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

ABSTRACT Whole-genome sequence analysis of a macrolide, lincosamide, streptogramin B (MLSB)-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 MLSB 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.

T rueperella pyogenes, a commensal Gram-positive bacterium of the skin and mucous membranes of animals, can cause suppurative infections in multiple animal species and rarely humans (1). Despite macrolides and lincosamides being antibiotics used as second-line treatment for these infections, their usage may contribute to the selection of antimicrobial resistances. So far, acquired resistances to macrolide, lincosamide, and streptogramin B (MLS B ) antibiotics in T. pyogenes have been associated with the presence of erythromycin ribosome methylase (erm) genes, specifically erm(B) and erm(X), that prevent the binding of the MLS B antibiotics to the 23S rRNA (2)(3)(4). In T. pyogenes, these genes have been reported within mobile genetic elements as either interposed between insertion sequence (IS) elements (5) or integrated in a class 1 integron together with other antimicrobial resistance genes (6).

Detection and characterization of erm(56)
T. pyogenes strain 09KM1269, isolated from an abscess of a dog in 2009 in Switzerland, exhibited constitutive resistance to erythromycin and clindamycin as determined by Clinical and Laboratory Standards Institute (CLSI) criteria (7), suggesting the presence of an MLS B methylase (Erm) ( Table 1). The absence of erm (B) and erm(X) as determined using previously described PCR assays (8) prompted us to search for the underly ing resistance mechanism by whole-genome sequence analysis. Genomic DNA was extracted using MasterPure Complete DNA and RNA Purification Kit (Lucigen, Middleton, WI), sequenced on a PacBio Sequel IIe system (Next-Generation Sequencing Platform, University of Bern), and the resulting reads were de novo assembled using Flye 2.9.1 (9). Analysis of the complete genome using ResFinder 4.1 (Center for Genomic Epidemiology, Denmark) and blast search showed the absence of any so-far known erm gene listed in the Nomenclature Center for MLS B Genes (https://faculty.washington.edu/marilynr/) (10). GenBank query using tblastx (https://blast.ncbi.nlm.nih.gov) and erm(X) as subject sequence (GenBank accession number NC_005206) identified a novel putative erm gene. This gene encoded a 267-aa 23S rRNA methylase and showed the closest relatedness to the Erm(X) determinant of plasmid pAP2 from T. pyogenes with 58% amino acid (aa) and 54% nucleotide (nt) identity ( Fig. 1) and was designated erm(56) (http://faculty.washing ton.edu/marilynr/) (10). Putative -35 (TTGACC) and -10 (TGCTAATGT) promoter sequen ces were identified using BPROM (11) 31 and 11 bp upstream of the putative guanine transcription start located 153 bp upstream of erm(56). This 153 bp upstream region contained the ribosomal binding sites and imperfect inverted repeats capable of folding into stem-loops which may play a role in the translational attenuation of the Erm(56) methylase (12) (Fig. S1).
MIC values of erythromycin (macrolide), clindamycin (lincosamide) (Sigma-Aldrich, St-Louis, MO, USA), pristinamycin IA (streptogramin B), and pristinamycin IIA (streptogramin A) (Molcan Corporation, Richmond Hill, ON, Canada) of E. coli and T. pyogenes strains were determined by broth microdilution using Mueller-Hinton broth supplemented with 5% lysed horse blood and 48 h incubation for Trueperella following CLSI recommendations (7) ( Table 1). When erm(56) was expressed from plasmid pJEM1269 in T. pyogenes 13OD0707, the MIC of MLS B antibiotics increased by more than 256-fold for erythromycin and clindamycin and by 16-fold for pristinamycin IA, while no difference was seen for the streptogramin A pristinamycin IIA as assumed for an Erm methylase. Increased MICs of erythromycin (64-fold) and clindamycin (8-fold) were also measured for AG100A containing pJEM1269, but no conclusion could be drawn for pristinamycin IA, for which the MIC was already high for the recipient strain and remained unchanged in the presence of erm(56) ( Table 1).

Genomic location of erm(56) and detection in additional bacteria
The erm(56) gene of T. pyogenes 09KM1269 was preceded by a hypothetical protein (hp) and integrated into the chromosome between two identical IS6100 elements located in the same orientation, each flanked by 14 bp inverted repeats (IR-L, GGCTCTGTTGCAAA; IR-R, TTTGCAACAGAGCC). The IS6100-hp-erm(56)-IS6100 element was situated next to a class 1 integron containing the sulfonamide resistance gene sul1, which was also delimited by a copy of IS6100 (Fig. 2). GenBank searches also identified erm(56) associ ated with IS6100 in T. pyogenes strain TP1 isolated from bovine lung in China (GenBank Observation mSphere accession number CP033902) and in Rothia nasimurium strain E1706032 isolated from duck brain in China (GenBank accession number CP056080) (Fig. 2). In T. pyogenes TP1, the IS6100-hp-erm(56)-IS6100 element was found on a large 27.4 kb fragment which was related to the Lactobacillus phage PLE2 (GenBank accession number NC031036) using PHASTER (17). The sequence of the IS6100 situated upstream of erm(56) in T. pyogenes TP1 had a cytosine deletion at position 164 leading to a frameshift and an early stop codon in the IS6100 transposase. In R. nasimurium E1706032, erm(56) was only preceded by one IS6100 and integrated next to tet(Z) and a class 1 integron containing aac(6')-Ib3, qacEΔ1, and sul1; this resistance element was delimited by two copies of IS6100 (Fig. 2). Although a circular conformation containing one copy of IS6100 was detected by PCR using primers pointing outward from erm(56) [erm56(159)-F, 5′-GGGACGATCTCTCAC AGCTG and hp-erm56(45)-R, 5′-GAGAACTCGACCCAAACGAGGATCA; annealing temper ature, 60°C; extension time, 2 min] and subsequent Sanger sequencing (Fig. 2), the erm(56) gene could not be transferred by either filter mating (19)   Although erm(56) could not be transferred in vitro, its detection on different elements in unrelated bacteria suggests that it has a potential for broader dissemination. Given its detection in bacteria from different animal sources and geographical origins, it is likely that it has been independently selected by the use of antibiotics in animals.

DATA AVAILABILITY
The nucleotide sequence of erm(56) of T. pyogenes 09KM1269 was deposited into the GenBank/ENA/DDBJ databases under accession number OQ326498. The complete genome sequences of T. pyogenes 09KM1269, 13OD0707, and 13KM1326 were deposited into the GenBank under accession numbers CP123393, CP123403, and CP123400.

ADDITIONAL FILES
The following material is available online.

Supplemental Material
Figure S1 (mSphere00239-23 S0001.pdf). Schematic minimum free energy (MFE) structure encoding base pair probabilities and predicting secondary structure of the 153-bp upstream DNA sequence upstream of the start codon of erm(56).