Abstract House flies (Musca domestica L. [Diptera: Muscidae]) can act as a mechanical vector for food-borne pathogens including Shiga toxin-producing Escherichia coli (Migula; Enterobacteriales: Enterobacteriaceae) (STEC) in and around cattle feedlots. The present study assessed the prevalence of STEC in house flies from a restaurant area of a town in northeastern Kansas. Two hundred twenty-four house flies were collected over 10 wk, surface sterilized, individually homogenized, and cultured by a multifaceted approach of direct plating on selective media and an enrichment broth, followed by the immunomagnetic separation. Bacterial isolates were screened for eight serogroups of E. coli: O103, O104, O26, O111, O45, O145, O121, and O157 using multiplex polymerase chain reaction (PCR). Furthermore, O-serogroup-positive isolates were tested for virulence genes stx1, stx2, eae, and ehxA by PCR. The majority (91.5%) of flies carried enteric bacteria, and the mean value of enteric concentration on the modified Possé agar was 6.7 ± 1.1 × 106 colony forming units per fly. Thirty-nine of the 224 flies (17.4%) were positive for one or more E. coli serogroup of interest; with the majority O103 (10.7%), followed by O26 (3.1%), O121 (1.3%), O45 (1.3%), and O104 (0.9%). However, none of the serogroup-positive isolates carried any of the virulence genes tested. Results of our study show that house flies in the urban environment do not carry STEC. Nevertheless, detection of E. coli O-serogroups with the potential to acquire virulence traits indicates that house flies in an urban environment represent a public health risk. house fly, Escherichia coli, Shiga-toxin, STEC, urban environment House flies (Musca domestica L. [Diptera: Muscidae]) are a nuisance pest of people and animals and a mechanical vector for various microorganisms (Graczyk et al. 2001). House flies build large populations on animal facilities during warm months and can fly freely among animal facilities and surrounding urban areas (Chakrabarti et al. 2010). As residential areas encroach further into agricultural habitats, house flies represent a possible risk as vectors of zoonotic and food-borne pathogens and antibiotic-resistant strains (Graczyk et al. 2001, Zurek and Ghosh 2014). For example, beef cattle are the primary asymptomatic reservoirs of Shiga-toxigenic Escherichia coli (Migula; Enterobacteriales: Enterobacteriaceae) (STEC) (Callaway et al. 2013). Infected animals shed the bacteria in their feces where bacteria come in contact with other animals and can spread throughout a facility (Persad and LeJeune 2014). STEC is recognized as one of the leading causes of human food-borne illness, responsible for hundreds of cases of diarrheal infections and hemolytic uremic syndrome each year (Bettelheim 2000). The number of human cases is typically highest in summer months (CDC 2011), and this very well coincides with the peak house fly populations. House flies from animal farms were reported to carry E. coli O157:H7, and this serotype was found to proliferate in fly mouthparts resulting in bio-enhanced transmission (Kobayashi et al. 1999, Alam and Zurek 2004). In addition, house flies were shown to transfer E. coli O157:H7 to cattle and their drinking water in the confined cattle environment (Ahmad et al. 2007). Recently, we have shown that house flies in the confined cattle environment also carry non-O157 STEC serotypes (Puri-Giri et al. 2017). However, the prevalence of STEC in house flies in an urban environment has not been evaluated, and the potential of house flies to disseminate STEC from animal facilities to nearby urban areas may represent a public health risk. The present study assessed the prevalence of STEC in house flies from a restaurant area of a town in northeastern Kansas. Materials and Methods Collection of House Flies House flies were collected weekly from a restaurant area of a town in northeastern Kansas in summer (June–August) for 10 wk. A minimum of 18 house flies were collected and processed from each sampling. Flies were captured with sweep nets, placed into previously unused plastic Ziploc bags, transported to the laboratory on wet ice, and processed within 2 h. Isolation and Detection of STEC Direct Plating House flies were surface sterilized using 0.5% sodium hypochlorite, 70% ethanol, and sterile water as described by Zurek et al. (2000) and individually homogenized using sterile plastic pestles in 1.0-ml phosphate-buffered saline (MP Biomedicals, Solon, OH). An aliquot of the fly homogenate (100 µl) was spread plated on modified Possé agar (mP) which is a selective and differentiating medium for targeted E. coli serotypes. The mP agar was prepared according to the protocol of Possé et al. (2008) with minor modifications in the concentrations of supplements: novobiocin (5.0 mg/l) and potassium tellurite (0.5 mg/liter) as described by Dewsbury et al. (2015). Colony forming units (CFU) were determined after 24-h incubation at 37°C and expressed as the mean ± standard error of mean per fly. Up to eight phenotypically different colonies (some serotypes of E. coli have distinct colony morphology on MP agar; Possé et al. 2008) were selected from mP agar and cultured overnight at 37°C on Tryptic Soy Agar (TSA; Bacto-TSA, Becton Dickinson, Sparks, MD) to obtain fresh cells for polymerase chain reaction (PCR) detection of non-O157 serogroups. In addition, for specific detection and isolation of the E. coli O157 serotype, another 100 μl of each fly homogenate was directly spread plated on cefixime tellurite supplemented sorbitol MacConkey agar (CT-SMAC) and incubated at 37°C overnight. Typical transparent colonies from CT-SMAC were screened for E. coli O157 serogroup by a latex-based agglutination test (Oxoid, Basingstoke, UK). Enrichment To culture the bacteria present in too low numbers to be detected by direct plating, an aliquot of 700 µl of each fly homogenate was added to 10 ml of E. coli broth (EC broth; Oxoid) and incubated at 40°C for 6 h with shaking at 50 rpm. Based on the CFU counts on mP agar and the turbidity in EC broth, fly samples with good growth (>100 CFU per fly) and/or high turbidity were subjected to the immunomagnetic separation (IMS) following the manufacturer’s instructions (Dynal Biotech, Lake Success, NY). The serogroup-specific IMS beads (Abraxis LLC, Warminister, PA) were pooled into two groups: A = serogroups O26 + O45 + O111 + O104, and B = serogroups O103 + O145 + O121. One milliliter of the enriched house fly sample was then added to the pooled beads for groups A and B (20 µl of each serogroup) for IMS. Following the IMS, 50 µl of IMS suspension was spread plated on mP agar at a 100-fold dilution to obtain isolated colonies. After 24 h incubation at 37°C, up to eight phenotypically different colonies were selected from each group (A and B), streaked onto TSA, and cultured overnight at 37°C to obtain fresh cells for PCR. Detection of Non-O157 Serogroups and Virulence Traits Up to 24 colonies (eight from the direct plating and 16 from the enrichment) from each house fly sample were analyzed for seven non-O157 STEC serogroups and virulence traits by multiplex PCRs as described in detail in the study by Puri-Giri et al. (2017). E. coli JB1-95 O111:H-, CDC 96–3285 O45:H2, CDC 90–3128 O103:H2, CDC 97–3068 O121:H19, 83–75 O145:NM, H30 O26:H11, and ATCC BAA-2326 O104:H4 were used as positive controls for each PCR. Results and Discussion Of the 224 house flies, 205 (91.5%) were positive for bacterial growth on mP agar. This high prevalence of flies carrying enteric bacteria in an urban restaurant area is noteworthy. With increasing encroachment of residential areas into agricultural sites, house flies have free access to both areas and likely transmit microbes either way. For 205 flies positive for bacterial growth on mP agar, the mean CFU/fly was 6.7 ± 1.1 × 106 and this fluctuated from week to week (Fig. 1). This finding is similar to the mean enteric concentration (3.6 ± 1.1 × 106 CFU) found in individual stable flies (Puri-Giri et al. 2016) and (1.5 ± 0.3 × 106 CFU) in individual house flies (Puri-Giri et al. 2017) collected from cattle feedlots using the same culturing approach. High enteric counts from flies in cattle feedlots are expected as this environment provides a suitable larval developmental habitat with a large and diverse microbial community (cattle manure) as well as ad libitum food source (cattle manure and nonsterile cattle feed) for adult flies. However, it is surprising to find comparable CFU counts in flies from an urban area that may provide a habitat for larval development (food waste and ill-managed garbage) but it typically does not offer access to large sources of E. coli and other fecal coliforms for adult flies. This indicates that as some house flies disperse from animal farms to the surrounding nonagricultural environment, they have the capacity to maintain a high concentration of bacteria in their digestive tract that likely originate from manure. Survival and proliferation of fecal bacteria in adult house flies have been reported in earlier studies (Kobayashi et al. 1999, Doud and Zurek 2012), and our findings from house flies from an urban site corroborate these studies. Fig. 1. View largeDownload slide Prevalence and concentration (mean ± SEM) of enteric bacteria in house flies collected from the urban environment. Asterisk indicates the number of flies positive for enteric bacteria out of total flies processed. Fig. 1. View largeDownload slide Prevalence and concentration (mean ± SEM) of enteric bacteria in house flies collected from the urban environment. Asterisk indicates the number of flies positive for enteric bacteria out of total flies processed. Thirty-nine of the 224 flies (17.4%) were positive for at least one E. coli O-serogroup of interest. Two flies were positive for a combination of two different serotypes (Table 1). Of the positive flies, the serogroup most frequently detected was O103 (10.7%), followed by O26 (3.1%), O121 (1.3%), O45 (1.3%), and O104 (0.9%) (Table 1). None of the flies carried E. coli O145, O111, and O157. Importantly, none of the serogroup-positive isolates were found to carry any of the tested virulence genes (stx1, stx2, eae, and ehxA). Therefore, while E. coli O-serogroups were detected, no STEC was found in house flies in this study. Table 1. Escherichia coli serogroup-positive isolates from house flies collected from an urban environment Serogroup No. of positive flies (%) No. of serogroup-positive isolates O103 24 (10.7) 42 O26 7 (3.1) 16 O45 3 (1.3) 4 O121 3 (1.3) 3 O104 2 (0.9) 3 O103 + O26 1 (0.5) 2 O103 + O45 1 (0.5) 2 Serogroup No. of positive flies (%) No. of serogroup-positive isolates O103 24 (10.7) 42 O26 7 (3.1) 16 O45 3 (1.3) 4 O121 3 (1.3) 3 O104 2 (0.9) 3 O103 + O26 1 (0.5) 2 O103 + O45 1 (0.5) 2 View Large The prevalence of O-serogroup positive flies was approximately a half of that (34.3%) found in house flies from the dairy and feedlot environment (Puri-Giri et al. 2017). In that study, we detected a low prevalence of the O145 serogroup 1.3% (6 out of 463), while in the present study, we did not detect this serogroup at all. The feedlot study also showed a relatively high prevalence of the serogroup O104 (12.7%), and this was much lower (0.9%) in the flies from an urban site. In contrast, the detection frequency of the serogroup O103 in flies from an urban area was about twofold higher compared to that from house flies from a cattle feedlot (10.7% vs 5.6%; Puri-Giri et al. 2017). Differences among the E. coli serogroups carried by house flies from two different environments indicate that some serogroups are likely better adapted to temporally survive and possibly multiply in the house fly digestive tract. It is also possible, although less likely (due to low STEC prevalence), that house flies acquired targeted E. coli serogroups from other sources than cattle, e.g., manure of other farm animals (sheep, goats) that were in a similar proximity to that of cattle farms or feces of pet animals in a town (e.g., dogs) or urban wildlife (e.g., rabbits) (Persad and LeJeune, 2014). Clearly, the potential fitness differences among STEC serogroups in house flies need to be tested in laboratory bioassays. Dewsbury et al. (2015) reported that STEC E. coli serogroups O157, O103, O26, and O145 were commonly detected in the fecal samples from cattle feedlots in summer. Our recent study provided evidence that house flies in cattle feedlots carry STEC O103, O104, and O45 with stx1+ eae and/or ehxA (Puri-Giri et al. 2017). The urban area sampled in the present study was about 5.0 to 9.0 km from the closest dairy and the feedlot, respectively. Because house flies are known to be able to actively fly up to 32 km (Levine and Levine 1991), it is possible that some of the house flies and their associated bacteria collected in this study originated from nearby animal facilities. Source tracking studies are warranted to provide a risk assessment for this possibility. In conclusion, STEC was not detected in house flies collected around restaurants in an urban site in this study. However, a high concentration of enteric bacteria carried by the majority of house flies and a relatively high prevalence of E. coli O-serogroups of interest with the potential to acquire Shiga toxins and other virulence traits merit the need to incorporate the house fly management into the food safety programs and strategies. Acknowledgment This study was supported by the USDA-NIFA-CAP grant 2012-68003-30155. This is contribution no. 18-184-J from the Kansas Agricultural Experiment Station. References Cited Ahmad, A., T. G. Nagaraja, and Zurek L.. 2007. Transmission of Escherichia coli O157:H7 to cattle by house flies. Prev. Vet. Med . 80: 74– 81. Google Scholar CrossRef Search ADS PubMed Alam, M. J., and Zurek L.. 2004. Association of Escherichia coli O157:H7 with houseflies on a cattle farm. Appl. Environ. Microbiol . 70: 7578– 7580. Google Scholar CrossRef Search ADS PubMed Bettelheim, K. 2000. Role of non-O157 VTEC. J. Appl. Microbiol . 88: 38S– 50S. 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Summer and winter prevalence of Shiga toxin-producing Escherichia coli (STEC) O26, O45, O103, O111, O121, O145, and O157 in feces of feedlot cattle. Foodborne Pathog. Dis . 12: 726– 732. Google Scholar CrossRef Search ADS PubMed Doud, C. W., and Zurek L.. 2012. Enterococcus faecalis OG1RF:pMV158 survives and proliferates in the house fly digestive tract. j. Med. Entomol . 49: 150– 155. Google Scholar CrossRef Search ADS PubMed Graczyk, T. K., R. Knight, R. H. Gilman, and Cranfield M. R.. 2001. The role of non-biting flies in the epidemiology of human infectious diseases. Microbes Infect . 3: 231– 235. Google Scholar CrossRef Search ADS PubMed Kobayashi, M., T. Sasaki, N. Saito, K. Tamura, K. Suzuki, H. Watanabe, and Agui N.. 1999. Houseflies: not simple mechanical vectors of enterohemorrhagic Escherichia coli O157:H7. Am. j. Trop. Med. Hyg . 61: 625– 629. Google Scholar CrossRef Search ADS PubMed Levine, O. S., and Levine M. M.. 1991. Houseflies (Musca domestica) as mechanical vectors of shigellosis. Rev. Infect. Dis . 13: 688– 696. Google Scholar CrossRef Search ADS PubMed Persad, A. K., and LeJeune J. T.. 2014. Animal reservoirs of Shiga toxin-producing Escherichia coli. Microbiol. Spectr . 2: EHEC-0027-2014. Google Scholar CrossRef Search ADS PubMed Possé, B., L. De Zutter, M. Heyndrickx, and Herman L.. 2008. Novel differential and confirmation plating media for Shiga toxin-producing Escherichia coli serotypes O26, O103, O111, O145 and sorbitol-positive and -negative O157. fems Microbiol. Lett . 282: 124– 131. Google Scholar CrossRef Search ADS PubMed Puri-Giri, R., A. Ghosh, J. L. Thomson, and Zurek L.. 2017. House flies in the confined cattle environment carry non-O157 shiga toxin-producing Escherichia coli. j. Med. Entomol . 54: 726– 732. Google Scholar PubMed Puri-Giri, R., A. Ghosh, and Zurek L.. 2016. Stable flies (Stomoxys calcitrans L.) from confined beef cattle do not carry shiga-toxigenic Escherichia coli (STEC) in the digestive tract. Foodborne Pathog. Dis . 13: 65– 67. Google Scholar CrossRef Search ADS PubMed Zurek, L., and Ghosh A.. 2014. Insects represent a link between food animal farms and the urban environment for antibiotic resistance traits. Appl. Environ. Microbiol . 80: 3562– 3567. Google Scholar CrossRef Search ADS PubMed Zurek, L., C. Schal, and Watson D. W.. 2000. Diversity and contribution of the intestinal bacterial community to the development of Musca domestica (Diptera: Muscidae) larvae. j. Med. Entomol . 37: 924– 928. Google Scholar CrossRef Search ADS PubMed © The Author(s) 2017. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: email@example.com.
Journal of Medical Entomology – Oxford University Press
Published: Mar 1, 2018
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