Monitoring Changes in the Antimicrobial-Resistance Gene Set (ARG) of Raw Milk and Dairy Products in a Cattle Farm, from Production to Consumption

Abstract

Raw milk and dairy products can serve as potential vectors for transmissible bacterial, viral and protozoal diseases, alongside harboring antimicrobial-resistance genes. This study monitors the changes in the antimicrobial-resistance gene pool in raw milk and cheese, from farm to consumer, utilizing next-generation sequencing. Five parallel sampling runs were conducted to assess the resistance gene pool, as well as phage or plasmid carriage and potential mobility. In terms of taxonomic composition, in raw milk the Firmicutes phylum made up 41%, while the Proteobacteria phylum accounted for 58%. In fresh cheese, this ratio shifted to 93% Firmicutes and 7% Proteobacteria. In matured cheese, the composition was 79% Firmicutes and 21% Proteobacteria. In total, 112 antimicrobial-resistance genes were identified. While a notable reduction in the resistance gene pool was observed in the freshly made raw cheese compared to the raw milk samples, a significant growth in the resistance gene pool occurred after one month of maturation, surpassing the initial gene frequency. Notably, the presence of extended-spectrum beta-lactamase (ESBL) genes, such as OXA-662 (100% coverage, 99.3% identity) and OXA-309 (97.1% coverage, 96.2% identity), raised concerns; these genes have a major public health relevance. In total, nineteen such genes belonging to nine gene families (ACT, CMY, EC, ORN, OXA, OXY, PLA, RAHN, TER) have been identified. The largest number of resistance genes were identified against fluoroquinolone drugs, which determined efflux pumps predominantly. Our findings underscore the importance of monitoring gene pool variations throughout the product pathway and the potential for horizontal gene transfer in raw products. We advocate the adoption of a new approach to food safety investigations, incorporating next-generation sequencing techniques.

Keywords: ESBL; NGS; antimicrobial-resistance genes; cattle; dairy products; food safety; next-generation sequencing; public health; raw milk; taxonomy.

Figure 1

Frequency of antimicrobial-resistance genes identified in raw milk samples, by drug group and resistance mechanism. PEN—penicillins; CEF—cephalosporins; MON—monobactams; CAR—carbapenems; TET—tetracyclines; AMG—aminoglycosides; FEN—phenicols; MAC—macrolides; LIN—lincosamides; PLE—pleuromutilins; PEP—peptide antibiotics; FOS—phosphomicin; FLQ—fluoroquinolones; SUL—sulphonamides; DIA—diaminopyrimidines; RIF—rifamicins; NIT—nitroimidazoles; DIS—disinfectants; AMC—aminocoumarins. The numbers in the bar chart represent the exact number of pieces of each resistance mechanism.

Figure 2

Frequency of antimicrobial-resistance genes identified in fresh cheese samples, by drug group and resistance mechanism. PEN—penicillins; CEF—cephalosporins; MON—monobactams; CAR—carbapenems; TET—tetracyclines; AMG—aminoglycosides; FEN—phenicols; MAC—macrolides; LIN—lincosamides; PLE—pleuromutilins; PEP—peptide antibiotics; FOS—phosphomicin; FLQ—fluoroquinolones; SUL—sulphonamides; DIA—diaminopyrimidines; RIF—rifamicins; NIT—nitroimidazoles; DIS—disinfectants; AMC—aminocoumarins. The numbers in the bar chart represent the exact number of pieces of each resistance mechanism.

Figure 3

Frequency of antimicrobial-resistance genes identified in matured cheese samples, by drug group and resistance mechanism. PEN—penicillins; CEF—cephalosporins; MON—monobactams; CAR—carbapenems; TET—tetracyclines; AMG—aminoglycosides; FEN—phenicols; MAC—macrolides; LIN—lincosamides; PLE—pleuromutilins; PEP—peptide antibiotics; FOS—phosphomicin; FLQ—fluoroquinolones; SUL—sulphonamides; DIA—diaminopyrimidines; RIF—rifamicins; NIT—nitroimidazoles; DIS—disinfectants; AMC—aminocoumarins. The numbers in the bar chart represent the exact number of pieces of each resistance mechanism.

Figure 4

The abundances of core-bacteriome (genera present in at least 10% of samples with a minimum abundance of 1%) varied among samples, including raw milk, raw cheese made from it and cheese examined after one month of aging. It is noticeable that following the inoculation of raw cheese, there is a selection for Gram-positive genera, followed by dominance of the Streptococcus genus during maturation. Az űrlap teteje.