The prevalence of antibiotic resistant (ART) bacteria in ready-to-eat foods including cheese products has been a major public health concern. The objectives of this study were to determine critical control points (CCP) in cheese fermentation for effective mitigation, and to characterize an ART isolate from the dairy environment to reveal potential mechanism(s) involved in the development, maintenance and dissemination of antibiotic resistance (AR).
Pilot plant studies indicated that pasteurization effectively reduced ART bacteria in milk and AR can be effectively minimized and controlled with proper sanitation and processing controls. ART bacteria were not significantly amplified during ripening. However, antibiotic facilitated the proliferation of ART bacteria by inhibiting the growth of starter cultures, suggesting rapid and sufficient growth of starter cultures for acid production was important to control AR during chess making process. Results of assessing samples from commercial cheese manufacturing facilities indicated that approximately 104 CFU/ml of Tetr bacteria were found in a manufacturer-maintained starter/adjunct culture vat and representative isolates were identified to be Streptococcus thermophilus (tet(S)). Additional identified AR gene (tet(S), tet(M) and tet(L)) carriers included Streptococcus spp., Leuconostoc sp., Staphylococcus sp., Lactococcus sp., and Lactobacillus sp., from cheese samples collected during the manufacturing process. Results from a small scale survey indicated that the overall quantities of AR genes by real-time PCR in retail cheese products purchased in 2010 reduced compared to those from 2006, suggesting the effectiveness of targeted AR mitigation in related products.
A dairy Enterococcus faecium isolate M7M2 carrying both tet(M) and tet(L) genes was further characterized and both tet(M) and tet(L) genes were found to be located on a 19.6 kb plasmid, which was stable (99% retaining rate) in the absence of antibiotic selective pressure after consecutive transfer for more than 500 generations. The tet(M)-tet(L) gene cluster was successfully transferred to and lead to acquired resistance in Enterococcus faecalis OG1RF by electroporation and Streptococcus mutans UA159 by natural transformation. DNA sequence analysis revealed that the 19.6kb plasmid contained 21 ORFs, which included a 10.6 kb backbone, which was highly homologous (99.9%) to the reported plasmid pRE25, but without the identified toxin-antitoxin (TA) plasmid stabilization mechanism. The derived backbone plasmid without the tetracycline resistance determinants exhibited 100% retention rate in the presence of acridine orange, suggesting the presence of a TA-independent, plasmid stabilization mechanism, with its impact on the persistence of a broad spectrum of resistance-encoding traits to be elucidated. Quantitative real-time RT-PCR indicated induced transcription of both tet(M) and tet(L) genes by tetracycline in pM7M2. E. faecium M7M2 also formed biofilms on stainless steel coupons as assessed by scanning electron microscopy, suggesting potential persistence of the strain in the environment.
The CCP identified in this study included pasteruzation, rapid growth of starter culture and proper maintenance of manufacturer-maintained cultures, which provided important information for controlling AR in dairy industry and reducing AR exposure from fermented dairy foods. Characteristics of E. faecium M7M2 provided essential information for the development of counter strategies for AR mitigation.