Characterization of Staphylococci Isolated from Bears in the Great Smoky Mountains National Park

Bacteria exist in essentially all habitats and can serve as key indicators of human influence. Bacteria naturally acquire resistance to antibiotics, however, the transfer of genes conferring resistance to therapeutically important antibiotics is a major concern in human and veterinary medicine.  In particular, methicillin resistance has increased rapidly in some staphylococcal species.[1] For reasons that are not yet fully understood methicillin resistance is usually accompanied with multi-drug resistance. A high proportion of staphylococci isolated from humans and animals with human contact are antibiotic resistant. On the other hand, animals with minimal human contact, such as polar bears living in the arctic, have a very low level of antibiotic resistance. [2] Thus bacterial flora may serve as an indicator of human influence on wild animals. This study was undertaken to determine which species of staphylococci colonize black bears in the (GSMNP), to search for previously unidentified species of staphylococci, and to determine the extent of antibiotic resistance occurring in staphylococci isolated from bears to gain a better understanding of the influence of human-bear interactions on bacterial flora and to serve as a time point reference for future studies.

For this study samples were collected by swabbing up to five sites on 14 nuisance bears. Samples were collected by park personnel, transferred to the University of Tennessee College of Veterinary Medicine Bacteriology Laboratory and cultured. They were tentatively identified using standard biochemical techniques and antibiotic susceptibility was determined for staphylococcal species using the Kirby-Bauer disk diffusion method on Mueller-Hinton agar plates.[1, 3-5] DNA was extracted from all samples and their 16S rRNA genes were amplified by PCR and the genes sequences. The sequence information was compared to deposits in a public database (GenBank) to determine the identity of the isolates at the genus or species level.  Among 108 isolates  61 were identified as staphylococci  from genetic testing. These included  S.saprophyticus (10),  S.sciuri (2), S.cohnii (2), S.muscae (2), S.gallinerum (1), S.aureus (3), S.pseudintermedius (6), S.schleiferi (28), S.warneri (3), and 2 unidentified and potentially novel staphylococcal species.   Eleven isolates exhibited resistance to methicillin and all of these isolates were resistant to multiples classes of antibiotics.  No resistance was seen to chloramphenicol, erythromycin, gentamicin, or trimethoprim sulfa.  Intermediate resistance only was found among the following percentages of isolates: clindamycin (3.6%), and marbofloxacin (1.8%). Resistance was found to amoxicillin/clavulanic acid (18.5%), ampicillin (16.7 %), penicillin (24.1%), tetracycline (1.8%), and methicillin (20.3 %). Resistance was found only in S.sciuri, S.saprophyticus, S.gallinerum, and S.pseudintermedius. Multilocus sequence typing of the three S.aureus samples determined that they were ST6 (clonal complex (CC) 5), ST 8 (CC 8), and ST 789 (CC 705).  S.aureus within CC5 are associated with bacteremia with hematogenous complications. [6] CC8 is a successful clone with broad distribution [7] and CC 705 is most often associated with bovine milk. [8] None of the MLST of the S.pseudintermedius isolates has been previously assigned or reported. 

S.aureus, the species of staphylococcus most closely associated with human colonization and disease did not exhibit resistance to commonly prescribed antibiotics.  S.sciuri and S.saprophyticus are widespread in nature  whereas S.gallinerum and S.pseudintermedius are often associated with domestic animals. The mechanism of antibiotic resistance and lateral gene in these organisms could be further elucidated by determining their plasmid profiles and presence of bacteriophage. These data may serve as an important reference for future studies to determine trends of human influence on bacteria in the GSMNP.