Evaluation of bacteriophage amplification assay for rapid detection of Shigella boydii in food systems
Lae-Seung Jung1 & Juhee Ahn1
Received: 6 June 2015 /Accepted: 28 October 2015 # Springer-Verlag Berlin Heidelberg and the University of Milan 2015
Abstract This study was designed to investigate the possibility of using bacteriophages for the detection of viable
Shigella boydii in food products. A Shigella bacteriophage belonging to a member of the Siphoviridae family was isolated from swine fecal samples. The free bacteriophages were highly stable against pH 4.0 to 9.0 and temperature change (z-value=17.1 °C). The bacteriophage amplification assay was able to selectively detect S. boydii in a bacterial mixture of Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella Typhimurium. The number of S. boydii bacteriophages enumerated by the amplification assay was highly correlated with the number of viable S. boydii in single (r=0.987) and mixed (r=0.969) cultures. The bacteriophage-based detection of S. boydii was highly reproducible in lettuce (6.3 log CFU/ml and 4.9 log PFU/ml) and cooked chicken breasts (6.1 log
CFU/ml and 6.0 log PFU/ml). These results suggest that the bacteriophage amplification assay can be used as an alternative method for rapid, selective, and cost-effective detection of S. boydii in food products, and provides useful information for designing a quick and simple detection kit.
Keywords Shigella boydii . Bacteriophage . Amplification assay . Lettuce . Chicken breast
As microbiological safety continues to be a top priority in the food industry, research endeavors over the last few decades have led to the development of rapid, sensitive, and selective detection methods (Gracias and McKillip 2004; Mandal et al. 2011). The timely determination of bacterial contamination in food is critically important for appropriate treatment and effective prevention of serious bacterial contamination. The traditional pathogen detection platforms are generally time-consuming, labor-intensive, and costly, and require several steps including bacterial enrichment, biochemical identification, and serological confirmation (Lleo et al. 2005; Javed et al. 2013). A number of immunological, genomic, and proteomic techniques have been applied for the accurate detection and identification of bacterial pathogens (Mandal et al. 2011;
Riahi et al. 2011). Although advanced detection tools have been proven powerful in terms of sensitivity and selectivity for the identification of pathogens contaminating foods, they require extensive training, technical experience, expensive instruments, and substantial sample preparation (McNerney et al. 1998; Oliveira et al. 2012; Javed et al. 2013). Therefore, the development of a simple and practical detection tool for microbiological analysis has been an area of interest for many researchers.
Bacteriophage-based detection technologies using engineered, reporter, and whole-type bacteriophages have recently received great attention due to their high specificity and low cross-reactivity (Smartt et al. 2012). Unlike engineered bacteriophage-based detection, the bacteriophage amplification assay is a relatively simple and easy method, enabling the quantitative detection of viable pathogens within a few hours (Stewart et al. 1998; Park et al. 2003; Botsaris et al. 2010). This technique is based on the principle of specific interactions between bacteriophage and host bacteria, and * Juhee Ahn firstname.lastname@example.org 1 Department of Medical Biomaterials Engineering and Institute of
Bioscience and Biotechnology, Kangwon National University,
Chuncheon, Gangwon 200-701, Republic of Korea
DOI 10.1007/s13213-015-1178-y consists of four steps: bacteriophage infection of the target host, inactivation of free bacteriophages, neutralization, and amplification (de Siqueira et al. 2006). Small-scale food processing facilities commonly have difficulty implementing new technologies due to a lack of technical experience and resources. The simple protocol of the bacteriophage amplification assay can be applied in small-scale food processing units as an easy and rapid method for confirming foodborne pathogens in food systems. Therefore, the objective of this study was to evaluate bacteriophage amplification as a practical approach for detecting and confirming the presence of Shigella boydii in lettuce and cooked chicken breasts.
Materials and methods
Bacterial strains and bacteriophage isolation
Strains of Escherichia coli O157:H7 KACC 11598, Shigella boydii KACC 10792, and Listeria monocytogenes KACC 12671 were kindly provided by the Korean Agricultural Culture Collection (KACC; Suwon, Korea). The Salmonella
Typhimurium KCCM 40253 strain was obtained from the
Korean Culture Center of Microorganisms (KCCM; Seoul,
Korea). The strains were cultured in Trypticase soy broth (TSB; Becton, Dickinson and Company [BD], Franklin
Lakes, NJ, USA) at 37 °C for 20 h and harvested by centrifugation at 3000 × g for 20 min at 4 °C. To isolate Shigella bacteriophages, swine fecal samples (10 g each) were mixed with 20 ml of TSB, and the mixtures were inoculated with the bacterial host S. boydii (106 CFU/ml) and incubated at 37 °C for 20 h. After centrifugation at 5000 × g for 10 min, the supernatant was filtered using a 0.2-μm membrane filter to completely eliminate fecal particles. The filtrates (100 μl each) were gently suspended in TSB with 0.5 % agar and poured on the lawn of S. boydii. The plates were incubated at 37 °C for 24 to 48 h. Shigella bacteriophages were isolated from the clear zone on the top agar.
Bacteriophage preparation and plaque assay
The isolated bacteriophages were propagated at 37 °C for 20 h in TSB containing the S. boydii bacterial host strain. After propagation, the cell-free culture supernatants were collected by centrifugation at 3000 × g for 20 min and then filtered through a 0.2-μm sterilized filter. The filtrates were further purified by polyethylene glycol (PEG) precipitation and cesium chloride (CsCl) gradient ultracentrifugation. The purified phage stocks were enumerated by a soft-agar overlay method (Bielke et al. 2007). In brief, the phages were serially (1:10) diluted with phosphate buffered saline (PBS, pH 7.2) and gently mixed with S. boydii cells in 0.5 % TSB soft-agar.