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Association of Enterococci with Stored Products and Insects: Public Health Implications

Explore the link between antibiotic-resistant enterococci in stored-products, insects, and implications for public health. The study investigates antibiotic resistance in stored-product insects to understand possible vectors of resistant bacteria.

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Association of Enterococci with Stored Products and Insects: Public Health Implications

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  1. Association of Enterococci with Stored Products and Stored-Product Insects: Public Health Importance and Implications H. C. Lakshmikantha, Bh. Subramanyam, Z. A. Larson, and L. Zurek† Department of Grain Science and Industry †Department of Entomology Kansas State University, Manhattan 66506, USA Presented at the 9th International Working Conference on Stored-Product Protection, 15-18 October 2006, Campinas, Brazil

  2. Introduction • Enterococci are ubiquitous, gram-positive, catalase negative, cocci • Enterococci are normal flora of the gastro-intestinal tract of humans and animals • Enterococci are not food-borne pathogens • Reservoirs for drug-resistant genes Enterococcus spp.

  3. Medical importance of enterococci • Readily develop resistance to antibiotics and can transfer resistance genes to other bacteria • Second most common cause of hospital-acquired infections in the United States • Documented resistance to numerous antibiotics, including vancomycin • Vancomycin resistant enterococci (VRE) and VRE infections have become a worldwide public health threat • In the United States, the prevalence of antibiotic-resistant enterococci (ARE) may be, in part, due to the use of antibiotics in feed as growth promoters for farm animals

  4. Antibiotics and the US feed industry • In the United States, about 3,000 feed mills produce 121 million tons of feeds annually for various domestic animals • An estimated 70% (nearly 2.5 million tons) of the antibiotics used in the United States each year are used as feed additives for chickens, hogs, and beef cattle • 11 antimicrobials are commonly used in animal feeds • Examples: bacitracin, chlortetracycline, dynafac, mycostatin, oxytetracycline, oleandomycin, penicillin, streptomycin, bambermycins, tilmicosin, and tylosin • Antibiotic use in feed may lead to development of resistance in bacterial strains of clinical importance

  5. Association of stored-product insects with bacteria • Stored-product insects are cosmopolitan in distribution and occur in diverse habitats from the farm to the consumer’s home • The digestive tract of these insects provides a microclimate for bacterial multiplication • Stored-product insects have been reported to harbor potentially pathogenic bacteria (Apuya et al. 1994; Larson 2004) • Stored-product insects may be of medical and veterinary interest, because they could serve as vectors for antibiotic resistant bacteria (Larson 2004)

  6. Importance of stored products and stored-product insects in the food web http://res2.agr.ca/winnipeg/storage/images/lo-res/control/ecosys-l.jpg

  7. Materials and methods ● ● ● ● ● • Insects: 392 • Mar-Nov, 2003 [298] • Apr-Jun, 2006 [94] • Stored products: 28 ● Stored products were collected from a feed mill and two livestock facilities in Kansas.

  8. Materials and methods Insect/feed Isolation Quantification Antibiotic resistance Specific primers; 16S rRNA gene sequences PCR Species level identification

  9. Specific media used for bacterial isolation • Trypticase soy broth agar: for general bacteria • MacConkey sorbitol agar: for Enterobacteriaceae bacteria • mEnterococcus agar: for Enterococcus spp. Antibiotic resistance assays conducted using the diffusion disk assay • Tetracycline (30 µg) • Ampicillin (10 µg) • Erythromycin (15 µg) • Vancomycin (30 µg) • Chloramphenicol (30 µg) • Ciprofloxacin (5 µg)

  10. Measuring antibiotic resistance Resistance is determined by the bacterial carpet growing up to the diffusion disk No resistance and intermittence is determined by measuring the zone of inhibition (dotted line) created by the antibiotic around the disk

  11. Primers and multiplex PCR conditions used for species identification of Enterococcus a F, forward; R, reverse.

  12. Results

  13. Percentage distribution of stored-product insects collected from various facilities for this study (n= 392)

  14. Number of stored product insects sampled from each mill and number of colony forming units (CFU) per insect on three media aSamples in mills 1-6 were collected during March to November, 2003 (Larson, 2004); samples in mills 7-12 were collected by H. C. Lakshmikantha during April to June, 2006. bN = Total number of insects sampled from each mill. cn = Number of positive samples (% of positive samples).

  15. Number of stored products sampled from each mill/facility and number of colony forming units (CFU) per g sample on three media aSites 1 and 3 are swine farms in Salina, Kansas; site 2 is the Kansas State University pilot feed mill and site 4 is the swine farm at Kansas State University bN = Total number of stored products sampled from each site cn= Number of positive samples (% of positive samples)

  16. Fig 1. Percentage of enterococcal isolates (n = 67) from stored-product insects with antibiotic resistance

  17. Fig 2. Percentage of enterococcal isolates (n = 125) from stored products with antibiotic resistance

  18. Prevalence and identification of enterococci isolated from stored products and stored-product insects collected during April to June, 2006

  19. Prevalence, identification, and distribution of enterococci isolated from stored products and stored-product insects collected during April to June, 2006

  20. Conclusions • Our findings provide evidence that stored products and stored-product insects harbor antibiotic resistant and potentially virulent enterococci • Our data reinforces the need for pest management to reduce the availability of insect vectors, particularly stored-product insects, in the feed mill and livestock environments

  21. Acknowledgements • Dr. Aqeel Ahmad • Dr. Keith Behnke • Consortium for Integrated Management of Stored Product Insect Pests (CIMSPIP) • Research reported here was funded by the United States Department of Agriculture, Cooperative State Research and Education, and Extension Service’s Risk Assessment and Mitigation Program under Agreement Number 2002-3438-112187.

  22. References Apuya, L. C., S. M. Stringham, J. J. Arends, and W. M. Brooks.1994 Prevalence of protozoan infections in darkling beetles from poultry houses in North Carolina. Journal of Invertebrate Pathology. 63: 255-9 Larson, A.Z. 2004. Diversity and Medical importance of stored- product insects in feed mills. M.S Thesis, Kansas state university, USA. http://bmj.bmjjournals.com/content/vol324/issue7338/twib.shtml http://service.merck.de/microbiology/tedisdata/prods/4973-1_05392_0500-1.jpg http://www.ebtron.com/images/map/usa-map.gif

  23. Thank you

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