Functional ccreening of antibiotic resistance genes in soil Chryseobacterium spp. genomic DNA libraries

Background: Species of the genus Chryseobacterium are abundantly found in soil and water and have recently emerged as opportunistic nosocomial pathogens in humans. Due to multidrug-resistant phenotype, displayed by some species, and well documented examples of gene exchange between the environmental and the clinical strains, the more detailed picture of Chryseobacterium resistance mechanisms is needed. Therefore, functional genomic screening was applied in order to identify antibiotic resistance determinants in environmental Chryseobacterium spp. isolates. Materials and Methods: Functional screening of Chryseobacterium spp. genomic DNA library from soil isolates were carried out with clinically important antibacterial agents which include: aminoglycosides (gentamicin, kanamycin, streptomycin), beta-lactam antibiotics (imipenem, cefuroxime), tetracycline, chloramfenicol, benzalkonium chloride and ciprofloxacin. Minimum inhibitory concentrations of clones were determined and interpreted according to EUCAST breakpoints. The inserts displaying resistance phenotype were sequenced and analyzed using bioinformatic approach. Results: Selections with streptomycin, tetracycline and cefuroxime resulted in single resistant clone per antibiotic. Two resistant clones were identified using imipenem and the same resistant clone was identified in separate selections with kanamycin or gentamycin. Analysis of sequences revealed that in 4 out of 6 inserts contained genes coding directly antibiotic modifying enzymes- streptomycin adenylyltransferase, tetracycline MFS transporter, metallo-like beta-lactamase and carbapenem-hydrolyzing IND beta-lactamase. The remaining inserts did not contain obvious resistance genes. Resistance to imipenem may be determined by the GNAT family N-acetyltransferase or monooxygenase, while the lipid A biosynthesis acyltransferase may be responsible for the resistance to gentamycin and kanamycin. Conclus[...]