Isolation and Characterization of Antibiotics Resistant Bacteria from Raw Milk

Objective: Isolation and identification of antibiotics resistant bacteria in raw milk. Methods: Luria-Bertani plates containing ampicillin (32 μg/mL), clindamycin (4 μg/mL), or chloramphenicol (32 μg/mL) were used in isolating the antibiotics resistant bacteria from samples; 16S rRNA analysis method was used to identify the isolates; blood agar plates were used in hemolysis assay; K-B disk diffusion method was used to confirm the resistant phenotypes. Results: 60 raw milk samples collected from Zhangjiakou were screened for antibiotic resistant bacteria. The screening results showed that 48 (80%) samples had isolates resistant to more than one antibiotic, 39 (65%) samples had isolates resistant to more than two antibiotics, and 14 (23.33%) samples had isolates resistant to three antibiotics tested. Totally, 48 strains were isolated resistant to ampicillin, 46 strains were isolated resistant to clindamycin, and 21 strains were isolated resistant to chloramphenicol. Ten strains (resistant to three antibiotics) were randomly selected for 16S rRNA analysis and they were identified as Serratia marcescens (1), Pseudomonas aeruginosa (8), and Enterobacter cloacae (1), respectively. Hemolysis test results showed those 2 strains with α-hemolytic phenotype, 2 strains with β-hemolytic phenotype, and 6 strains with γ-hemolytic phenotype. Furthermore, with K-B disk diffusion method, all 10 isolates were validated resistant to 3 antibiotics tested except that one isolate intermediated to chloramphenicol and two isolates susceptible to chloramphenicol. Conclusion: Raw milk is the reservoir of a variety of antibiotics resistant bacteria; antibiotic plates can be used for preliminary screening of antibiotics resistant bacteria from raw milk samples. Keywords-milk; resistant bacteria; isolation and identification; K-B disk diffusion method