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Debate exists about whether agricultural versus medical antibiotic use drives increasing antibiotic resistance (AR) across nature. Both sectors have been inconsistent at antibiotic stewardship, but it is unclear which sector has most influenced acquired AR on broad scales. Using qPCR and soils archived since 1923 at Askov Experimental Station in Denmark, we quantified four broad-spectrum β-lactam AR genes (ARG;  bla  TEM ,  bla  SHV ,  bla  OXA  and  bla  CTX-M ) and class-1 integron genes ( int 1) in soils from manured (M) versus inorganic fertilised (IF) fields. “Total” β-lactam ARG levels were significantly higher in M versus IF in soils post-1940 (paired-t test; p < 0.001). However, dominant individual ARGs varied over time;  bla  TEM  and  bla  SHV  between 1963 and 1974,  bla  OXA  slightly later, and  bla  CTX-M  since 1988. These dates roughly parallel first reporting of these genes in clinical isolates, suggesting ARGs in animal manure and humans are historically interconnected. Archive data further show when non-therapeutic antibiotic use was banned in Denmark,  bla  CTX-M  levels declined in M soils, suggesting accumulated soil ARGs can be reduced by prudent antibiotic stewardship. Conversely,  int 1 levels have continued to increase in M soils since 1990, implying direct manure application to soils should be scrutinized as part of future stewardship programs.

Appearance of β-lactam Resistance Genes in Agricultural Soils and Clinical Isolates over the 20th Century