Performing Department
ENTOMOLOGY
Non Technical Summary
House flies (HF) develop in the manure of domestic animals including those that are frequently fed antibiotics for growth promotion and prophylaxis. In the United States, due to heavy use of antimicrobial agents in the agriculture, antibiotic resistance genes in the gastro-intestinal microbial communities of domestic animals and their feces/manure have become common. Antibiotic resistance in clinical isolates has become a serious problem because of the limited number of effective antibiotics available for treatment of human bacterial infections. The connection between antibiotic resistances of food animal origin and that of clinical isolates has been suggested in several studies; however, the ecology of bacterial antibiotic resistance and virulence genes in the environment is poorly understood. Due to their developmental habitats, mode of feeding, unrestricted movement, and attraction to residential areas, the manure-borne insects, primarily HF, likely play an
important role in the ecology and dissemination of virulence and antibiotic resistance genes in agricultural and urban environment. To assess this role, enterococci have been chosen as a bacterial model system because of their medical importance (virulence factors), frequent and diverse antibiotic resistance genes, great variety of mobile genetic elements (pheromone responsive plasmids, conjugative plasmids, transposons), and their consistent presence in animal and human feces as well as in the digestive tract of manure-borne flies.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
1) Assessment of the antibiotic resistance profiles of enterococci from the gastro-intestinal community of adult house flies associated with the manure of animals frequently exposed to antibiotics (feedlot cattle) and animals with no or very limited exposure to antibiotics (American bison) 2) Characterization of selected enterococcal isolates from manure and flies by: a) Assessment of the diversity of tetracycline, vancomycin, and erythromycin resistance genes b) Screening for virulence factors including, cytolysin (cylA, cylB), gelatinase (gelE) and selected adhesins (as, esp).
Project Methods
Fresh fecal material of feedlot cattle (K-State Agricultural Experimental Station) and American bison (Konza Prairie Biological Station) and associated adults of HF will be collected three times per year (spring, summer, fall) for two consecutive years. Feedlot cattle receive tylosin and monensin as growth promoters 120 days before slaughter and oxytetracycline, ceftiofur (cephalosporin), and tilmycosin (fluoronquinolone) therapeutically. The herd of American bison has not received any antibiotics for the past 10 years. Ten grams of fecal material from each sample will be mixed in 50 ml of PBS buffer and serially diluted. Adult flies will be identified under the dissection microscope and fifty randomly selected HF from each collection time will be used for the bacterial analysis. Flies will be surface sterilized by sodium hypochlorite and ethanol and individually homogenized in 5 ml of PBS and the homogenate serially diluted. Serially diluted samples in PBS (to 10-8)
will be drop plated in triplicates on mENT agar. The plates will be cultured aerobically at 37oC for 48 hours. Red, burgundy, and pink colonies will be counted using a colony counter to assess the enterococcal population size in each sample. Five colonies will be randomly selected from each sample, streaked on TSBA and stored at 4oC until further analysis. The genus level will be confirmed phenotypically. Isolates will be identified identified to species level by multiplex using species-specific primers. A customized 96 well panel of antimicrobials for the NARMS program will be used for antibiotic resistance testing. Results will be interpreted according to NCCLS guidelines. Antibiotics selected for this study include: ampicillin, penicillin, high level-gentamicin, high-level streptomycin, vancomycin, teicoplanin, erythromycin, lincomycin, virginiamycin, quinopristin/dalfopristin, tetracycline, chloramphenicol, neomycin, norfloxacin, nitrofurantoin, levofloxacin and ciprofloxacin. A
comparable resistance profile between ruminant and fly populations can be an indicator of strain transfer between mammals and flies and/or vice versa. Resistance genes to tetracycline [tet (O), tet(S), tet (W), tet (Q), tet (K), tet (C), tet (L)]; to erythromycin [erm (A), erm (B), erm (C) ], and vancomycin [van (A), and van (B)] will detected by specific PCR primers. Detection of genes of five virulence factors, including cylA, cylB, as, esp, and gelE will be performed using multiplex PCR.