to results. Immunocapture IC to concentrate target pathogen from food samples offers a better alternative to traditional pre-enrichment and enrichment steps for routine analysis in microbiology laboratories [ 6 , 7 ].

Antibody attached to solid surface can capture bacteria through attachment to cell surface proteins and allow specific isolation of the bacteria from samples with high background flora [ 8 ]. IC can be used in combination with culture or molecular methods for isolation and detection of the pathogens. One form of IC, immune-magnetic separation IMS that uses magnetic beads coated with antibody has been developed against many pathogens such as Salmonella spp.

IMS has been investigated for the concentration and purification of bacterial pathogens from food samples [ 13 ] and are reportedly more sensitive than comparable conventional culture methods [ 14 ]. By using IMS, PCR inhibitors inherent to fecal samples were successfully eliminated [ 15 ], but this approach is limited in routine laboratories of underdeveloped countries due to instrumentation cost. This study focused on three important foodborne pathogens: Vibrio cholerae , Salmonella Typhi and Shigella flexneri.

Many previous reports have showed prevalence of Vibrio cholerae , Salmonella spp. Salmonella Typhi and Shigella spp. Shigella flexneri in food samples in Bangladesh. Shammi [ 16 ] reported contamination of Vibrio spp. Mrityunjoy et al. Noor et al. Fatema et al. Aktar et al. Prevalence of Vibrio cholerae , Salmonella spp.

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This study evaluates microplate IC wells of 96 well polystyrene plate coated with antibody as a better alternative to conventional methods for detection of foodborne pathogens. This trend was similar for all three pathogens. This may be due to limited space and bound antibody in the wells of 96 well plate. On the basis of the results of Figs. To determine the specificity of microplate IC, detection of other related pathogens has been tested. CE was zero or very low in case of other bacteria tested Fig.

One of the potential disadvantages of this method is the chance of cross reaction with closely related species, as it occurs in this experiment. Typhi from In contrast, the traditional culture method was unable to detect any pathogen in these samples and direct PCR can detect S.

Performance evaluation of the microplate IC method with the conventional culture based method showed comparable performance of the method for detection of the pathogens Table 2. Improvements in the microbiological safety of foods have been largely driven by public demand in response to disease outbreaks [ 25 ]. The ability to analyze food products for the presence of pathogenic bacteria is essential for verifying the safety of foods, identifying agents of foodborne illness and determining sources of foodborne outbreaks.

Conventionally, the microbiological analysis of food involves culture enrichment followed by isolation on selective media [ 14 ]. Such methods suffer from a number of drawbacks. In this study, we assessed the suitability of antibody coated 96 well microplates for the detection of three foodborne pathogen, namely Vibrio cholerae , Salmonella Typhi, and Shigella flexneri.

Publication details

Specificity of the IC method was evaluated and the method showed significant specificity for detection of the target pathogens, Salmonella Typhi, Shigella flexneri, and Vibrio cholerae. The method can discriminate the pathogens from closely related species, too Fig. Suitability of the IC method for detection of pathogens in food samples was tested with artificially contaminated food samples. Food samples were spiked with different concentration of pathogens, and the IC method and culture method were employed to detect the pathogens. Results of the comparison of IC and culture method have been summarized in Table 1.

In the case of minced beef, S. Typhi in In minced chicken, S. In minced fish samples, V.

Action of Antibodies: Neutralization, Opsonization, Complement Activation and ADCC (FL-Immuno/37)

In the case of minced shrimp, three pathogens S. Typhi, S. Finally, comparison of performance parameters of IC and culture method showed that the IC method offers better performance than the culture method for detection of pathogens from food in terms of the detection limit, specificity, sensitivity, and accuracy Table 2. Different variations of IC have been developed and reported earlier, most of which are based on immune-magnetic separation IMS.

Xiong et al. Wang et al. Conceicao et al. The microplate immunocapture MIC method developed in this study offers a better alternative to the previously reported, IMS methods in terms of ease of operation as it obviates the use of magnetic beads and separation systems. The MIC method also showed competitive performance sensitivity, specificity and accuracy with the above mentioned IMS methods. Future research is needed to resolve some shortcomings of this method, such as the effect of high microbial background in food samples.

More advanced methods such as whole genome sequencing may provide a more accurate and precise identification scheme for detection of bacteria from food, but in resource-limited laboratories, simpler PCR based method is still the preferable method.

This study showed that antibody coated microplate can be used for detection of foodborne pathogens from a consortium of non-target organisms with high specificity and sensitivity. This method provides an alternative to the conventional culture based microbiological detection method. The microplate IC method is simple, expensive equipment is not needed and can be modified to detect multiple pathogens simultaneously.

Background

From preliminary screening, the method showed promise, but further detailed studies are needed to validate the method for use as a routine method for food laboratories. All the media used were purchased from HiMedia India. Antibodies used were as follows- V. The list of other strains used in this study is provided in Table 3. On the next day, antibody solution was discarded and the microplate was washed 3 times with sterile PBS. Capture efficiency was defined as the percentage fraction of the total bacteria captured by the antibodies in the well and was calculated using a method based on the cells unbound in the well or left in the supernatant.

Following equation was used for CE calculation-. Number of unbound cells in the well were counted based on optical density.

Brazilian Journal of Microbiology

To test the specificity as well as the validity of the IC method, and to ensure that the method does not amplify closely related pathogens, a number of closely related and non-target organisms were included in the study Table 3. Immunocapture was performed and capture efficiency was determined as described above. A negative control well was used to check cross-contamination.

see url After incubation, the medium was discarded and the wells were washed with sterile PBS and fresh LB broth was added. Bacteria from the well were scraped with sterile loop and streaked onto selective agar plate for respective bacteria TCBS for Vibrio cholerae ; SS agar for Salmonella and Shigella and the plates were incubated at 37 0 C overnight.

Colonies developed on the agar were sub-cultured onto LB agar and identification was done either by culture method or PCR. Spiking was done to obtain different concentration of target bacteria S. Typhi in beef, S. For IC-culture, the well was washed with the original media vigorously and the media were inoculated onto selective media by spread plating method.

In case of spiked shrimp sample, all three antibodies were tested together. In other samples, a single antibody was used. For detection of Salmonella spp. For detection of Shigella spp. DNA concentration was measured by Nanodrop spectrophotometer. Primers used for PCR are listed in Table 4.

PCR reaction was performed according to the references provided [ 21 , 22 , 23 ].