Enterococcus faecalis is an important nosocomial pathogen and house flies have been implicated in the dissemination of this bacterium. In this study, GFP-expressing E. faecalis OG1RF:pMV158 was used ...to track the fate of the bacterium in the digestive tract of the house fly, Musca domestica (L.) to assess the vector potential of this insect for E. faecalis. Colony forming unit (CFU) counts were obtained from viable fluorescing E. faecalis recovered from mouthparts and digestive tract regions (labelum, foregut, midgut, and hindgut) at 1, 4, 8, 24, 48, 72, and 96 h after the bacterial exposure. Bacterial counts were significantly highest in the midgut at 1 h and 4 h and declined during the first 24 h. In the labelum, E. faecalis concentrations were low within the first 24 h and then greatly increased. Bacterial counts and direct observations of the digestive tract under a dissecting microscope with ultra violet light revealed that E. faecalis peaked in the crop after 48 h and remained high until the end of the experiment. Concentrations of E. faecalis in the hindgut were low when compared with other parts of the digestive tract. Microscopy and CFU counts suggest that E. faecalis was digested in the midgut but proliferated in the crop. Both drinking water and feed (flaked corn) sampled at the end of the assay (96 h) were contaminated by fluorescing E. faecalis, demonstrating that the flies disseminated E. faecalis. Our data support the notion that house flies can act as a bioenhanced vector for bacteria.
Aims
The winter beehive debris containing bodies of honeybee parasitic mite Varroa destructor is used for veterinary diagnostics. The Varroa sucking honeybee haemolymph serves as a reservoir of ...pathogens including bacteria. Worker bees can pick up pathogens from the debris during cleaning activities and spread the infection to healthy bees within the colony. The aim of this study was to detect entomopathogenic bacteria in the Varroa collected from the winter beehive debris.
Methods and Results
Culture‐independent approach was used to analyse the mite‐associated bacterial community. Total DNA was extracted from the samples of 10 Varroa female individuals sampled from 27 different sites in Czechia. The 16S rRNA gene was amplified using universal bacterial primers, cloned and sequenced, resulting in a set of 596 sequences representing 29 operational taxonomic units (OTU97). To confirm the presence of bacteria in Varroa, histological sections of the mites were observed. Undetermined bacteria were observed in the mite gut and fat tissue.
Conclusion
Morganella sp. was the most frequently detected taxon, followed by Enterococcus sp., Pseudomonas sp., Rahnella sp., Erwinia sp., and Arsenophonus sp. The honeybee putative pathogen Spiroplasma sp. was detected at one site and Bartonella‐like bacteria were found at four sites. PCR‐based analysis using genus‐specific primers enabled detection of the following taxa: Enterococcus, Bartonella‐like bacteria, Arsenophonus and Spiroplasma.
Significance and Impact of the Study
We found potentially pathogenic (Spiroplasma) and parasitic bacteria (Arsenophonus) in mites from winter beehive debris. The mites can be reservoirs of the pathogenic bacteria in the apicultures.
House flies, Musca domestica L., develop within and feed upon microbe-rich substrates such as manure, acquiring and potentially disseminating pathogenic bacteria. Because adult female flies frequent ...manure due to oviposition or nutrition requirements, we hypothesized females would consume more manure than males even in the presence of additional food sources (e.g. sugar), resulting in measurable differences in bacterial load between sexes. House fly acquisition of bacteria from manure inoculated with GFP-expressing E. coli or Salmonella sp. was examined for both sexes over 24 h in assays where 1) inoculated manure was the only food source and 2) both inoculated manure and sugar water were provided. We conducted assays with mated male and female flies separately to determine sex-specific effects on bacterial acquisition. Over 24 h, bacterial abundance increased in manure inoculated with S. Typhimurium, but not E. coli. In flies, bacterial abundance increased within sex only in S. Typhimurium assays. Overall, female flies harbored more bacteria than males; however, differences in abundance were only significant at early time points. In the E. coli manure–sugar assays, male and female colony-forming units (CFU) abundance differed at 4 and 12 h, while CFU abundance differed at 4 and 12 h in all S. Typhimurium assays. Fly digestive tract observations from manure–sugar assays supported these initial differences especially at 4 h where females contained manure and fly food, while males contained only sugar water. Identifying sex-specific effects on house fly acquisition and carriage of bacteria from manure facilitates risk assessment of pathogen transmission on farms.
Cattle manure is one of the primary larval developmental habitats of house flies, Musca domestica (L.). Cattle serve as asymptomatic reservoirs of Shiga toxin-producing Escherichia coli (STEC), and ...bacteria are released into the environment in cattle feces. The USDA-FSIS declared seven STEC serogroups (O157, O26, O45, O103, O145, O121, and O111) as adulterants in beef products. In addition, the serogroup O104 was a culprit of a large outbreak in Germany in 2011. Our study aimed to assess the prevalence of seven non-O157 STEC (O26, O45, O145, O103, O121, O111, and O104) serogroups in adult house flies. Flies (n = 463) were collected from nine feedlots and three dairy farms in six states in the United States and individually processed. This involved a culturing approach with immunomagnetic separation followed by multiplex polymerase chain reactions for detection of individual serogroups and virulence traits. The concentration of bacteria on modified Possé agar ranged between 1.0 × 101 and 7.0 × 107 (mean: 1.5 ± 0.3 × 106) CFU/fly. Out of 463 house flies, 159 (34.3%) carried one or more of six E. coli serogroups of interest. However, STEC was found in 1.5% of house flies from feedlots only. These were E. coli O103 and O104 harboring stx1 and ehxA and E. coli O45 with stx1, eae, and ehxA. This is the first study reporting the isolation of non-O157 STEC in house flies from the confined cattle environment and indicating a potential role of this insect as a vector and reservoir of non-O157 STEC in confined beef cattle.
The main reservoir of
Escherichia coli O157:H7 is the digestive tract of cattle; however, the ecology of this food-borne pathogen is poorly understood. House flies (
Musca domestica L.) might play a ...role in dissemination of this pathogen in the cattle environment. In our study, eight calves were individually exposed to house flies that were orally inoculated with a mixture of four strains of nalidixic acid-resistant
E. coli O157:H7 (
Nal
REcO157) for 48
h. Another eight calves were individually exposed to uninoculated flies and served as the control. Fresh cattle feces (rectal sampling) and drinking water were periodically sampled and screened for
Nal
REcO157 up to 19 days after the exposure. At the end of the experiment, all calves were euthanized and the lumen contents of rumen, cecum, colon, and rectum as well as swab samples of gall-bladder mucosa and the recto-anal mucosa were screened for
Nal
REcO157. On day 1 after the exposure, fecal samples of all eight calves and drinking-water samples of five of eight calves exposed to inoculated flies tested positive for
Nal
REcO157. The concentration of
Nal
REcO157 in feces ranged over time from detectable only by enrichment (<10
2) to up to 1.1
×
10
6
CFU/g. Feces of all calves remained positive for
Nal
REcO157 up to 11 days after the exposure and 62% were positive until the end of experiment. Contamination of drinking water was more variable and all samples were negative on day 19. At necropsy, the highest prevalence of
Nal
REcO157 was in the recto-anal mucosa region, followed by rectal and colonic contents.
Enterococci are important nosocomial pathogens, with Enterococcus faecalis most commonly responsible for human infections. In this study, we used several measures to test the hypothesis that house ...flies, Musca domestica (L.), acquire and disseminate antibiotic-resistant and potentially virulent E. faecalis from wastewater treatment facilities (WWTF) to the surrounding urban environment. House flies and sludge from four WWTF (1–4) as well as house flies from three urban sites close to WWTF-1 were collected and cultured for enterococci. Enterococci were identified, quantified, screened for antibiotic resistance and virulence traits, and assessed for clonality. Of the 11 antibiotics tested, E. faecalis was most commonly resistant to tetracycline, doxycycline, streptomycin, gentamicin, and erythromycin, and these traits were intra-species horizontally transferrable by in vitro conjugation. Profiles of E. faecalis (prevalence, antibiotic resistance, and virulence traits) from each of WWTF sludge and associated house flies were similar, indicating that flies successfully acquired these bacteria from this substrate. The greatest number of E. faecalis with antibiotic resistance and virulence factors (i.e., gelatinase, cytolysin, enterococcus surface protein, and aggregation substance) originated from WWTF-1 that processed meat waste from a nearby commercial meat-processing plant, suggesting an agricultural rather than human clinical source of these isolates. E. faecalis from house flies collected from three sites 0.7–1.5 km away from WWTF-1 were also similar in their antibiotic resistance profiles; however, antibiotic resistance was significantly less frequent. Clonal diversity assessment using pulsed-field gel electrophoresis revealed the same clones of E. faecalis from sludge and house flies from WWTF-1 but not from the three urban sites close to WWTF-1. This study demonstrates that house flies acquire antibiotic-resistant enterococci from WWTF and potentially disseminate them to the surrounding environment.
Antibiotic-resistant bacterial infections result in higher patient mortality rates, prolonged hospitalizations, and increased health care costs. Extensive use of antibiotics as growth promoters in ...the animal industry represents great pressure for evolution and selection of antibiotic-resistant bacteria on farms. Despite growing evidence showing that antibiotic use and bacterial resistance in food animals correlate with resistance in human pathogens, the proof for direct transmission of antibiotic resistance is difficult to provide. In this review, we make a case that insects commonly associated with food animals likely represent a direct and important link between animal farms and urban communities for antibiotic resistance traits. Houseflies and cockroaches have been shown to carry multidrug-resistant clonal lineages of bacteria identical to those found in animal manure. Furthermore, several studies have demonstrated proliferation of bacteria and horizontal transfer of resistance genes in the insect digestive tract as well as transmission of resistant bacteria by insects to new substrates. We propose that insect management should be an integral part of pre- and postharvest food safety strategies to minimize spread of zoonotic pathogens and antibiotic resistance traits from animal farms. Furthermore, the insect link between the agricultural and urban environment presents an additional argument for adopting prudent use of antibiotics in the food animal industry.
Stable flies, Stomoxys calcitrans (L.), are the most important pests of cattle in the United States. However, adequate management strategies for stable flies, especially for pastured cattle, are ...lacking. Microbial/symbiont-based approaches offer novel venues for management of insect pests and/or vector-borne human and animal pathogens. Unfortunately, the fundamental knowledge of stable fly-microbial associations and their effect on stable fly biology is lacking. In this study, stable flies laid greater numbers of eggs on a substrate with an active microbial community (> 95% of total eggs oviposited) than on a sterilized substrate. In addition, stable fly larvae could not develop in a sterilized natural or artificial substrate/medium. Bacteria were isolated and identified from a natural stable fly oviposition/developmental habitat and their individual effect on stable fly oviposition response and larval development was evaluated in laboratory bioassays. Of nine bacterial strains evaluated in the oviposition bioassays, Citrobacter freundii stimulated oviposition to the greatest extent. C. freundii also sustained stable fly development, but to a lesser degree than Serratia fanticola. Serratia marcescens and Aeromonas spp. neither stimulated oviposition nor supported stable fly development. These results demonstrate a stable fly bacterial symbiosis; stable fly larval development depends on a live microbial community in the natural habitat, and stable fly females are capable of selecting an oviposition site based on the microbially derived stimuli that indicate the suitability of the substrate for larval development. This study shows a promising starting point for exploiting stable fly-bacterial associations for development of novel approaches for stable fly management.
Cattle are an asymptomatic reservoir of Escherichia coli O157:H7, but the bacterial colonization and shedding patterns are poorly understood. The prevalence and shedding of this human pathogen have ...been reported to be seasonal with rates typically increasing during warm months. The objectives of this study were (i) to assess the prevalence of E. coli O157:H7 in feces of feedlot cattle in Kansas during summer, fall, and winter months, and (ii) to characterize E. coli O157:H7 by screening for virulence factors. Of 891 fecal samples collected, 82 (9.2%) were positive for E. coli O157:H7. No significant differences in prevalence were detected among summer, fall, and winter months. The highest monthly prevalence (18.1%) was detected in February. All tested isolates were positive for stx2 (Shiga toxin 2) and eaeA (intimin) genes; 14 isolates (12.8%) also carried stx1. Our results indicate the prevalence of E. coli O157:H7 in beef cattle feces is not necessarily season dependent.
Stable flies (Stomoxys calcitrans Diptera: Muscidae L.) are blood-feeding synanthropic pests, which cause significant economic losses in livestock. Stable fly antennae contain olfactory sensilla ...responsive to host and host environment-associated odours. Field observation indicated that the abundance of stable flies increased significantly in grasslands or crop fields when cattle manure slurry was applied. Major volatile compounds emanating from manure slurry were collected and identified. Behavioural responses of stable flies to those compounds were investigated in laboratory bioassays and field-trapping studies. Results from olfactometer assays revealed that phenol, p-cresol and m-cresol were attractive to adult stable flies. When tested individually, attraction was higher with lower dosages. Stable flies were most attracted to blends of phenol and m-cresol or p-cresol. Traps with binary blend lures caught more stable flies in field trials as well.
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