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.

BACKGROUND: The mechanical transmission of pathogenic bacteria by synanthropic filth flies is widely recognized. While many studies report the fate and the temporospatial distribution of ingested foodborne bacteria by filth flies, there is little evidence about the transmission dynamics of ingested foodborne bacteria by adult house flies (Musca domestica) to their progeny. In this study, we fed parental house fly adults with food contaminated with low, medium, and high concentrations of Salmonella enterica, Cronobacter sakazakii, Escherichia coli O157:H7, and Listeria monocytogenes and evaluated the probability of transmission of these pathogens to house fly eggs and the surface and the alimentary canal of their first filial (F1) generation adults. RESULTS: All foodborne pathogens were present in samples containing pooled house fly eggs. The probability of transmission was higher after parental house flies ingested food containing medium bacterial loads. Cronobacter sakazakii was 16, 6, and 3 times more likely to be transmitted to house fly eggs than S. enterica, E. coli O157:H7, and L. monocytogenes, respectively. Only S. enterica and C. sakazakii were transmitted to F1 generation adults and their presence was 2.4 times more likely on their body surfaces than in their alimentary canals. The highest probabilities of finding S. enterica (60 %) and C. sakazakii (28 %) on newly emerged F1 adults were observed after parental house flies ingested food containing medium and high levels of these pathogens, respectively. CONCLUSION: Our study demonstrates that adult house flies that fed from food contaminated with various levels of foodborne bacteria were able to transmit those pathogens to their eggs and some were further transmitted to newly emerged F1 generation adults, enhancing the vector potential of these insects. Understanding the type of associations that synanthropic filth flies establish with foodborne pathogens will help to elucidate transmission mechanisms and possible ways to mitigate the spread of foodborne pathogens.

Detection Limits of Insect Fragments in Spiked Whole Wheat Flour Using Multiplex Polymerase Chain Reaction (PCR).

The need for a sensitive molecular method to detect specific species of insect contaminants in food products remains a significant challenge in the food industry. This study evaluated the detection limit of a multiplex end-point PCR assay for detecting insects in food. The assay amplifies two fragments of the cytochrome oxidase subunit I gene (COI-Fa and COI-Fb) and one fragment of the protein-coding wingless (wg) gene found in insects. Five insect species, comprising three vectors of foodborne pathogens (the housefly, Musca domestica, the American cockroach, Periplaneta americana, and the pharaoh ant, Monomorium pharaonis) and two storage insect pests (the red flour beetle, Tribolium castaneum and the Indian meal moth, Plodia interpunctella), were spiked separately and in combination at levels of 1, 0.1, 0.01, and 0.001% in whole wheat flour. At spike levels greater than 0.01%, amplicon bands of expected sizes were seen in 100% of samples containing fragments from distinct insect species. At least 25% of spiked samples at the lowest spike level had amplicon bands, except for samples spiked with M. domestica. Results showed an 18.9% probability (with 11.3% and 30% lower and upper confidence limits, respectively) of detecting insect fragments at the lowest spike level (0.001%, corresponding to 3-22 fragments), which is far below the FDA's regulatory level of less than 75 fragments per 50 g of wheat flour. The intensity of amplicon bands in the gel images was higher at higher spike levels. However, this method is not quantitative enough to extrapolate the intensity of the amplicon bands to the number of insect fragments present in a sample. This multiplex assay was also evaluated in a variety of market food samples derived from plants and animals, showing its potential use in various food types. Overall, the sensitivity and specificity of this molecular approach suggest that it could be used in the future as a screening tool for detecting insect contaminants in food.

Development of a Multiplex Polymerase Chain Reaction (PCR) Assay for the Potential Detection of Insect Contaminants in Food.

Molecular methods can potentially be used to detect insect contaminants of food products. In this study, we used three sets of group-specific primers, two of them targeting the amplification of two regions of the insect's mitochondrial cytochrome c oxidase subunit I (COI-Fa and COI-Fb) and the other targeting a region of the nuclear protein-coding wingless (wg) gene. Using singleplex and multiplex polymerase chain reaction (PCR), we evaluated the three sets of primers using genomic DNA (gDNA) from 48 insect species including food storage insect pests and known vectors of foodborne pathogens. Seven plant-based food matrices were also evaluated for exclusivity testing. Additionally, we spiked fragments from five insect species in a selected food matrix (whole wheat flour). Singleplex and multiplex PCR amplified single specific bands (401-449 bp), corresponding to the wg gene, from insect species belonging to families Blattidae and Formicidae, and in Plodia interpunctella (Hübner) (Lepidoptera: Pyralidae). The COI-Fa primers amplified specific bands (171-188 bp) in all Dipteran species and the COI-Fb primers amplified a specific band (∼140 bp) in DNA from Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) and P. interpunctella. However, the presence of specific bands in most Coleopterans was not consistent. No amplicon bands were observed in any of the food matrixes tested and the expected pattern of amplicon bands was seen in multiplex reactions using gDNA from spiked food samples. Our multiplex PCR assay targeted specific groups of insects that commonly contaminate foods without amplifying bands from the food matrixes tested; thus, molecular methods may be suitable for detecting insects or their fragments in foods.

Prevalence and relative risk of Cronobacter spp., Salmonella spp., and Listeria monocytogenes associated with the body surfaces and guts of individual filth flies.

Although flies are important vectors of food-borne pathogens, there is little information to accurately assess the food-related health risk of the presence of individual flies, especially in urban areas. This study quantifies the prevalence and the relative risk of food-borne pathogens associated with the body surfaces and guts of individual wild flies. One hundred flies were collected from the dumpsters of 10 randomly selected urban restaurants. Flies were identified using taxonomic keys before being individually dissected. Cronobacter spp., Salmonella spp., and Listeria monocytogenes were detected using the PCR-based BAX system Q7. Positive samples were confirmed by culture on specific media and through PCR amplification and sequencing or ribotyping. Among collected flies were the housefly, Musca domestica (47%), the blowflies, Lucilia cuprina (33%) and Lucilia sericata (14%), and others (6%). Cronobacter species were detected in 14% of flies, including C. sakazakii, C. turicensis, and C. universalis, leading to the proposal of flies as a natural reservoir of this food-borne pathogen. Six percent of flies carried Salmonella enterica, including the serovars Poona, Hadar, Schwarzengrund, Senftenberg, and Brackenridge. L. monocytogenes was detected in 3% of flies. Overall, the prevalence of food-borne pathogens was three times greater in the guts than on the body surfaces of the flies. The relative risk of flies carrying any of the three pathogens was associated with the type of pathogen, the body part of the fly, and the ambient temperature. These data enhance the ability to predict the microbiological risk associated with the presence of individual flies in food and food facilities.

Detection of foodborne bacterial pathogens from individual filth flies.

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.

Effect of orthoses on postural stability in asymptomatic subjects with rearfoot malalignment during a 6-week acclimation period.

OBJECTIVE: To determine the effect of custom-fitted orthoses on postural sway over a 6-week acclimation period. DESIGN: Repeated-measures analysis of variance on postural sway measures with factors being group (control, malaligned), time (initial, 2 wk, 4 wk, 6 wk postintervention), and condition (with orthoses, without orthoses). For single-limb stance, side (right, left) was analyzed to determine bilateral differences. SETTING: Biodynamics laboratory. PARTICIPANTS: Twenty-one subjects, 11 asymptomatic with rearfoot malalignment and 10 asymptomatic with normal rearfoot alignment. INTERVENTIONS: Orthoses were prescribed and worn for 6 weeks. Balance testing was performed on 4 different dates with each subject tested in both orthotic conditions. Postural control was measured with three 10-second eyes-closed trials for single-limb stance, one 20-second eyes-closed bilateral stance with the platform moving, and one 20-second eyes-open bilateral stance with the platform and surroundings moving. MAIN OUTCOME MEASURES: Sway velocity (in deg/s) for single-limb stance and equilibrium score for bilateral stance. RESULTS: Postural sway measures were significantly decreased during single-limb testing with orthoses versus without orthoses, regardless of group. The orthotic intervention significantly improved bilateral stance equilibrium score in the malaligned group at weeks 2, 4, and 6 when compared with measures at the initial week. Equilibrium score of the malaligned group with orthoses at initial week was significantly lower (worse) than the control group with orthoses at initial week; however, these results were not repeated during measurements taken at weeks 2, 4, or 6. CONCLUSIONS: The application of orthoses decreased sway velocity for single-limb stance, improving postural stability regardless of group when visual feedback was removed. During bilateral stance, postural stability was initially worse for the malaligned group with and without orthoses when compared with the control group; however, improvements were seen by week 2 and continued throughout the remainder of testing. Clinically, the application of orthoses appears to improve postural control in people with rearfoot malalignment, particularly when vision is removed.