Detection of Foodborne Bacterial Pathogens from Individual Filth Flies

Abstract

There is unanimous consensus that insects are important vectors of foodborne pathogens. However, linking insects as vectors of the pathogen causing a particular foodborne illness outbreak has been challenging. This is because insects are not being aseptically collected as part of an environmental sampling program during foodborne outbreak investigations and because there is not a standardized method to detect foodborne bacteria from individual insects. To take a step towards solving this problem, we adapted a protocol from a commercially available PCR-based system that detects foodborne pathogens from food and environmental samples, to detect foodborne pathogens from individual flies.Using this standardized protocol, we surveyed 100 wild-caught flies for the presence of Cronobacter spp., Salmonella enterica, and Listeria monocytogenes and demonstrated that it was possible to detect and further isolate these pathogens from the body surface and the alimentary canal of a single fly. Twenty-two percent of the alimentary canals and 8% of the body surfaces from collected wild flies were positive for at least one of the three foodborne pathogens. The prevalence of Cronobacter spp. on either body part of the flies was statistically higher (19%) than the prevalence of S. enterica (7%) and L.monocytogenes (4%). No false positives were observed when detecting S. enterica and L. monocytogenes using this PCR-based system because pure bacterial cultures were obtained from all PCR-positive results. However, pure Cronobacter colonies were not obtained from about 50% of PCR-positive samples, suggesting that the PCR-based detection system for this pathogen cross-reacts with other Enterobacteriaceae present among the highly complex microbiota carried by wild flies. The standardized protocol presented here will allow laboratories to detect bacterial foodborne pathogens from aseptically collected insects, thereby giving public health officials another line of evidence to find out how the food was contaminated when performing foodborne outbreak investigations.

Ingested Salmonella enterica, Cronobacter sakazakii, Escherichia coli O157:H7, and Listeria monocytogenes: transmission dynamics from adult house flies to their eggs and first filial (F1) generation adults

“…We have previously reported that C. sakazakii colonies could not be recovered from some PCR-positive samples while using this combined approach, likely due to the PCR being positive when other closely related bacterial genera , such as Citrobacter freundii , are present in the samples [ 7 , 27 ]. Additionally, besides C. sakazakii a number of other Enterobacteriaceae are α-glucosidase positive, therefore the co-isolation of those organisms from samples with highly complex microbiota (such as the fly’s alimentary canal) could lower the efficiency of recovery of C. sakazakii from the chromogenic media used [ 28 ].…”

“…Immobilized flies were then transferred to a disposable Petri dish containing 70 % alcohol for 2 min. Using a dissecting scope, three adult female house flies were randomly sub-sampled per each glass jar (n = 48 per each foodborne bacterium) and individually transferred to an autoclaved two ml tube to be surface-disinfected and their alimentary canals dissected as described by Pava-Ripoll , et al [ 27 ]. The alimentary canals of maternal house flies were individually evaluated for the presence of the target bacteria as described in sections below.…”

“…Under a dissecting scope, three females were randomly sub-sampled per each Petri dish (n = 48 per each foodborne bacterium) and individually transferred to autoclaved two ml tubes containing one ml of enrichment media specific for the target pathogen (see Additional file 4 ) to collect microbiota from the surface of the newly emerged house fly. Each house fly was then removed from the enrichment media, surface-disinfected and their alimentary canals aseptically dissected as described by Pava-Ripoll , et al [ 27 ]. Tubes with enrichment media containing microbiota from the surface (s) and the alimentary canal (ac) of each F 1 adult house fly were incubated at times and temperatures recommended for each bacterial pathogen (see Additional file 4 ).…”

“…Enriched samples were assessed for the presence/absence of the target bacteria using a combined molecular and culture approach. The molecular approach was performed using a commercial PCR cycler/detector system (BAX® System Q7, DuPont Qualicon, Wilmington, DE) and assay kits specific for each bacteria (see Additional file 4 ) following manufacturer’s instructions and as described by Pava-Ripoll , et al [ 27 ]. Each assay kit contains PCR-ready tablets with an intercalating dye that emits a fluorescence signal when binding to the target double-stranded DNA.…”

Risk Factors and Level of Listeria monocytogenes Contamination of Raw Pork in Retail Markets in China

“…Subsequently 1 ml of the above culture was transferred into 9 ml Fraser (Oxoid Ltd.) for 48 h at 37°C with shaking (220 rpm). Flies and cockroach samples were pulverized before culturing because that L. monocytogenes could exist on the body surface as well as the alimentary canal of a single fly ( Pava-Ripoll et al, 2015 ). Enriched broth was then spread onto brilliance listeria agar (Oxoid Ltd.) for 24–36 h at 37°C.…”