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- Materials and methods
A rapid method based on bacterial adhesion was developed for the detection of Salmonella in an enriched meat system. Minced beef samples inoculated with Salm. enteritidis (10 cfu g−1) were incubated overnight (18 h) at 37 °C in buffered peptone water. Salmonella enteritidis cells were isolated from the enriched meat sample by surface adhesion onto a polycarbonate membrane attached to a glass slide. The organisms attached to this polycarbonate membrane were subsequently visualized using immunofluorescent microscopy. The technique had a detection level of log10 3·5 Salmonella ml−1. The surface adhesion immunofluorescent technique correlated well with Salmonella plate counts (r2 = 0·99). Application of the rapid method to retail beef and poultry samples (n = 100) confirmed the correlation between this technique and traditional microbiological procedures. Thirty-one retail samples were reported positive for Salmonella species. No false positives or negatives were recorded for the rapid method.
Currently available cultural methods for the detection of Salmonella in food are time consuming, taking up to 4–6 d to detect the pathogen, and are generally unsuitable for use in an industrial based laboratory. This is clearly unsatisfactory and a range of alternative rapid methods has been developed (Tietjen & Fung 1995). Approaches to rapid methodology for detection of Salmonella and other pathogens of concern to the food industry include electrical methods (impedance and conductance) and methods based on immunological and DNA hybridization techniques. While these tests are considerably faster than the traditional plate count methods, they still rely on lengthy enrichment (48 h) to increase very low initial Salmonella numbers to detectable levels. A novel rapid method for the detection of Listeria monocytogenes has been reported which takes 20 h to carry out (Sheridan et al. 1997). This method involved an overnight enrichment in buffered peptone water (BPW), isolation of the L. monocytogenes by attachment to a polycarbonate membrane immersed in the culture, followed by detection of the pathogen using immunofluorescent microscopy. The basic principles of this method would appear to have the potential to be applied to other pathogens of concern to the food industry, such as Salmonella.
It is already widely reported in the literature that Salmonella can attach to a wide range of inert surfaces such as stainless steel, glass and polymeric substrates, and to biological surfaces such as skin, muscle and cell membranes (Dickson & Daniels 1991; Mafu et al. 1991; Absolom et al. 1983). This suggests that a surface adhesion membrane extraction system may prove suitable for isolation of this pathogen.
The purpose of this study was to develop a rapid method for the detection of Salmonella based on an 18 h enrichment step, isolation by membrane surface adhesion and detection by immunofluorescence microscopy. This paper describes the application of this novel rapid technique to inoculated meat samples and it's correlation with a traditional cultural method. This novel rapid method was also applied to a small number of commercial meat samples to investigate the presence of naturally occurring Salmonella.
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- Materials and methods
This study found that the use of a selective media containing inhibitory agents offered no advantages in terms of an enrichment step as part of a rapid technique. In addition, using a non-selective broth such as BPW allows the recovery of injured cells (Bailey & Cox 1992), which is of particular importance in the analyses of processed foods.
The second phase of this study showed that Salm. enteritidis could be isolated from an enriched culture using a surface adhesion technique. The investigation found that maximum attachment of Salm. enteritidis to the polycarbonate membrane occurred after 15 min immersion in the culture, while immersion for an additional time of up to 60 min did not result in a significant increase in Salm. enteritidis attachment. This is similar to the attachment rates recorded for other pathogens, including L. monocytogenes and Yersinia enterocolitica, to a polycarbonate membrane in an enriched culture (Sheridan et al. 1997, 1998).
In this study, similar attachment rates were recorded for Salm. enteritidis enriched in broth and meat culture systems, indicating that the attachment of the pathogen was not affected by the presence of naturally occurring meat microflora. Other workers have also reported that attachment of one bacterial species was unaffected by other species present (Farber & Idziak 1984; Sheridan et al. 1997). Adherence is mainly dependent on physiochemical properties, including the structure of the attachment surface and environmental conditions such as pH (Dickson & Koohmaraie 1989; Mafu et al. 1991; Duffy & Sheridan 1997a).
The surface adhesion-based extraction of Salmonella gives a rapid and clean deposit of the bacteria onto the membrane surface from which they can then be detected using FITC-labelled antibodies. The membrane surface adhesion technique and immunomagnetic beads for the isolation of L. monocytogenes from enrichment broths were compared and the surface adhesion method was reportedly quicker while achieving similar extraction rates (Duffy et al. 1997b). It is assumed that a similar scenario would be recorded for the isolation of Salmonella.
Studies using inoculated meat determined that a level of log10 3·50 cfu ml−1 of Salm. enteritidis was necessary for detection by the SAIF technique. Data generated in the early part of this study on the modelling of Salmonella growth in BPW predicted that if very low initial levels of Salm. enteritidis (1 cfu 25 g−1) were present in the sample, an enrichment period of 18 h would be sufficient to allow the pathogen to reach a detectable level. Retail samples naturally contaminated with Salmonella spp. have been shown in this study to contain levels of approximately <log10 2·18 cfu g−1. Therefore, a shorter enrichment period might be possible for many samples. This enrichment period is considerably shorter than the 48 h enrichment period needed for commercial ELISA and DNA based technologies, which require a detection level of 105–106 cfu ml−1 target cells in the enriched sample (Foster et al. 1992; Felsine et al. 1993).
In inoculated meat samples, an excellent correlation was reported between Salm. enteritidis traditional plate counts and the numbers detected by the SAIF (r2 = 0·99). This indicates that it would be possible to predict accurately numbers of Salmonella in the enrichment broth from those detected by the SAIF technique. Predictive modelling techniques could then be applied to estimate the initial level of contamination in the product. This type of information would be invaluable in the development and application of risk assessment models (Buchanan & Whiting 1996).
Correlation of the SAIF assay with traditional methods was carried out using a small amount of naturally contaminated retail samples. A total of 31 samples tested positive for Salmonella by both methods, with no false positives or negatives being recorded for the rapid technique. After an 18 h enrichment, experimental plate counts obtained compared very favourably with predicted plate counts as determined from the SAIF method.
Salmonella bredeney was the most commonly isolated serotype in this study. No published data are available regarding the most prevalent Salmonella serotypes in foods in Ireland. However, clinical reports from the southern regions of the country noted that Salm. typhimurium was the most frequently isolated serotype, followed by Salm. enteritidis (Anonymous 1997 Infoscan. Communicable Disease Report. Salmonella. Quarterly Reports, 7, 1–4.
In conclusion, the SAIF technique is easy to perform, ensuring rapid isolation and identification of Salmonella from enriched samples. It requires minimal time and labour and would have considerable potential for on-line use in an HACCP system in an industrial based laboratory where the rapidity of a result is vital.