The tva(A) Gene from Brachyspira hyodysenteriae Confers Decreased Susceptibility to Pleuromutilins and Streptogramin A in Escherichia coli

Brachyspira hyodysenteriae is the etiological agent of swine dysentery, a bacterial disease which is controlled by the use of pleuromutilins (1). However, pleuromutilin resistance in B. hyodysenteriae has been observed in several countries in association with single point mutations in the 23S rRNA, in the ribosomal proteins L2 and L3, and in the elongation factor G FusA (2–5). In addition, resistance to pleuromutilins in B. hyodysenteriae has been suggested to be also associated with the presence of the so-called tiamulin-valnemulin antibiotic resistance gene tva(A) (5). The 1,518-bp tva(A) gene is located on the chromosome of many pleuromutilin-resistant B. hyodysenteriae strains, and it encodes a predicted ATP-binding cassette subfamily F (ABC-F) ribosomal protection protein (5). Whole-genome comparative analysis suggested that reduced susceptibility to pleuromutilins is correlated with the carriage of tva(A), which, in combination with single point mutations, leads to higher-level resistance in B. hyodysenteriae strains (5). Nevertheless, a direct association of the tva(A) gene with pleuromutilin resistance has not yet been demonstrated by recombinant gene expression. Due to the lack of simple genetic tools in B. hyodysenteriae, we determined whether tva(A) confers decreased susceptibility to ribosome-targeting antimicrobials in the highly antibiotic-susceptible Escherichia coli strain AG100A (ΔacrAB::Tn903 Kan^r) (6) and Staphylococcus aureus strain RN4220 (7).

The tva(A) gene was cloned from the chromosome of the pleuromutilin-resistant B. hyodysenteriae strain BH718 (8) into the E. coli-S. aureus shuttle vector pBUS1-HC (9). It was placed under the control of either its native promoter (pAGS2) or the strong constitutive S. aureus type 1 capsule gene 1A promoter P_cap (pAGS2-P_cap) that has been shown to promote gene expression in both E. coli and S. aureus (9). For pAGS2 construction, the tva(A) gene and its native promoter (position 821793 to 823659 in GenBank accession number CP019600.1) were amplified from B. hyodysenteriae BH718 DNA (8) by PCR using the Phusion Hot Start II High-Fidelity DNA polymerase (Thermo Scientific) and primers tva(A)-Kpn-I-F and tva(A)-XhoI-R (Table 1) and inserted into KpnI/XhoI sites of pBUS1-HC (9). Plasmid pAGS2-P_cap was generated through PCR-based mutagenesis of pAGS2 using PrimeStar GXL DNA polymerase (TaKaRa Bio) and primers Pcap-tva(A)-F1 and Pcap-tva(A)-R1 that excluded amplification of the tva(A) promoter region and contained the P_cap sequence in their partially overlapping 5′ overhangs (Table 1). The parental pAGS2 template was digested using DpnI (Roche Diagnostics). Both constructs pAGS2 and pAGS2-P_cap were obtained in E. coli DH5α and subsequently transformed into E. coli AG100A by heat shock (10) and into S. aureus RN4220 by electroporation (11). Transformants were selected on Luria-Bertani and NYE (1% casein hydrolysate, 0.5% yeast extract, 0.5% sodium chloride) agar plates supplemented with 10 μg/ml of tetracycline (10, 11). Transcription of the tva(A) gene from pAGS2 and pAGS2-P_cap in the transformed E. coli AG100A and S. aureus RN4220 was analyzed by reverse transcriptase PCR (RT-PCR). RNA was extracted using the RNeasy mini kit (Qiagen), treated with RQ1 (RNA qualified 1) RNase-free DNase (Promega), and converted to cDNA using Moloney murine leukemia virus reverse transcriptase (Roche Diagnostics) and tva(A)-RT-F1 primer (Table 1). A second-round PCR amplification was performed using Taq polymerase (FIREPol DNA polymerase; Solis BioDyne) and primers tva(A)-RT-F1 and tva(A)-RT-R1, generating a specific fragment of 130 bp (Fig. 1). Samples which were not incubated with the reverse transcriptase were included as negative controls to ensure absence of genomic DNA. Transcription of the tva(A) gene was observed in both E. coli AG100A and S. aureus RN4220 strains with plasmid pAGS2 as well as with pAGS2-P_cap (Fig. 1).

Antimicrobial susceptibility of E. coli AG100A and S. aureus RN4220 for ribosome-targeting antibiotics of the pleuromutilin, macrolide, lincosamide, streptogramin A and B, tetracycline, phenicol, aminoglycoside, and oxazolidinone classes was assessed by broth dilution in Müller-Hinton according to the guidelines of the Clinical and Laboratory Standard Institute in 2018 (12) using Sensititre EUST plates (Thermo Fisher Scientific). The MICs of tiamulin, valnemulin, and lincomycin were also determined using VetMIC Brachy panels (National Veterinary Institute of Sweden) and 500 μl of E. coli or S. aureus bacterial suspensions (1× 10⁵ to 2 × 10⁵ CFU/ml). The concentration range of each of the tested antibiotic is indicated in Table S1 in the supplemental material. Additionally, MICs of the streptogramins virginiamycin M1, pristinamycin IIA, and pristinamycin IA for E. coli and S. aureus were determined in 96-well microplates using serial 2-fold dilution method (12). MICs of pleuromutilins for B. hyodysenteriae strains were determined using the VetMIC Brachy panels according to the manufacturer’s instructions (13), and those of streptogramins were obtained by broth dilution method in brain heart infusion broth supplemented with 10% fetal bovine serum using 24-well plates and 1 ml per well. B. hyodysenteriae suspensions were prepared as described previously (13), and MIC plates were incubated at 37°C for 4 days under anaerobic conditions (14). S. aureus ATCC 29213 and B. hyodysenteriae CCUG 46668^T were used as quality control strains of antimicrobial susceptibility testing. Only the MICs of the antibiotics that were affected by expression of the tva(A) gene are presented in Table 2. Reduced susceptibility to the pleuromutilins tiamulin and valnemulin and to streptogramin A virginiamycin M1 and pristinamycin IIA was observed for E. coli AG100A expressing the tva(A) from pAGS2 as well as from pAGS2-P_cap (Table 2). The measured MIC values were higher with plasmid pAGS2 than pAGS2-P_cap. Compared to E. coli AG100A cells containing no plasmid or the empty plasmid pBUS1-HC, E. coli AG100A cells containing pAGS2 showed at least a 2-fold increase in MIC for tiamulin (from 0.5 to 2 μg/ml), a 1-fold increase for valnemulin (from 0.5 to 1 μg/ml), a 2-fold increase for virginiamycin M1 (from 2 to 8 μg/ml), and a 3-fold increase for pristinamycin IIA (from 2 to 16 μg/ml). This phenotype was also seen (1-fold increase) for tiamulin, virginiamycin M1, and pristinamycin IIA in S. aureus when the tva(A) gene was expressed from pAGS2 but not from pAGS2-P_cap (Table 2). Expression of the tva(A) gene in E. coli and S. aureus seems not to affect streptogramin B activity (Table 2), indicating that strains carrying tva(A) should remain susceptible to the synergistic action of streptogramin A and B.

We experimentally demonstrated that expression of the tva(A) gene of B. hyodysenteriae leads to an increase in the MIC for pleuromutilins and for the structurally unrelated streptogramin A, which have overlapping binding sites on the 50S ribosomal subunit (15), in E. coli AG100A and, to a lesser extent, in S. aureus. Cross-resistance to pleuromutilins (P) and streptogramin A (S_A), in combination with lincosamides (L) (LS_AP phenotype), has been described in Gram-positive bacterial strains associated with the presence of ABC-F proteins of the vga, lsa, and sal gene families (16). These genes were reported to only be functional in Gram-positive bacteria, and their expressions were not found to mediate resistance in E. coli (16). In contrast, the tva(A) gene confers cross-resistance to pleuromutilins and streptogramin A in E. coli, and its presence has a low effect in S. aureus. Increased MICs of pleuromutilins and streptogramin A were also observed in B. hyodysenteriae BH718 compared to that in strain CCUG 46668^T, likely due to the presence of tva(A) in strain BH718 (Table 2). B. hyodysenteriae BH718 also exhibited an increased MIC to the streptogramin B pristinamycin IA, which may be associated with an A2058T gene mutation in the 23S rRNA of B. hyodysenteriae BH718 (8). Mutations at position 2058 in the 23S rRNA gene are known to confer resistance to macrolide-lincosamide-streptogramin B, but not to streptogramin A, in many bacteria, including Brachyspira (17, 18). Selection of B. hyodysenteriae containing both tva(A) and 23S rRNA mutations could be triggered by the use of virginiamycin, a mixture of virginiamycin M1 (streptogramin A) and virginiamycin S1 (streptogramin B), which is still used as a growth promoter in pigs in some countries around the world (19). The presence of these combined resistance mechanisms in B. hyodysenteriae would lead to coresistance to macrolides, lincosamides, streptogramins, and pleuromutilins, thus seriously limiting therapeutic options for treatment in case of swine dysentery.

This study showed that ABC-F ribosomal protection proteins can also alter antibiotic susceptibility of Gram-negative bacteria. It provides evidence that the tva(A) gene from B. hyodysenteriae specifies for pleuromutilin resistance as well as for streptogramin A as demonstrated by heterologous gene expression in E. coli.

We thank Silvio De Luca (Istituto Zooprofilattico Sperimentale dell’Umbria e delle Marche, Perugia, Umbria, Italy) for providing the B. hyodysenteriae BH718 strain and Fabien Labroussaa and Pamela Nicholson (Institute of Veterinary Bacteriology, University of Bern, Bern, Switzerland) for advice.

This study was financed by grant number 1.16.04 (antimicrobial susceptibility situation and evaluation of the sanitation of Brachyspira hyodysenteriae in Swiss pig herds) from the Swiss Federal Food Safety and Veterinary Office.