Isolation and characterization of tetracycline-resistant Citrobacter spp. from catfish

Fifty-two tetracycline-resistant Citrobacter spp. strains were isolated from farm-raised catfish. Morphological and biochemical characteristics indicated that 38 of the 52 citrobacters were Citrobacter freundii, 7 were C. amalonaticus and 7 were C. braakii. All isolates were resistant to multiple antibiotics. Polymerase chain reaction (PCR) protocols were developed to detect the presence of 3 tetracycline-resistance genes (tetA, tetB and tetG) from Citrobacter isolates. Oligonucleotide primers specifically targeting a 967-bp region of tetB successfully amplified the PCR amplicons from 3238 (85.0%) of C. freundii strains, 57 (71.0%) of C. amalonaticus and 47 (57%) from C. braakii. Oligonucleotide primers specific for the detection of tetA gene amplified the 417-bp PCR amplicons from 738 (18.0%) of tetracycline-resistant C. freundii only. The assay failed to amplify tetA genes from C. brakii or C. amalonaticus. Plasmids (2.0-16.0kb) were isolated from 14 of the 38 strains of C. freundii. Strains of C. amalonaticus and C. brakii did not contain any plasmids. Dendrogram analysis of the SpeI pulsed field gel electrophoresis (PFGE) results identified 23 distinct macrorestriction patterns (mrps) among the 36 strains of C. freundii, 3 distinct mrps among the 7 strains of C. braakii and 4 unique mrps among the 7 strains of C. amalonaticus. Our results indicate that citrobacters from catfish could serve as reservoirs of tetracycline-resistance determinants.