Streptococcus gallolyticus subsp. pasteurianus is an under-recognized pathogen and zoonotic agent causing opportunistic infections in humans. Despite increasing recognition of this subspecies as a cause for human infectious diseases, limited information is known about its antibiotic resistance mechanism. In this study, we aim to identify the molecular mechanism underlying the high macrolide resistance of six S. gallolyticus subsp. pasteurianus isolates from dead ducklings collected in several natural outbreaks in China during 2010–2013. All isolates exhibited multi-drug resistance including high macrolide resistance (MIC ≥ 1024 mg/L for erythromycin, and 512 mg/L for clarithromycin). Efflux-encoding mefA and mefE genes were not detectable in these isolates. The presence of 23S rRNA mutations in specific isolates did not significantly change macrolide MICs. No nucleotide substitutions were found in genes encoding ribosomal proteins L4 or L22. The ermB and ermT genes were found in the genomes of all isolates. These two genes were acquired independently in one highly virulent isolate AL101002, and clustered with Tn916 and IS1216, respectively. The expression of both ermB and ermT in all isolates was erythromycin inducible and yielded comparable macrolide MICs in all six isolates. Taken together, inducible expression of both ermB and ermT conferred high macrolide resistance in these S. gallolyticus subsp. pasterianus isolates. Our findings reveal new macrolide resistance features in S. gallolyticus subsp. pasteurianus by both ermB and ermT.

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