Novel macrolide-lincosamide-streptogramin B resistance gene erm(56) in Trueperella pyogenes

Abstract

Whole-genome sequence analysis of a macrolide, lincosamide, streptogramin B (MLS_B)-resistant Trueperella pyogenes from a dog revealed a new 23S ribosomal RNA methylase gene erm(56). Expression of the cloned erm(56) confers resistance to MLS_B in T. pyogenes and Escherichia coli. The erm(56) gene was flanked by two IS6100 integrated on the chromosome next to a sul1-containing class 1 integron. GenBank query revealed additional erm(56)-containing elements in another T. pyogenes and in Rothia nasimurium from livestock. IMPORTANCE A novel 23S ribosomal RNA methylase gene erm(56) flanked by insertion sequence IS6100 was identified in a Trueperella pyogenes isolated from the abscess of a dog and was also present in another T. pyogenes and in Rothia nasimurium from livestock. It was shown to confer resistance to macrolide, lincosamide, streptogramin B antibiotics in T. pyogenes and E. coli, indicating functionality in both Gram-positive and Gram-negative bacteria. The detection of erm(56) on different elements in unrelated bacteria from different animal sources and geographical origins suggests that it has been independently acquired and likely selected by the use of antibiotics in animals.

Keywords: DNA sequencing; gene expression; macrolides-lincosamides-streptogramin B; mechanisms of resistance.

Fig 1

Relationship tree of all known Erm methylases, including the novel Erm(56) detected in T. pyogenes 09KM1269. Amino acid (aa) and nucleotide (nt) identity was obtained by sequence alignment using Clustal Omega 1.2.2 (Geneious Prime 2022.2.2, Biomatters Ltd., Auckland, New Zealand). The tree was constructed using BioNumerics 8.1.1 (BioMérieux, Marcy-l'Étoile, France) and the following settings: standard algorithm for pairwise alignment; open gap penalty, 100%; unit gap penalty, 0%; and unweighted pair group method using average linkages. The protein sequences are indicated by their GenBank accession numbers. The Erm(I) amino acid (aa) sequence is not available (NA) in the GenBank and was obtained from its original publication (13).

Fig 2

Schematic gene map showing the erm(56)-containing elements and flanking region of T. pyogenes 09KM1269 (GenBank accession number CP123393) compared to the chromosomal region of erm(56)-negative strain 13OD0707 (GenBank accession number CP123403), as well as the integration of erm(56) in T. pyogenes TP1 (GenBank accession number CP033902) and R. nasimurium E1706032 (GenBank accession number CP056080). Black and dark-gray areas represent regions showing 100% and 85% similarity at nucleotide level, respectively. Arrows represent open reading frames (ORFs). The ORF of erm(56) is indicated by a red arrow, and the erm(56)-preceding ORF (hypothetical protein gene, hp) is in purple. ORFs of other antibiotic resistance genes are shown in orange: sul1, dihydropteroate synthase gene for sulfonamide resistance; aac(6')-Ib3, aminoglycoside N-acetyltransferase gene; qacEΔ1, quaternary ammonium compound efflux transporter gene; tet(Z), tetracycline efflux transporter gene. Other ORFs of hypothetical proteins are represented by gray arrows. ORFs of transposase genes associated with IS and integrase (intI1) are indicated with green arrows. Asterisk (*) within IS6100 of T. pyogenes TP1 indicates a cytosine deletion at position 164 leading to a frameshift of the transposase of IS6100. ORFs of phage protein genes are indicated in dark blue. The 3′-end truncated part (gnat′) and the 5′-end truncated part (′gnat) of the GNAT N-acetyltransferase gene are indicated in light blue. ORFs of core genome genes are indicated as dark gray arrows. Inverted repeats of the IS6100 elements are indicated by yellow arrow heads (IR-L, GGCTCTGTTGCAAA; IR-R, TTTGCAACAGAGCC). Circular conformation of the IS6100-erm(56) element obtained by PCR using primers erm56(159)-F and hp-erm56(45)-R (indicated by small arrows 1 and 2) is represented by a circle containing the respective ORFs. The figure was generated using Clinker (18) and Adobe Illustrator.