The worldwide increase in the emergence and spread of antibiotic resistance has become a major public health concern, with economic, social and political ramifications. Clearly, the prevalence of antibiotic resistant bacteria in the gastro-intestinal microbial communities of domestic food animals and their feces/manure has become high in the United States likely due to extensive use of antibiotics in food animal production [3, 6, 10, 34–36]. Although a connection between antibiotic resistance in bacterial isolates from healthy food animals and clinical isolates of human and animal origins has been suggested, this is a controversial issue because little is known about the amplification and spread of antibiotic resistant bacteria and genes in the environment [12–14, 16, 37–41].
The two groups of insects most frequently screened for food borne-pathogens are house flies and cockroaches. These insects have been implicated as mechanical or biological vectors for bacterial pathogens including Salmonella spp., Campylobacter spp; Pseudomonas aeruginosa, Listeria spp., Shigella spp ., Aeromonas spp ., Yersinia pseudotuberculosis, Escherichia coli O157:H7, and E. coli F18 that can cause diseases in humans and/or animals [17, 18]. Multi-antibiotic resistant enterococci have been reported from house flies collected from fast-food restaurants . In addition, the horizontal transfer of tet(M) among E. faecalis in the house fly digestive tract as well as the great capacity of house flies to contaminate human food with enterococci have been demonstrated [42, 43]. Organic wastes in and around animal production facilities including swine farms provide excellent habitats for house flies and German cockroaches. Several features of house flies and cockroaches, including their dependence on live microbial communities, active dispersal ability and human-mediated transport, attraction to places where food is prepared and stored, developmental sites, and mode of feeding/digestion make these insects an important "delivery vehicle" for transport of bacteria including antibiotic resistant enterococci from reservoirs (animal manure), where they pose minimal hazard to people, to places where they pose substantial risk (food) [17, 18, 44]. Several reports showed a positive correlation between the incidence of food-borne diarrhea and the density of house fly or cockroach populations. For example, suppression of flies in military camps in the Persian Gulf resulted in an 85% decrease in Shigellosis and a 42% reduction in the incidence of other diarrheal disease . Esrey  reported a 40% reduction in the incidence of diarrheal infections in children after suppression of a fly population. Another study showed that fly control could reduce trachoma and diarrhoea among children in Gambia . An outbreak of gastro-enteritis caused by S. typhimurium in the children's ward of a Belgian hospital dropped as soon as the German cockroach infestation had been controlled . Tarshis  recorded that control of cockroaches was accompanied by a decrease in the incidence of endemic infectious hepatitis. The German cockroach was also shown as a potential mechanical vector of the piglet pathogen Escherichia coli F18 .
To our knowledge, surveillance for resistance to antibiotics in enterococci from insects associated with swine production environments has not been previously conducted. Recently, Graham et al.  reported that flies may be involved in the transmission of drug resistant enterococci and staphylococci from confined poultry farms. In our study, enterococci were detected in the digestive tracts of house flies, cockroach fecal samples and pig fecal samples collected from two different swine farms with enterococci recovered from 93.7% of 364 samples analyzed. High concentrations of enterococci in the digestive tract of house flies and cockroaches suggest that enterococci are common commensals of these insects intestinal microbiota. Among the four most frequently identified species, E. faecalis and E. faecium are the most important enterococcal species from a clinical perspective [20, 22, 27]. However, infections caused by E. hirae and E. casseliflavus may also occur and warrant attention . In addition, enterococci are regarded as important reservoirs of antibiotic resistance and virulence genes that are often found on mobile genetic elements [22, 27, 30, 52].
The most frequently encountered enterococcal species in the intestines of farm animals are E. faecalis, E. faecium, E. hirae, and E. durans; however, culture methods may influence the recovery and selection of enterococcal species [36, 53]. The dominance of E. hirae in pig feces in our study is consistent with studies of the enterococcal community of swine [32, 33]. E. faecalis was observed more frequently from the digestive tract of insects and these results are also in agreement with previous studies [19, 54]. The favorable conditions in the fly and cockroach digestive tract may serve to select and amplify environmentally acquired E. faecalis, including those carrying antibiotic resistance genes.
High frequency of resistance to tetracycline, erythromycin, streptomycin, kanamycin, and ciprofloxacin in our study likely reflects use of tetracyclines, macrolides, aminoglycosides and fluoroquinolones for swine in the USA . Unfortunately, we were unable to obtain any specific information on the use of antibiotics in the two commercial farms in this study. Similar results were reported on antimicrobial resistant phenotypes and resistance genes in enterococci from animals and insects [10, 19, 51]. The patterns of antibiotic resistance observed in Enterococcus spp. recovered from the pig fecal samples were similar to those observed in isolates recovered from digestive tracts of house flies and cockroach fecal samples indicating that insects acquired enterococci from the pig manure. PFGE analysis of selected E. faecalis and E. faecium isolates confirmed that both insect species carried some of the same clones that were detected in the swine manure. This supports our data indicating that insects acquired the drug-resistant and potentially virulent enterococci from the swine feces although the opposite route cannot be ruled out. However, our previous study  showed that the prevalence of antibiotic resistant enterococci in house flies decreases with increasing distance from the likely source (cattle feedlot). This indicates that the source of antibiotic resistant enterococci in house flies and cockroaches in this study was the swine manure due to very high prevalence of antibiotic resistant enterococci in all three sources. The absence of VRE in this study is in agreement with previous findings and reflects a relationship between extensive use of specific antibiotics as growth promoters and presence of VRE [32, 35, 57]. Since avoparcin has not been used as a growth promoter in the United States, and VRE are rarely isolated from US food animal production environments. In contrast, VRE have been frequently isolated from food animal production environments in Europe where vancomycin was extensively used for farm animals .
Our findings are in agreement with the results of other studies which showed that tet (M) and erm (B) are the most widespread resistance genes among enterococci from food animals or foods [10, 15, 19, 24, 59, 60]. Furthermore, a strong association of the tet (M) and erm (B) genes with the conjugative transposon family Tn 1545/Tn 916 was also detected in many isolates in our study, indicating that antibiotic resistant enterococci associated with the confined swine environment could be a reservoir of transferable tetracycline and erythromycin resistance. The similar prevalence of resistance determinants and Tn 1545/Tn 916 transposons among isolates from pig feces, house flies and cockroach feces indicates exchange of resistant strains or their resistance genes. This is important because the Tn 1545/Tn 916 family has a very broad host range and members of this family of transposons can be transferred by conjugation to numerous bacterial species in the human gastrointestinal microbial community [61–63].
The highest incidence of multiple virulence factors was detected in E. faecalis with similar virulence profiles from the digestive tract of house flies, cockroach feces and pig feces. The gelE gene was detected frequently in E. faecalis (63.0%) and was the most common of the virulence factors. Prevalence of the gelE gene has been frequently documented in E. faecalis, and rarely in E. faecium and E. durans [12, 27]. The presence of gelE was, however, not strictly correlated with the phenotype suggesting that some gelE genes are silent which is likely due to a 23.9-kb chromosomal deletion involving the fsr locus that regulates gelE expression [64, 65]. We found little correlation between the clumping phenotype in vitro and the presence of the asa1 gene in E. faecalis showing that asa1 is not commonly expressed under these in vitro conditions. The phenotypic test for β-hemolysis (cytolysin production) with E. faecalis, E. faecium and E. casseliflavus showed a strong correlation between cylA and β-hemolysis on human blood. However, 8.1% of the E. faecalis from house flies were positive for β-hemolysis but negative for cylA, suggesting the presence of unknown determinant(s). Some of the genes encoding virulence determinants, including cytolysin and aggregation substance, are known to be present on pheromone-responsive plasmids, such as pAD1 and therefore transferable to other E. faecalis strains .
The data presented in this study offer evidence that should be helpful for future research initiatives aimed at reducing the dissemination of antibiotic resistant and virulent bacteria. It is likely that the high prevalence of resistant and potentially virulent enterococci in house flies and German cockroaches associated with confined swine environments reflects an extensive use of antibiotics by the swine industry. However, the degree to which these resistant and virulent enterococci hamper the efficacy of medically important antibiotics and thus pose risks to humans is unknown. The gastrointestinal tracts of mini-pigs, humans, and mice provide favorable environments for intra- and interspecies transfer of antibiotic resistance genes, but these processes have not been investigated in the digestive tract of insects and related arthropods with few exceptions [42, 66–71]. Knowing the sources of enterococci harboring in house flies and German cockroaches is also important to accurately assess risk, to identify and implement management plans for fecal waste, and to establish insect management practices that prevent the spread of antibiotic resistant strains and other potential human and animal pathogens. Further studies are warranted to pinpoint the potential sources of fecal contamination of insects, their subsequent contamination of food and feed, and for a detailed understanding gene transfer in the digestive tract of insects.