Marta Mikš-Krajnik

Real-time PCR method combined with immunomagnetic separation for detecting healthy and heat-injured Salmonella Typhimurium on raw duck wings

Conventional culture detection methods are time consuming and labor-intensive. For this reason, an alternative rapid method combining real-time PCR and immunomagnetic separation (IMS) was investigated in this study to detect both healthy and heat-injured Salmonella Typhimurium on raw duck wings. Firstly, the IMS method was optimized by determining the capture efficiency of Dynabeads(®) on Salmonella cells on raw duck wings with different bead incubation (10, 30 and 60 min) and magnetic separation (3, 10 and 30 min) times. Secondly, three Taqman primer sets, Sal, invA and ttr, were evaluated to optimize the real-time PCR protocol by comparing five parameters: inclusivity, exclusivity, PCR efficiency, detection probability and limit of detection (LOD). Thirdly, the optimized real-time PCR, in combination with IMS (PCR-IMS) assay, was compared with a standard ISO and a real-time PCR (PCR) method by analyzing artificially inoculated raw duck wings with healthy and heat-injured Salmonella cells at 10(1) and 10(0) CFU/25 g. Finally, the optimized PCR-IMS assay was validated for Salmonella detection in naturally contaminated raw duck wing samples. Under optimal IMS conditions (30 min bead incubation and 3 min magnetic separation times), approximately 85 and 64% of S. Typhimurium cells were captured by Dynabeads® from pure culture and inoculated raw duck wings, respectively. Although Sal and ttr primers exhibited 100% inclusivity and exclusivity for 16 Salmonella spp. and 36 non-Salmonella strains, the Sal primer showed lower LOD (10(3) CFU/ml) and higher PCR efficiency (94.1%) than the invA and ttr primers. Moreover, for Sal and invA primers, 100% detection probability on raw duck wings suspension was observed at 10(3) and 10(4) CFU/ml with and without IMS, respectively. Thus, the Sal primer was chosen for further experiments. The optimized PCR-IMS method was significantly (P=0.0011) better at detecting healthy Salmonella cells after 7-h enrichment than traditional PCR method. However there was no significant difference between the two methods with longer enrichment time (14 h). The diagnostic accuracy of PCR-IMS was shown to be 98.3% through the validation study. These results indicate that the optimized PCR-IMS method in this study could provide a sensitive, specific and rapid detection method for Salmonella on raw duck wings, enabling 10-h detection. However, a longer enrichment time could be needed for resuscitation and reliable detection of heat-injured cells.

Antibacterial effect and mechanism of high-intensity 405 ± 5 nm light emitting diode on Bacillus cereus, Listeria monocytogenes, and Staphylococcus aureus under refrigerated condition.

This study investigated the antibacterial effect of 405 ± 5 nm light emitting diode (LED) on Bacillus cereus, Listeria monocytogenes, and Staphylococcus aureus, and examined its antibacterial mechanism by determining the bacterial membrane and DNA damages. A 405 ± 5 nm LED illuminated the Gram-positive pathogens until 486 J/cm(2) at 4 °C. Weibull model was used to calculate reliable life (tR) to compare bacterial sensitivities to LED illumination. The membrane damage was determined by NaCl and LIVE/DEAD® assay, while comet assay and DNA ladder analysis were conducted to determine DNA degradation. The illumination resulted in 1.9, 2.1, and 1.0 log reductions for B. cereus, L. monocytogenes, and S. aureus at 486 J/cm(2), respectively. The comparison of tR values revealed that L. monocytogenes was identified as the most susceptible strain to LED illumination. The percentage of the bacterial sensitivity to NaCl remarkably increased in LED-illuminated cells compared to non-illuminated cells. Moreover, loss of membrane integrity was confirmed for LED-illuminated cells by LIVE/DEAD® assay, whereas no DNA breakage was indicated by comet assay and DNA ladder analysis. Thus, these findings suggest that the antibacterial effect of 405 ± 5 nm LED illumination on these pathogens might be due to physical damage to bacterial membrane rather than DNA degradation.

Evaluation of real-time PCR coupled with immunomagnetic separation or centrifugation for the detection of healthy and sanitizer-injured Salmonella spp. on mung bean sprouts

Fresh mung bean sprouts have been identified as a source of many Salmonella outbreaks worldwide. The aim of this study was to develop a rapid and accurate detection methodology for low levels of healthy and sanitizer-injured Salmonella on mung bean sprouts using real-time PCR coupled with either immunomagnetic separation (PCR-IMS) or centrifugation (PCR-cen). Initially, three parameters of IMS; specificity/sensitivity, bacterial concentration and bead incubation time were optimized. Secondly, limit of detection (LOD) was determined for the optimized PCR-IMS and PCR-cen. These two methods were compared against PCR alone (PCR) and the standard culture method (ISO) for their ability to detect Salmonella using inoculated and uninoculated sprouts. Under optimum IMS conditions (10(5)CFU/ml for 30 min), capture efficiency of Salmonella in sprout suspensions was lower than 40%, most probably due to the non-specific binding of the background microbiota. PCR-IMS and PCR-cen had a similar LOD at 10(3)CFU/ml, which was one log unit lower than PCR. Enrichment of 10h was sufficient to detect 100% of the inoculated sprouts with both PCR-IMS and PCR-cen, which was significantly faster compared to PCR and the ISO method. Moreover, the validation study using uninoculated sprouts revealed that PCR-IMS and PCR-cen were equally effective on Salmonella detection, showing 98.3% accuracy. These results suggest that PCR-cen would be the effective and less costly method for the detection of both healthy and sanitizer-injured Salmonella on mung bean sprouts.

Detection of volatile organic compounds as markers of chicken breast spoilage using HS-SPME-GC/MS-FASST

Volatile organic compounds (VOCs) of naturally aerobically spoiled chicken breast at ambient temperature were analyzed to identify volatiles that can be used as spoilage markers. The headspace solid-phase micro-extraction (HS-SPME) technique coupled with gas GC/MS running in Fast Automated Scan/SIM Type (FASST) mode was applied using 4 SPME fibers of different polarity. All of fibers were able to detect the sulfides methanethiol (MeSH), dimethyl disulfide (DMDS), and dimethyl trisulfide (DMTS), the alcohols ethanol (EtOH), 1- and 2-butanol, and 1-butanol isomers, and free fatty acids (FFAs) in the range of C2 to C5. Principal component analysis (PCA) revealed that spoilage in chicken meat is 2-step process. Initially, an increase in amounts of alcohols and FFAs was observed (primary spoilage), followed by an increase in the sulfide content (secondary spoilage). The most promising volatile spoilage markers for chicken breast were EtOH and 3-methyl-1-butanol, followed by acetic acid (C2) and sulfides.

Evaluation of commercial kit based on loop-mediated isothermal amplification for rapid detection of low levels of uninjured and injured Salmonella on duck meat, bean sprouts, and fishballs in Singapore.

The objective of this study was to evaluate performance of the commercial kit based on loop-mediated isothermal amplification (LAMP) in comparison with the International Organization for Standardization method for detecting uninjured and sublethally injured Salmonella cells artificially inoculated at levels of 10(0) and 10(1) CFU/25 g on raw duck wing, raw mung bean sprouts, and processed fishballs. Injured cells were prepared by a heat treatment for duck wings and fishball samples and a chlorine treatment for bean sprout samples. Additionally, a validation study was performed on naturally contaminated food samples sold in Singapore. A total of 110 samples of each commodity were analyzed in this study. Regardless of inoculum levels, the detection by the commercial LAMP kit showed 100% sensitivity and specificity for both inoculated and uninoculated samples compared with the International Organization for Standardization method, with the exception of bean sprout samples. Only 20% of bean sprout samples inoculated with 10(0) CFU/25 g injured Salmonella cells were positive by using the commercial LAMP-based kit. However, all negative samples became positive following a secondary enrichment in Rappaport-Vassiliadis medium with soy broth or after concentration by centrifugation. These results suggest that secondary enrichment or centrifugation should be considered as an additional step to increase the sensitivity of the commercial LAMP-based kit with low numbers of injured target cells in samples with high background microflora (such as mung bean sprouts). The validation study also showed that the commercial LAMP-based kit provided 91% sensitivity and 95% specificity for naturally contaminated samples. Thus, this study demonstrates that the commercial LAMP-based kit might be a cost-effective method, as this system could provide rapid, accurate detection of both uninjured and injured Salmonella cells on raw duck wings, raw mung bean sprouts, and processed fishballs in less than 26 h.

ENSURING FOOD SECURITY THROUGH ENHANCING MICROBIOLOGICAL FOOD SAFETY

Food safety and food security are interrelated concepts with a profound impact on the quality of human life. Food security describes the overall availability of food at different levels from global to individual household. While, food safety focuses on handling, preparation and storage of foods in order to prevent foodborne illnesses. This review focuses on innovative thermal and non-thermal technologies in the area of food processing as the means to ensure food security through improving food safety with emphasis on the reduction and control of microbiological risks. The antimicrobial efficiency and mechanism of new technologies to extend the shelf life of food product were also discussed.