ENVIRONMENTAL RAOULTELLA ORNITHINOLYTICA WITH MULTIPLE ANTIBIOTIC RESISTANCE GENES

Raoultella ornithinolytica (Enterobacteriaceae) are aquatic bacteria in a new proposed genus that was originally classified as Klebsiella. A recent study describes the rising role of R. ornithinolytica in hospital acquired human infections, with 71% of cases demonstrating resistance to antibiotics. In particular, resistance to three classes of antibiotics that are commonly used to treat Raoultella infections was found: 3^rd generation cephalosporin, quinolone, and aminoglycoside. No specific antibiotic resistance genes have been reported for R. ornithinolytica, but the following have been reported to be found in the genus: aminoglycoside (ant(3”)-I), β-lactams (bla_CTX-M, bla_SHV-2, and bla_TEM-1), and trimethoprim (dfrA1). The objectives of this study were 1) to analyze the antibiotic resistance genes found in an R. ornithinolytica collected in the environment and 2) to determine if the antibiotic resistance genes are located on plasmids or chromosomes. An R. ornithinolytica isolate was collected from Rock Creek Park in Kensington, Maryland and identified with mass-spectrometry. Antibiotic resistance profile was determined with disk diffusion tests for eight classes of antibiotics: β-lactam, aminoglycoside, phenicol, quinolone, fosfomycin, tetracycline, sulfonamide, and trimethoprim. Plasmids were isolated and ran on an agarose gel to determine their number and size. Plasmid DNA was sequenced by next generation sequencing. Twelve antibiotic resistance genes were found from seven different categories: four aminoglycosides (aac(6’)Ib-cr, aac(3)-IIa, strB, and strA), three β-lactams (bla_TEM-1B, bla_CTX-M-15, and bla_OXA-1), two fluoroquinolones (qnrB1 and aac(6’)Ib-cr), one phenicol (catB3), one sulfonamide (sul2), one tetracycline (tetA), and one trimethoprim (dfrA14). The disk diffusion test showed resistance to five of the classes of antibiotics: aminoglycoside, β-lactam (1^st – 3^rd generation cephalosporin, monobactam, and 3^rd and 4^th generation penicillin), sulfonamide, tetracycline, and trimethoprim. It also showed intermediate resistance to quinolone, tetracycline, and β-lactam (4^th generation cephalosporin). The isolated plasmid is 205,220 bp, belongs to the incompatibility group IncFIIK, and has all twelve of the antibiotic resistance genes located in it. This is the first report of the aac(6’)Ib-cr, bla_TEM-1B, and bla_CTX-M-15 genes being found in the species R. ornithinolytica, and the first report of the aac(3)-IIa, strB, strA, bla_OXA-1, qnrB1, catB3, sul2, tetA, and dfrA14 genes found in the genus Raoultella. This study demonstrates the prevalence of antibiotic resistance genes in the environment with R. ornithinolytica being resistant to multiple antibiotics commonly used to treat infections caused by these bacteria.