Occurrence of the family Enterobacteriaceae (sensu stricto) in cow’s milk and their antimicrobial resistance

Objectives: The Enterobacteriaceae family is a group of bacteria that serve as indicators of milk collection hygiene at the farm level. The objective of this study was to detect and identify members of this family in raw cow’s milk and pasteurized milk and to evaluate their antimicrobial susceptibility and the presence of resistance genes. Materials and Methods: A total of 14 samples of raw bulk milk and 14 samples of pasteurized milk were collected from different dairy plants in Slovakia. Isolates were identified at the family level by PCR and at the species level by MALDI-TOF MS. Ten antibiotics (ampicillin, cefoxitin, ceftizoxime, cefazolin, kanamycin, streptomycin, tetracycline, gentamicin, ofloxacin, and chloramphenicol) were tested using the disc diffusion method. Genes that confer antimicrobial resistance to beta-lactam antibiotics were detected by PCR using gene-specific primers. Results: Klebsiella oxytoca was the most frequently identified isolate (30.0%), followed by Enterobacter cloacae (13.3%) and Citrobacter gillenii (13.3%). According to the CLSI criteria, 22 of the 30 isolates (73.3%) were classified as cefoxitin-resistant, whereas EUCAST classified 26/30 (86.7%) as cefoxitin-resistant. The presence of resistance genes (at least one of the genes tested) was confirmed in 26 isolates. In 20 isolates, blaTEM was found; in 11, blaCTX–M; and in 10, ampC genes. Conclusions: Our findings highlight the absence of Enterobacteriaceae in pasteurized milk, discrepancies in evaluation between the CLSI and EUCAST systems, a high level of beta-lactam resistance, and the presence of resistance genes detected only in raw milk samples.