Characterization of poxtA, a novel phenicol–oxazolidinone–tetracycline resistance gene from an MRSA of clinical origin

Characterization of poxtA, a novel phenicol–oxazolidinone–tetracycline resistance gene from... Abstract Objectives To characterize a novel phenicol–oxazolidinone–tetracycline resistance gene, named poxtA, identified in a previously described MRSA strain that was highly resistant to linezolid and also carried the cfr gene. Methods The poxtA gene was identified by bioinformatic analysis of the whole genome sequence of Staphylococcus aureus AOUC-0915. The poxtA gene was cloned in a shuttle plasmid vector and expressed in Escherichia coli, S. aureus and Enterococcus faecalis to investigate the protein function. Comparative sequence analyses at the protein and genetic levels were carried out using standard procedures. Results The poxtA gene encodes a protein that is 32% identical to OptrA and exhibits structural features typical of the F lineage of the ATP-binding cassette (ABC) protein superfamily that cause antibiotic resistance by ribosomal protection. Expression of poxtA in E. coli, S. aureus and E. faecalis was able to decrease susceptibility to phenicols, oxazolidinones and tetracyclines. A database search identified the presence of poxtA in E. faecalis, Enterococcus faecium and Pediococcus acidilactici strains, mostly of animal origin, and revealed the presence of poxtA homologues in the genomes of some Clostridiales. Analysis of the genetic context revealed that poxtA was located in a composite transposon-like structure containing two IS1216 elements. Conclusions A novel resistance gene, named poxtA, encoding a protein of the antibiotic resistance (ARE) ABC-F lineage, was identified in the genome of an MRSA of clinical origin. PoxtA can confer decreased susceptibility to phenicols, oxazolidinones and tetracyclines and is associated with a putative mobile element that could contribute to its horizontal dissemination. Introduction The ATP-binding cassette (ABC) superfamily includes a large, diverse and ubiquitous group of proteins detected in all kingdoms of life. Some of these proteins are able to confer resistance to various classes of antibiotics in prokaryotes. The ABC proteins associated with antibiotic resistance (ARE) belong to the F lineage of the ABC superfamily (ARE ABC-F) and include single polypeptides containing two conserved nucleotide binding domains (NBDs) separated by a linker of ∼80 amino acids of variable composition, with no transmembrane domains (TMDs).1,2 The mechanism by which the ARE ABC-F proteins mediate resistance has been debated for a long time and attributed to either efflux or ribosomal protection.3,4 Recently, the resistance mechanism of ARE ABC-F proteins has been clarified and shown to be mediated by ribosomal protection.5 The ARE ABC-F proteins can mediate resistance to several different classes of anti-ribosomal antibiotics, including tetracyclines, macrolides, ketolides, lincosamides, phenicols, pleuromutilins, oxazolidinones and streptogramins.5 Many of them can actually mediate resistance to multiple antibiotic classes, e.g. Msr(A), conferring resistance to macrolides and group B streptogramins, or the recently described OptrA protein, conferring resistance to phenicols and oxazolidinones.6 The ARE ABC-F proteins are often encoded by genes carried on mobile genetic elements, which can disseminate these resistance determinants horizontally among bacterial pathogens, although some are encoded by resident genes in a number of antibiotic-producing Streptomyces spp.6–8 We recently reported an MRSA strain that exhibited high-level resistance to linezolid (32 mg/L).9 The strain had been isolated from a cystic fibrosis patient after linezolid treatment and was found to carry at least two different linezolid resistance mechanisms including a G152D substitution in the L3 ribosomal protein9 and the cfr gene, an acquired gene encoding a ribosomal rRNA methylase, which can mediate resistance to oxazolidinones and several other anti-ribosomal drugs.9–13 In this study, we describe the identification and characterization of a novel acquired resistance gene, named poxtA, found in the same MRSA strain. The poxtA gene encodes a protein of the ARE ABC-F family, which is distantly related to OptrA and able to confer reduced susceptibility to phenicols, oxazolidinones and tetracyclines. Materials and methods Bacterial strains AOUC-0915 is an ST5-MRSA-II strain isolated in September 2015 from the respiratory tract of a cystic fibrosis patient at Florence Careggi University Hospital (Florence, Italy).9 Antimicrobial susceptibility and resistome of this strain, in terms of known resistance genes, have previously been reported.9,Escherichia coli Mach1™ T1R (Thermo Fisher Scientific, Waltham, MA, USA), Staphylococcus aureus RN422014 and Enterococcus faecalis JH2-215 were used as hosts for cloning of the poxtA gene. Recombinant DNA methodology The poxtA gene, with its flanking regions, was cloned in the pMU1328 vector16 using the PIPE (Polymerase Incomplete Primer Extension) cloning method17 and E. coli Mach1™ T1R cells, to obtain the recombinant plasmid pMU-poxtA. Cloning was designed to replace the chloramphenicol acetyl transferase (cat) gene of the plasmid with the poxtA locus, using primers targeting the amplification of a region spanning from 863 bp upstream of the poxtA start codon to 60 bp downstream of the stop codon (Figure S1, available as Supplementary data at JAC Online). A plasmid derived from the pMU1328 vector, named pMU-E, and carrying a deletion of the cat gene, was also constructed using the PIPE cloning method and specific primers (Figure S1) to be used as a control. E. coli transformants, obtained by a heat-shock method,18 were selected on Mueller–Hinton agar (MHA) supplemented with erythromycin (100 mg/L). The authenticity of the cloned DNA fragment in pMU-poxtA and of the deletion in plasmid pMU-E were confirmed by Sanger sequencing both strands of plasmid DNA extracted from the Mach1™ T1R transformants. The recombinant plasmids were introduced into S. aureus RN4220 and E. faecalis JH2-2 by electrotransformation (2.5 kV, 200 Ω, 25 μF). Transformants were selected on MHA containing erythromycin (2 mg/L). Antimicrobial susceptibility testing MICs were determined by broth microdilution according to the M7-A10 standard of the CLSI,19 except for that of tedizolid, which was measured by MIC Test Strip (Liofilchem s.r.l., Roseto degli Abruzzi, Italy). Antimicrobial susceptibility testing was carried out in triplicate. The results were interpreted according to the EUCAST clinical breakpoint tables.20 Reference strains S. aureus ATCC 29213, E. faecalis ATCC 29212 and E. coli ATCC 25922 were used as quality controls for antimicrobial susceptibility testing. Bioinformatics and sequence analysis The presence of TMDs and of a signal peptide in PoxtA was predicted using the TMHMM Server v. 2.0 web service21 and the SignalP 4.1 server,22 respectively. The search for poxtA homologues was carried out using the BLAST web server, selecting alternatively the wgs or nr databases (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Amino acid sequence alignments of PoxtA with other ARE ABC-F proteins and the phylogenetic tree, generated using the maximum likelihood method with 1000 bootstrap replicates, were generated with the MEGA 6.0.6 software package.23 The genetic context of poxtA in S. aureus AOUC-0915 was investigated by PCR mapping and Sanger sequencing experiments on the regions unresolved by WGS assembly.9 A graphical comparison of the genetic context of poxtA homologues was generated using EasyFig.24 The nucleotide sequence of the poxtA gene and flanking regions has been deposited in the GenBank database under the accession number MF095097. Results Identification of a new ARE ABC-F resistance determinant in S. aureus AOUC-0915 Analysis of the genome sequence of S. aureus AOUC-0915 revealed the presence of an ORF encoding a protein of 542 amino acids that exhibited similarity (32% identical amino acid residues) with OptrA over most of its length (coverage of 95%) (Figure 1). This protein showed conserved features with members of the ARE ABC-F family,5,25 including two NBDs and the lack of detectable TMDs. Moreover, no leader peptide was detected by the two bioinformatics tools used for this analysis (Figure 1). Figure 1. View largeDownload slide Alignment of PoxtA and homologous proteins. The conserved regions of ABC-F proteins, including the Walker A and B motifs, the signature and the switch motifs, involved in ATP binding and hydrolysis are indicated. Residues conserved in all sequences are shown in bold. Protein accession numbers are as follows: Coprobacillus sp. CAG:605, CCZ88872.1; C. clostridioforme CAG:132, CDB61438.1; Clostridium sp. C105KSO14, CUX74470.1; C. bolteae 90B8, ENZ34498.1; Clostridiales bacterium 1_7_47FAA, EEQ60488.1; Clostridiales bacterium VE202-28, WP_025484560.1; Azospirillum sp. 51_20, OLA82622.1; B. producta ATCC 27340, WP_026255743.1; H. hathewayi 2789STDY5834916, WP_055650089.1; E. faecium 9-F-6, OZN12776.1; Sal(A), AGN74946.1; and OptrA, AKA86814.1. Figure 1. View largeDownload slide Alignment of PoxtA and homologous proteins. The conserved regions of ABC-F proteins, including the Walker A and B motifs, the signature and the switch motifs, involved in ATP binding and hydrolysis are indicated. Residues conserved in all sequences are shown in bold. Protein accession numbers are as follows: Coprobacillus sp. CAG:605, CCZ88872.1; C. clostridioforme CAG:132, CDB61438.1; Clostridium sp. C105KSO14, CUX74470.1; C. bolteae 90B8, ENZ34498.1; Clostridiales bacterium 1_7_47FAA, EEQ60488.1; Clostridiales bacterium VE202-28, WP_025484560.1; Azospirillum sp. 51_20, OLA82622.1; B. producta ATCC 27340, WP_026255743.1; H. hathewayi 2789STDY5834916, WP_055650089.1; E. faecium 9-F-6, OZN12776.1; Sal(A), AGN74946.1; and OptrA, AKA86814.1. To confirm its role in antibiotic resistance, the gene, with some flanking regions, was cloned in the pMU1328 shuttle plasmid vector, which can replicate in E. coli, in S. aureus and in E. faecalis. The antimicrobial susceptibilities of the E. coli, S. aureus and E. faecalis strains carrying the cloned gene were investigated and compared with those of strains carrying the empty pMU-E vector. The expression of the gene in S. aureus and E. faecalis demonstrated that it was associated with decreased susceptibility to phenicols (chloramphenicol and florfenicol), oxazolidinones (linezolid and tedizolid) and tetracyclines (tetracycline and doxycycline). Expression of the gene in E. coli was associated with decreased susceptibility to the same antibiotics (tedizolid was not tested) and also to tigecycline (Table 1). No differences in susceptibility to amikacin, gentamicin, ampicillin, ciprofloxacin, rifampicin and trimethoprim/sulfamethoxazole were detected following expression of the gene in the E. coli and S. aureus hosts (data not shown). Given the antibiotic resistance phenotype, the new resistance gene was designated poxtA (after phenicols, oxazolidinones and tetracyclines). Table 1. Antimicrobial susceptibilities of S. aureus, E. faecalis and E. coli strains carrying a cloned copy of the poxtA gene Antibiotic MIC (mg/L) S. aureusa E. faecalisb E. colic RN4220 (pMU-poxtA) RN4220 (pMU-E) JH2-2 (pMU-poxtA) JH2-2 (pMU-E) Mach1™ T1R (pMU-poxtA) Mach1™ T1R (pMU-E) Linezolid 2 1 4 1 1024 256 Tedizolid 0.5 0.25 0.5 0.25 — — Chloramphenicol 8 4 8 4 32 8 Florfenicol 16 2 16 2 128 8 Tigecycline 0.25 0.25 0.25 0.25 1 0.5 Tetracycline 0.25 0.125 0.25 0.125 2 0.5 Doxycycline 0.25 0.125 0.125 ≤0.06 8 2 Antibiotic MIC (mg/L) S. aureusa E. faecalisb E. colic RN4220 (pMU-poxtA) RN4220 (pMU-E) JH2-2 (pMU-poxtA) JH2-2 (pMU-E) Mach1™ T1R (pMU-poxtA) Mach1™ T1R (pMU-E) Linezolid 2 1 4 1 1024 256 Tedizolid 0.5 0.25 0.5 0.25 — — Chloramphenicol 8 4 8 4 32 8 Florfenicol 16 2 16 2 128 8 Tigecycline 0.25 0.25 0.25 0.25 1 0.5 Tetracycline 0.25 0.125 0.25 0.125 2 0.5 Doxycycline 0.25 0.125 0.125 ≤0.06 8 2 Susceptibilities of the same strains carrying the empty plasmid vector are also reported for comparison. The MIC measurements were performed in triplicate and results were fully reproducible, with no discrepancies. a The MICs were evaluated in medium supplemented with 50 mg/L erythromycin for plasmid maintenance. b The MICs were evaluated in medium supplemented with 2 mg/L erythromycin for plasmid maintenance. c The MICs were evaluated in medium supplemented with 100 mg/L erythromycin for plasmid maintenance. Table 1. Antimicrobial susceptibilities of S. aureus, E. faecalis and E. coli strains carrying a cloned copy of the poxtA gene Antibiotic MIC (mg/L) S. aureusa E. faecalisb E. colic RN4220 (pMU-poxtA) RN4220 (pMU-E) JH2-2 (pMU-poxtA) JH2-2 (pMU-E) Mach1™ T1R (pMU-poxtA) Mach1™ T1R (pMU-E) Linezolid 2 1 4 1 1024 256 Tedizolid 0.5 0.25 0.5 0.25 — — Chloramphenicol 8 4 8 4 32 8 Florfenicol 16 2 16 2 128 8 Tigecycline 0.25 0.25 0.25 0.25 1 0.5 Tetracycline 0.25 0.125 0.25 0.125 2 0.5 Doxycycline 0.25 0.125 0.125 ≤0.06 8 2 Antibiotic MIC (mg/L) S. aureusa E. faecalisb E. colic RN4220 (pMU-poxtA) RN4220 (pMU-E) JH2-2 (pMU-poxtA) JH2-2 (pMU-E) Mach1™ T1R (pMU-poxtA) Mach1™ T1R (pMU-E) Linezolid 2 1 4 1 1024 256 Tedizolid 0.5 0.25 0.5 0.25 — — Chloramphenicol 8 4 8 4 32 8 Florfenicol 16 2 16 2 128 8 Tigecycline 0.25 0.25 0.25 0.25 1 0.5 Tetracycline 0.25 0.125 0.25 0.125 2 0.5 Doxycycline 0.25 0.125 0.125 ≤0.06 8 2 Susceptibilities of the same strains carrying the empty plasmid vector are also reported for comparison. The MIC measurements were performed in triplicate and results were fully reproducible, with no discrepancies. a The MICs were evaluated in medium supplemented with 50 mg/L erythromycin for plasmid maintenance. b The MICs were evaluated in medium supplemented with 2 mg/L erythromycin for plasmid maintenance. c The MICs were evaluated in medium supplemented with 100 mg/L erythromycin for plasmid maintenance. Relatedness of PoxtA with other ARE ABC-F proteins Comparative sequence analysis of PoxtA with other members of the ARE ABC-F family confirmed that PoxtA is a new member of this protein family. The closest homologues were putative ARE ABC-F proteins encoded by the genomes of Enterococcus faecium 9-F-6 and Azospirillum sp. 51_20, (73% and 70% amino acid identity, respectively). Other homologues (66%–68% amino acid identity) were detected in the genomes of some Clostridiales, including Clostridium bolteae, Clostridium clostridioforme, Hungatella hathewayi (formerly Clostridium hathewayi) and Blautia producta, and of Coprobacillus sp. CAG:605 (Figure 1). Among known ARE ABC-F proteins, PoxtA belongs to a sublineage that also includes OptrA and Sal(A)26 (Figure 2). Figure 2. View largeDownload slide Phylogenetic tree of ARE ABC-F proteins constructed using the maximum likelihood method. The tree represents the consensus obtained after 1000 replicates and bootstrap values are indicated next to the branches. Amino acid sequences of the ARE ABC-F proteins were extracted from the ‘tetracycline and MLS nomenclature’ web site (https://faculty.washington.edu/marilynr/ermweb2.pdf), except for Lmo0919 (CAC98997.1), Var(M) (BAA96297.1), Vml(R) (WP_003234144.1) and LmrC (CAA55774.1). Antibiotics affected by the different proteins are indicated and the species in which the various ARE ABC-F have been described for the first time are also indicated.  SA, streptogramin A; SB, streptogramin B; P, pleuromutilins; L, lincosamides; M, macrolides; Ph, phenicols; O, oxazolidinones; T, tetracyclines. Antibiotic-producing species are indicated by an asterisk. Figure 2. View largeDownload slide Phylogenetic tree of ARE ABC-F proteins constructed using the maximum likelihood method. The tree represents the consensus obtained after 1000 replicates and bootstrap values are indicated next to the branches. Amino acid sequences of the ARE ABC-F proteins were extracted from the ‘tetracycline and MLS nomenclature’ web site (https://faculty.washington.edu/marilynr/ermweb2.pdf), except for Lmo0919 (CAC98997.1), Var(M) (BAA96297.1), Vml(R) (WP_003234144.1) and LmrC (CAA55774.1). Antibiotics affected by the different proteins are indicated and the species in which the various ARE ABC-F have been described for the first time are also indicated.  SA, streptogramin A; SB, streptogramin B; P, pleuromutilins; L, lincosamides; M, macrolides; Ph, phenicols; O, oxazolidinones; T, tetracyclines. Antibiotic-producing species are indicated by an asterisk. Detection of the poxtA gene in other strains and genetic context of poxtA A BLAST search in sequence databases using poxtA as a query revealed the presence of identical genes in a number of Gram-positive strains including E. faecalis 599 (accession no. EJU87034.1), E. faecalis 12 (accession no. KII46686.1), E. faecium P36 (accession no. KP834591.1) and Pediococcus acidilactici BCC1 (accession no. CP018763.1). This finding confirmed the mobile nature of poxtA, which is apparently able to spread among different Gram-positive cocci. Interestingly, these strains were of animal origin (except for E. faecalis 599, whose origin was not reported). The genetic context of poxtA in S. aureus AOUC-0915 was further investigated by PCR mapping and sequencing experiments. This analysis revealed that poxtA was flanked by two IS1216-like ISs (hereinafter referred to as IS1216_U and IS1216_D) in the same orientation (Figure 3a), suggesting that the resistance gene had been mobilized via a composite transposon constituted by the two IS1216-like elements. The IRL of the IS1216_D that is present at the 3′-end of the poxtA gene is actually part of the coding sequence (CDS) constituting the final 21 nucleotides of the CDS and including the poxtA stop codon (Figure 3b). This condition is conserved also in all the available sequences containing poxtA (e.g. in P. acidilactici BCC1 and in E. faecium P36) (Figure 3a), suggesting that the partial deletion of the poxtA gene may be an ancestral event that originated from the mobilization of this resistance gene by the IS1216-like elements. As a consequence a hybrid gene results, encoding a protein with a modified C-terminus. The IRR of the IS1216_D was disrupted by the insertion of an IS1252-like IS that apparently generated a duplicated region of three nucleotides abutting the left and right boundaries of the element. Remnants of the IS1216_D IRR are present downstream of the IS1252-like element, suggesting that the insertion of this IS was likely an independent event following poxtA mobilization (Figure 3b). Figure 3. View largeDownload slide Genetic context of the poxtA gene. (a) Comparison of the genetic context of poxtA in P. acidilactici BCC1 (region 1 547 068–1 552 271 of accession number CP018763.1) and in E. faecium P36 (region 1–3421 of accession number KP834591.1). Regions having >99% nucleotide identity are connected by grey zones. ISs are boxed and transposase-encoding genes are indicated by striped arrows, while the poxtA gene is represented by a dotted arrow. IS1216-like and IS1252-like IRs are indicated by black and grey triangles, respectively. In S. aureus AOUC-0915, the remnant of IS1216_D IRR is indicated by a black triangle downstream of the insertion point of the IS1252-like element. The region of overlap between the IS1216_D IRL and the poxtA CDS is indicated by an asterisk. (b) Nucleotide sequence showing the insertion points of IS1216_D and of the IS1252-like element. The regions encoding the IS1216_D and the IS1252-like transposases are indicated by arrows and are shown only partially; the numbers of amino acids that are not shown are indicated. The IS1252-like element is boxed and its IRs are underlined. An asterisk indicates the three-nucleotide duplication originated by the IS1252-like insertion event. IS1216_D IRs are highlighted in grey and the region of overlap between the IS1216_D IRL and the poxtA CDS is shown in capital letters. The C-terminus PoxtA sequence is shown under the nucleotide sequence. Figure 3. View largeDownload slide Genetic context of the poxtA gene. (a) Comparison of the genetic context of poxtA in P. acidilactici BCC1 (region 1 547 068–1 552 271 of accession number CP018763.1) and in E. faecium P36 (region 1–3421 of accession number KP834591.1). Regions having >99% nucleotide identity are connected by grey zones. ISs are boxed and transposase-encoding genes are indicated by striped arrows, while the poxtA gene is represented by a dotted arrow. IS1216-like and IS1252-like IRs are indicated by black and grey triangles, respectively. In S. aureus AOUC-0915, the remnant of IS1216_D IRR is indicated by a black triangle downstream of the insertion point of the IS1252-like element. The region of overlap between the IS1216_D IRL and the poxtA CDS is indicated by an asterisk. (b) Nucleotide sequence showing the insertion points of IS1216_D and of the IS1252-like element. The regions encoding the IS1216_D and the IS1252-like transposases are indicated by arrows and are shown only partially; the numbers of amino acids that are not shown are indicated. The IS1252-like element is boxed and its IRs are underlined. An asterisk indicates the three-nucleotide duplication originated by the IS1252-like insertion event. IS1216_D IRs are highlighted in grey and the region of overlap between the IS1216_D IRL and the poxtA CDS is shown in capital letters. The C-terminus PoxtA sequence is shown under the nucleotide sequence. Comparison between the genetic context of poxtA in S. aureus AOUC-0915, E. faecium P36 and P. acidilactici BCC1, for which some flanking sequences are available, showed an overall conserved genetic context, but with some differences. In particular, in P. acidilactici BCC1 the IS1216 present upstream of the poxtA gene was in the opposite orientation, while in E. faecium P36 a copy of IS1216 was present downstream of the gene (Figure 3a). Discussion A novel resistance determinant belonging to the ARE ABC-F family of proteins, named PoxtA, was identified in this work. PoxtA was initially detected by bioinformatic analysis of the genome of S. aureus AOUC-0915, a linezolid-resistant MRSA strain isolated from a cystic fibrosis patient, based on sequence homology with OptrA, a recently described member of the ARE ABC-F family of proteins that confers resistance to phenicols and oxazolidinones by means of ribosomal protection.5,6 Cloning and expression experiments revealed that the poxtA gene was functional in decreasing susceptibility to at least three classes of anti-ribosomal antibiotic classes, including phenicols, oxazolidinones and tetracyclines. Altogether, these results confirmed that PoxtA, which carries two recognizable NBDs and is apparently lacking TMDs and a signal peptide, is a new member of the ARE ABC-F family of proteins that can confer reduced susceptibility to phenicols, oxazolidinones and tetracyclines. To the best of our knowledge, no other members of the ARE ABC-F family show a comparable broad spectrum of activity for these three antibiotic classes. The decreased antimicrobial susceptibility conferred by PoxtA was observed in three different hosts, including S. aureus, E. faecalis and E. coli, with an overall similar pattern. Unexpectedly, when expressed in E. coli, poxtA conferred a more marked decrease in susceptibility to antibiotics than when expressed in the Gram-positive cocci. This could be due to differences in the gene expression level or to differences in the interaction of PoxtA with the ribosomal target in the different species. The genetic context and the scattered distribution among a few strains of different species (S. aureus, Enterococcus spp. and P. acidilactici) strongly support a mobile nature of poxtA. Indeed, the gene was found to be associated with ISs, in a structure resembling a composite transposon, which were likely responsible for its capture and mobilization as a hybrid form, due to an IS1216 insertion at the C-terminus, which nevertheless has conserved a ribosomal protection function. Since poxtA homologues were detected in the genomes of several clostridia, some members of Clostridiales could be the original source of poxtA-like genes. Notably, in the genomes of clostridia, the poxtA homologues are located between genes encoding putative MATE efflux proteins and antibiotic acetyltransferases. In particular, these antibiotic acetyltransferase homologues exhibit a significant similarity (amino acid identity >49%) with known acetyltransferases, including streptogramin A27 and chloramphenicol acetyltransferases,28 suggesting that ABC-F might act synergistically with these enzymes to protect the host from antibiotics. Since most of the other strains in which the gene was detected originated from animal sources, this suggests that selection of the poxtA gene could have occurred in the animal setting, as reported for other genes mediating resistance to phenicols and other anti-ribosomal drugs that are broadly used in veterinary medicine.29,30 The apparently limited distribution in the clinical setting and the relatively low impact on oxazolidinone susceptibility point to an overall limited clinical impact of the poxtA gene. However, the discovery of a novel transferable mechanism able to reduce the susceptibility to both linezolid and tedizolid among Gram-positive cocci is a matter of concern. In fact, the poxtA gene could also act synergistically with other oxazolidinone resistance mechanisms to further increase the level of resistance to these drugs in a stepwise manner. Indeed, the AOUC-0915 strain exhibited high-level resistance to linezolid (due also to the presence of the cfr gene and of a G152D substitution in the L3 ribosomal protein) and it will be interesting to ascertain the contribution of poxtA and cfr to this resistance phenotype by separate and sequential gene inactivation experiments. On the other hand, screening for the presence of the poxtA gene in collections of clinical and veterinary isolates would be of interest to assess the real prevalence of this gene. Funding This study was supported by internal funding. Transparency declarations None to declare. Supplementary data Figure S1 is available as Supplementary data at JAC Online. References 1 Kerr ID. Sequence analysis of twin ATP binding cassette proteins involved in translational control, antibiotic resistance, and ribonuclease L inhibition . Biochem Biophys Res Commun 2004 ; 315 : 166 – 73 . Google Scholar CrossRef Search ADS PubMed 2 Dean M , Rzhetsky A , Allikmets R. 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Google Scholar CrossRef Search ADS PubMed 23 Tamura K , Stecher G , Peterson D et al. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 2013 ; 30 : 2725 – 9 . Google Scholar CrossRef Search ADS 24 Sullivan MJ , Petty NK , Beatson SA. Easyfig: a genome comparison visualizer . Bioinformatics 2011 ; 27 : 1009 – 10 . Google Scholar CrossRef Search ADS PubMed 25 Wilson DN. The ABC of ribosome-related antibiotic resistance . MBio 2016 ; 7 : e00598-16. Google Scholar CrossRef Search ADS PubMed 26 Hot C , Berthet N , Chesneau O. Characterization of sal(A), a novel gene responsible for lincosamide and streptogramin A resistance in Staphylococcus sciuri . Antimicrob Agents Chemother 2014 ; 58 : 3335 – 41 . Google Scholar CrossRef Search ADS PubMed 27 Seoane A , García Lobo JM. Identification of a streptogramin A acetyltransferase gene in the chromosome of Yersinia enterocolitica . Antimicrob Agents Chemother 2000 ; 44 : 905 – 9 . 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This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Antimicrobial Chemotherapy Oxford University Press

Characterization of poxtA, a novel phenicol–oxazolidinone–tetracycline resistance gene from an MRSA of clinical origin

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Oxford University Press
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© The Author(s) 2018. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.
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0305-7453
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10.1093/jac/dky088
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Abstract

Abstract Objectives To characterize a novel phenicol–oxazolidinone–tetracycline resistance gene, named poxtA, identified in a previously described MRSA strain that was highly resistant to linezolid and also carried the cfr gene. Methods The poxtA gene was identified by bioinformatic analysis of the whole genome sequence of Staphylococcus aureus AOUC-0915. The poxtA gene was cloned in a shuttle plasmid vector and expressed in Escherichia coli, S. aureus and Enterococcus faecalis to investigate the protein function. Comparative sequence analyses at the protein and genetic levels were carried out using standard procedures. Results The poxtA gene encodes a protein that is 32% identical to OptrA and exhibits structural features typical of the F lineage of the ATP-binding cassette (ABC) protein superfamily that cause antibiotic resistance by ribosomal protection. Expression of poxtA in E. coli, S. aureus and E. faecalis was able to decrease susceptibility to phenicols, oxazolidinones and tetracyclines. A database search identified the presence of poxtA in E. faecalis, Enterococcus faecium and Pediococcus acidilactici strains, mostly of animal origin, and revealed the presence of poxtA homologues in the genomes of some Clostridiales. Analysis of the genetic context revealed that poxtA was located in a composite transposon-like structure containing two IS1216 elements. Conclusions A novel resistance gene, named poxtA, encoding a protein of the antibiotic resistance (ARE) ABC-F lineage, was identified in the genome of an MRSA of clinical origin. PoxtA can confer decreased susceptibility to phenicols, oxazolidinones and tetracyclines and is associated with a putative mobile element that could contribute to its horizontal dissemination. Introduction The ATP-binding cassette (ABC) superfamily includes a large, diverse and ubiquitous group of proteins detected in all kingdoms of life. Some of these proteins are able to confer resistance to various classes of antibiotics in prokaryotes. The ABC proteins associated with antibiotic resistance (ARE) belong to the F lineage of the ABC superfamily (ARE ABC-F) and include single polypeptides containing two conserved nucleotide binding domains (NBDs) separated by a linker of ∼80 amino acids of variable composition, with no transmembrane domains (TMDs).1,2 The mechanism by which the ARE ABC-F proteins mediate resistance has been debated for a long time and attributed to either efflux or ribosomal protection.3,4 Recently, the resistance mechanism of ARE ABC-F proteins has been clarified and shown to be mediated by ribosomal protection.5 The ARE ABC-F proteins can mediate resistance to several different classes of anti-ribosomal antibiotics, including tetracyclines, macrolides, ketolides, lincosamides, phenicols, pleuromutilins, oxazolidinones and streptogramins.5 Many of them can actually mediate resistance to multiple antibiotic classes, e.g. Msr(A), conferring resistance to macrolides and group B streptogramins, or the recently described OptrA protein, conferring resistance to phenicols and oxazolidinones.6 The ARE ABC-F proteins are often encoded by genes carried on mobile genetic elements, which can disseminate these resistance determinants horizontally among bacterial pathogens, although some are encoded by resident genes in a number of antibiotic-producing Streptomyces spp.6–8 We recently reported an MRSA strain that exhibited high-level resistance to linezolid (32 mg/L).9 The strain had been isolated from a cystic fibrosis patient after linezolid treatment and was found to carry at least two different linezolid resistance mechanisms including a G152D substitution in the L3 ribosomal protein9 and the cfr gene, an acquired gene encoding a ribosomal rRNA methylase, which can mediate resistance to oxazolidinones and several other anti-ribosomal drugs.9–13 In this study, we describe the identification and characterization of a novel acquired resistance gene, named poxtA, found in the same MRSA strain. The poxtA gene encodes a protein of the ARE ABC-F family, which is distantly related to OptrA and able to confer reduced susceptibility to phenicols, oxazolidinones and tetracyclines. Materials and methods Bacterial strains AOUC-0915 is an ST5-MRSA-II strain isolated in September 2015 from the respiratory tract of a cystic fibrosis patient at Florence Careggi University Hospital (Florence, Italy).9 Antimicrobial susceptibility and resistome of this strain, in terms of known resistance genes, have previously been reported.9,Escherichia coli Mach1™ T1R (Thermo Fisher Scientific, Waltham, MA, USA), Staphylococcus aureus RN422014 and Enterococcus faecalis JH2-215 were used as hosts for cloning of the poxtA gene. Recombinant DNA methodology The poxtA gene, with its flanking regions, was cloned in the pMU1328 vector16 using the PIPE (Polymerase Incomplete Primer Extension) cloning method17 and E. coli Mach1™ T1R cells, to obtain the recombinant plasmid pMU-poxtA. Cloning was designed to replace the chloramphenicol acetyl transferase (cat) gene of the plasmid with the poxtA locus, using primers targeting the amplification of a region spanning from 863 bp upstream of the poxtA start codon to 60 bp downstream of the stop codon (Figure S1, available as Supplementary data at JAC Online). A plasmid derived from the pMU1328 vector, named pMU-E, and carrying a deletion of the cat gene, was also constructed using the PIPE cloning method and specific primers (Figure S1) to be used as a control. E. coli transformants, obtained by a heat-shock method,18 were selected on Mueller–Hinton agar (MHA) supplemented with erythromycin (100 mg/L). The authenticity of the cloned DNA fragment in pMU-poxtA and of the deletion in plasmid pMU-E were confirmed by Sanger sequencing both strands of plasmid DNA extracted from the Mach1™ T1R transformants. The recombinant plasmids were introduced into S. aureus RN4220 and E. faecalis JH2-2 by electrotransformation (2.5 kV, 200 Ω, 25 μF). Transformants were selected on MHA containing erythromycin (2 mg/L). Antimicrobial susceptibility testing MICs were determined by broth microdilution according to the M7-A10 standard of the CLSI,19 except for that of tedizolid, which was measured by MIC Test Strip (Liofilchem s.r.l., Roseto degli Abruzzi, Italy). Antimicrobial susceptibility testing was carried out in triplicate. The results were interpreted according to the EUCAST clinical breakpoint tables.20 Reference strains S. aureus ATCC 29213, E. faecalis ATCC 29212 and E. coli ATCC 25922 were used as quality controls for antimicrobial susceptibility testing. Bioinformatics and sequence analysis The presence of TMDs and of a signal peptide in PoxtA was predicted using the TMHMM Server v. 2.0 web service21 and the SignalP 4.1 server,22 respectively. The search for poxtA homologues was carried out using the BLAST web server, selecting alternatively the wgs or nr databases (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Amino acid sequence alignments of PoxtA with other ARE ABC-F proteins and the phylogenetic tree, generated using the maximum likelihood method with 1000 bootstrap replicates, were generated with the MEGA 6.0.6 software package.23 The genetic context of poxtA in S. aureus AOUC-0915 was investigated by PCR mapping and Sanger sequencing experiments on the regions unresolved by WGS assembly.9 A graphical comparison of the genetic context of poxtA homologues was generated using EasyFig.24 The nucleotide sequence of the poxtA gene and flanking regions has been deposited in the GenBank database under the accession number MF095097. Results Identification of a new ARE ABC-F resistance determinant in S. aureus AOUC-0915 Analysis of the genome sequence of S. aureus AOUC-0915 revealed the presence of an ORF encoding a protein of 542 amino acids that exhibited similarity (32% identical amino acid residues) with OptrA over most of its length (coverage of 95%) (Figure 1). This protein showed conserved features with members of the ARE ABC-F family,5,25 including two NBDs and the lack of detectable TMDs. Moreover, no leader peptide was detected by the two bioinformatics tools used for this analysis (Figure 1). Figure 1. View largeDownload slide Alignment of PoxtA and homologous proteins. The conserved regions of ABC-F proteins, including the Walker A and B motifs, the signature and the switch motifs, involved in ATP binding and hydrolysis are indicated. Residues conserved in all sequences are shown in bold. Protein accession numbers are as follows: Coprobacillus sp. CAG:605, CCZ88872.1; C. clostridioforme CAG:132, CDB61438.1; Clostridium sp. C105KSO14, CUX74470.1; C. bolteae 90B8, ENZ34498.1; Clostridiales bacterium 1_7_47FAA, EEQ60488.1; Clostridiales bacterium VE202-28, WP_025484560.1; Azospirillum sp. 51_20, OLA82622.1; B. producta ATCC 27340, WP_026255743.1; H. hathewayi 2789STDY5834916, WP_055650089.1; E. faecium 9-F-6, OZN12776.1; Sal(A), AGN74946.1; and OptrA, AKA86814.1. Figure 1. View largeDownload slide Alignment of PoxtA and homologous proteins. The conserved regions of ABC-F proteins, including the Walker A and B motifs, the signature and the switch motifs, involved in ATP binding and hydrolysis are indicated. Residues conserved in all sequences are shown in bold. Protein accession numbers are as follows: Coprobacillus sp. CAG:605, CCZ88872.1; C. clostridioforme CAG:132, CDB61438.1; Clostridium sp. C105KSO14, CUX74470.1; C. bolteae 90B8, ENZ34498.1; Clostridiales bacterium 1_7_47FAA, EEQ60488.1; Clostridiales bacterium VE202-28, WP_025484560.1; Azospirillum sp. 51_20, OLA82622.1; B. producta ATCC 27340, WP_026255743.1; H. hathewayi 2789STDY5834916, WP_055650089.1; E. faecium 9-F-6, OZN12776.1; Sal(A), AGN74946.1; and OptrA, AKA86814.1. To confirm its role in antibiotic resistance, the gene, with some flanking regions, was cloned in the pMU1328 shuttle plasmid vector, which can replicate in E. coli, in S. aureus and in E. faecalis. The antimicrobial susceptibilities of the E. coli, S. aureus and E. faecalis strains carrying the cloned gene were investigated and compared with those of strains carrying the empty pMU-E vector. The expression of the gene in S. aureus and E. faecalis demonstrated that it was associated with decreased susceptibility to phenicols (chloramphenicol and florfenicol), oxazolidinones (linezolid and tedizolid) and tetracyclines (tetracycline and doxycycline). Expression of the gene in E. coli was associated with decreased susceptibility to the same antibiotics (tedizolid was not tested) and also to tigecycline (Table 1). No differences in susceptibility to amikacin, gentamicin, ampicillin, ciprofloxacin, rifampicin and trimethoprim/sulfamethoxazole were detected following expression of the gene in the E. coli and S. aureus hosts (data not shown). Given the antibiotic resistance phenotype, the new resistance gene was designated poxtA (after phenicols, oxazolidinones and tetracyclines). Table 1. Antimicrobial susceptibilities of S. aureus, E. faecalis and E. coli strains carrying a cloned copy of the poxtA gene Antibiotic MIC (mg/L) S. aureusa E. faecalisb E. colic RN4220 (pMU-poxtA) RN4220 (pMU-E) JH2-2 (pMU-poxtA) JH2-2 (pMU-E) Mach1™ T1R (pMU-poxtA) Mach1™ T1R (pMU-E) Linezolid 2 1 4 1 1024 256 Tedizolid 0.5 0.25 0.5 0.25 — — Chloramphenicol 8 4 8 4 32 8 Florfenicol 16 2 16 2 128 8 Tigecycline 0.25 0.25 0.25 0.25 1 0.5 Tetracycline 0.25 0.125 0.25 0.125 2 0.5 Doxycycline 0.25 0.125 0.125 ≤0.06 8 2 Antibiotic MIC (mg/L) S. aureusa E. faecalisb E. colic RN4220 (pMU-poxtA) RN4220 (pMU-E) JH2-2 (pMU-poxtA) JH2-2 (pMU-E) Mach1™ T1R (pMU-poxtA) Mach1™ T1R (pMU-E) Linezolid 2 1 4 1 1024 256 Tedizolid 0.5 0.25 0.5 0.25 — — Chloramphenicol 8 4 8 4 32 8 Florfenicol 16 2 16 2 128 8 Tigecycline 0.25 0.25 0.25 0.25 1 0.5 Tetracycline 0.25 0.125 0.25 0.125 2 0.5 Doxycycline 0.25 0.125 0.125 ≤0.06 8 2 Susceptibilities of the same strains carrying the empty plasmid vector are also reported for comparison. The MIC measurements were performed in triplicate and results were fully reproducible, with no discrepancies. a The MICs were evaluated in medium supplemented with 50 mg/L erythromycin for plasmid maintenance. b The MICs were evaluated in medium supplemented with 2 mg/L erythromycin for plasmid maintenance. c The MICs were evaluated in medium supplemented with 100 mg/L erythromycin for plasmid maintenance. Table 1. Antimicrobial susceptibilities of S. aureus, E. faecalis and E. coli strains carrying a cloned copy of the poxtA gene Antibiotic MIC (mg/L) S. aureusa E. faecalisb E. colic RN4220 (pMU-poxtA) RN4220 (pMU-E) JH2-2 (pMU-poxtA) JH2-2 (pMU-E) Mach1™ T1R (pMU-poxtA) Mach1™ T1R (pMU-E) Linezolid 2 1 4 1 1024 256 Tedizolid 0.5 0.25 0.5 0.25 — — Chloramphenicol 8 4 8 4 32 8 Florfenicol 16 2 16 2 128 8 Tigecycline 0.25 0.25 0.25 0.25 1 0.5 Tetracycline 0.25 0.125 0.25 0.125 2 0.5 Doxycycline 0.25 0.125 0.125 ≤0.06 8 2 Antibiotic MIC (mg/L) S. aureusa E. faecalisb E. colic RN4220 (pMU-poxtA) RN4220 (pMU-E) JH2-2 (pMU-poxtA) JH2-2 (pMU-E) Mach1™ T1R (pMU-poxtA) Mach1™ T1R (pMU-E) Linezolid 2 1 4 1 1024 256 Tedizolid 0.5 0.25 0.5 0.25 — — Chloramphenicol 8 4 8 4 32 8 Florfenicol 16 2 16 2 128 8 Tigecycline 0.25 0.25 0.25 0.25 1 0.5 Tetracycline 0.25 0.125 0.25 0.125 2 0.5 Doxycycline 0.25 0.125 0.125 ≤0.06 8 2 Susceptibilities of the same strains carrying the empty plasmid vector are also reported for comparison. The MIC measurements were performed in triplicate and results were fully reproducible, with no discrepancies. a The MICs were evaluated in medium supplemented with 50 mg/L erythromycin for plasmid maintenance. b The MICs were evaluated in medium supplemented with 2 mg/L erythromycin for plasmid maintenance. c The MICs were evaluated in medium supplemented with 100 mg/L erythromycin for plasmid maintenance. Relatedness of PoxtA with other ARE ABC-F proteins Comparative sequence analysis of PoxtA with other members of the ARE ABC-F family confirmed that PoxtA is a new member of this protein family. The closest homologues were putative ARE ABC-F proteins encoded by the genomes of Enterococcus faecium 9-F-6 and Azospirillum sp. 51_20, (73% and 70% amino acid identity, respectively). Other homologues (66%–68% amino acid identity) were detected in the genomes of some Clostridiales, including Clostridium bolteae, Clostridium clostridioforme, Hungatella hathewayi (formerly Clostridium hathewayi) and Blautia producta, and of Coprobacillus sp. CAG:605 (Figure 1). Among known ARE ABC-F proteins, PoxtA belongs to a sublineage that also includes OptrA and Sal(A)26 (Figure 2). Figure 2. View largeDownload slide Phylogenetic tree of ARE ABC-F proteins constructed using the maximum likelihood method. The tree represents the consensus obtained after 1000 replicates and bootstrap values are indicated next to the branches. Amino acid sequences of the ARE ABC-F proteins were extracted from the ‘tetracycline and MLS nomenclature’ web site (https://faculty.washington.edu/marilynr/ermweb2.pdf), except for Lmo0919 (CAC98997.1), Var(M) (BAA96297.1), Vml(R) (WP_003234144.1) and LmrC (CAA55774.1). Antibiotics affected by the different proteins are indicated and the species in which the various ARE ABC-F have been described for the first time are also indicated.  SA, streptogramin A; SB, streptogramin B; P, pleuromutilins; L, lincosamides; M, macrolides; Ph, phenicols; O, oxazolidinones; T, tetracyclines. Antibiotic-producing species are indicated by an asterisk. Figure 2. View largeDownload slide Phylogenetic tree of ARE ABC-F proteins constructed using the maximum likelihood method. The tree represents the consensus obtained after 1000 replicates and bootstrap values are indicated next to the branches. Amino acid sequences of the ARE ABC-F proteins were extracted from the ‘tetracycline and MLS nomenclature’ web site (https://faculty.washington.edu/marilynr/ermweb2.pdf), except for Lmo0919 (CAC98997.1), Var(M) (BAA96297.1), Vml(R) (WP_003234144.1) and LmrC (CAA55774.1). Antibiotics affected by the different proteins are indicated and the species in which the various ARE ABC-F have been described for the first time are also indicated.  SA, streptogramin A; SB, streptogramin B; P, pleuromutilins; L, lincosamides; M, macrolides; Ph, phenicols; O, oxazolidinones; T, tetracyclines. Antibiotic-producing species are indicated by an asterisk. Detection of the poxtA gene in other strains and genetic context of poxtA A BLAST search in sequence databases using poxtA as a query revealed the presence of identical genes in a number of Gram-positive strains including E. faecalis 599 (accession no. EJU87034.1), E. faecalis 12 (accession no. KII46686.1), E. faecium P36 (accession no. KP834591.1) and Pediococcus acidilactici BCC1 (accession no. CP018763.1). This finding confirmed the mobile nature of poxtA, which is apparently able to spread among different Gram-positive cocci. Interestingly, these strains were of animal origin (except for E. faecalis 599, whose origin was not reported). The genetic context of poxtA in S. aureus AOUC-0915 was further investigated by PCR mapping and sequencing experiments. This analysis revealed that poxtA was flanked by two IS1216-like ISs (hereinafter referred to as IS1216_U and IS1216_D) in the same orientation (Figure 3a), suggesting that the resistance gene had been mobilized via a composite transposon constituted by the two IS1216-like elements. The IRL of the IS1216_D that is present at the 3′-end of the poxtA gene is actually part of the coding sequence (CDS) constituting the final 21 nucleotides of the CDS and including the poxtA stop codon (Figure 3b). This condition is conserved also in all the available sequences containing poxtA (e.g. in P. acidilactici BCC1 and in E. faecium P36) (Figure 3a), suggesting that the partial deletion of the poxtA gene may be an ancestral event that originated from the mobilization of this resistance gene by the IS1216-like elements. As a consequence a hybrid gene results, encoding a protein with a modified C-terminus. The IRR of the IS1216_D was disrupted by the insertion of an IS1252-like IS that apparently generated a duplicated region of three nucleotides abutting the left and right boundaries of the element. Remnants of the IS1216_D IRR are present downstream of the IS1252-like element, suggesting that the insertion of this IS was likely an independent event following poxtA mobilization (Figure 3b). Figure 3. View largeDownload slide Genetic context of the poxtA gene. (a) Comparison of the genetic context of poxtA in P. acidilactici BCC1 (region 1 547 068–1 552 271 of accession number CP018763.1) and in E. faecium P36 (region 1–3421 of accession number KP834591.1). Regions having >99% nucleotide identity are connected by grey zones. ISs are boxed and transposase-encoding genes are indicated by striped arrows, while the poxtA gene is represented by a dotted arrow. IS1216-like and IS1252-like IRs are indicated by black and grey triangles, respectively. In S. aureus AOUC-0915, the remnant of IS1216_D IRR is indicated by a black triangle downstream of the insertion point of the IS1252-like element. The region of overlap between the IS1216_D IRL and the poxtA CDS is indicated by an asterisk. (b) Nucleotide sequence showing the insertion points of IS1216_D and of the IS1252-like element. The regions encoding the IS1216_D and the IS1252-like transposases are indicated by arrows and are shown only partially; the numbers of amino acids that are not shown are indicated. The IS1252-like element is boxed and its IRs are underlined. An asterisk indicates the three-nucleotide duplication originated by the IS1252-like insertion event. IS1216_D IRs are highlighted in grey and the region of overlap between the IS1216_D IRL and the poxtA CDS is shown in capital letters. The C-terminus PoxtA sequence is shown under the nucleotide sequence. Figure 3. View largeDownload slide Genetic context of the poxtA gene. (a) Comparison of the genetic context of poxtA in P. acidilactici BCC1 (region 1 547 068–1 552 271 of accession number CP018763.1) and in E. faecium P36 (region 1–3421 of accession number KP834591.1). Regions having >99% nucleotide identity are connected by grey zones. ISs are boxed and transposase-encoding genes are indicated by striped arrows, while the poxtA gene is represented by a dotted arrow. IS1216-like and IS1252-like IRs are indicated by black and grey triangles, respectively. In S. aureus AOUC-0915, the remnant of IS1216_D IRR is indicated by a black triangle downstream of the insertion point of the IS1252-like element. The region of overlap between the IS1216_D IRL and the poxtA CDS is indicated by an asterisk. (b) Nucleotide sequence showing the insertion points of IS1216_D and of the IS1252-like element. The regions encoding the IS1216_D and the IS1252-like transposases are indicated by arrows and are shown only partially; the numbers of amino acids that are not shown are indicated. The IS1252-like element is boxed and its IRs are underlined. An asterisk indicates the three-nucleotide duplication originated by the IS1252-like insertion event. IS1216_D IRs are highlighted in grey and the region of overlap between the IS1216_D IRL and the poxtA CDS is shown in capital letters. The C-terminus PoxtA sequence is shown under the nucleotide sequence. Comparison between the genetic context of poxtA in S. aureus AOUC-0915, E. faecium P36 and P. acidilactici BCC1, for which some flanking sequences are available, showed an overall conserved genetic context, but with some differences. In particular, in P. acidilactici BCC1 the IS1216 present upstream of the poxtA gene was in the opposite orientation, while in E. faecium P36 a copy of IS1216 was present downstream of the gene (Figure 3a). Discussion A novel resistance determinant belonging to the ARE ABC-F family of proteins, named PoxtA, was identified in this work. PoxtA was initially detected by bioinformatic analysis of the genome of S. aureus AOUC-0915, a linezolid-resistant MRSA strain isolated from a cystic fibrosis patient, based on sequence homology with OptrA, a recently described member of the ARE ABC-F family of proteins that confers resistance to phenicols and oxazolidinones by means of ribosomal protection.5,6 Cloning and expression experiments revealed that the poxtA gene was functional in decreasing susceptibility to at least three classes of anti-ribosomal antibiotic classes, including phenicols, oxazolidinones and tetracyclines. Altogether, these results confirmed that PoxtA, which carries two recognizable NBDs and is apparently lacking TMDs and a signal peptide, is a new member of the ARE ABC-F family of proteins that can confer reduced susceptibility to phenicols, oxazolidinones and tetracyclines. To the best of our knowledge, no other members of the ARE ABC-F family show a comparable broad spectrum of activity for these three antibiotic classes. The decreased antimicrobial susceptibility conferred by PoxtA was observed in three different hosts, including S. aureus, E. faecalis and E. coli, with an overall similar pattern. Unexpectedly, when expressed in E. coli, poxtA conferred a more marked decrease in susceptibility to antibiotics than when expressed in the Gram-positive cocci. This could be due to differences in the gene expression level or to differences in the interaction of PoxtA with the ribosomal target in the different species. The genetic context and the scattered distribution among a few strains of different species (S. aureus, Enterococcus spp. and P. acidilactici) strongly support a mobile nature of poxtA. Indeed, the gene was found to be associated with ISs, in a structure resembling a composite transposon, which were likely responsible for its capture and mobilization as a hybrid form, due to an IS1216 insertion at the C-terminus, which nevertheless has conserved a ribosomal protection function. Since poxtA homologues were detected in the genomes of several clostridia, some members of Clostridiales could be the original source of poxtA-like genes. Notably, in the genomes of clostridia, the poxtA homologues are located between genes encoding putative MATE efflux proteins and antibiotic acetyltransferases. In particular, these antibiotic acetyltransferase homologues exhibit a significant similarity (amino acid identity >49%) with known acetyltransferases, including streptogramin A27 and chloramphenicol acetyltransferases,28 suggesting that ABC-F might act synergistically with these enzymes to protect the host from antibiotics. Since most of the other strains in which the gene was detected originated from animal sources, this suggests that selection of the poxtA gene could have occurred in the animal setting, as reported for other genes mediating resistance to phenicols and other anti-ribosomal drugs that are broadly used in veterinary medicine.29,30 The apparently limited distribution in the clinical setting and the relatively low impact on oxazolidinone susceptibility point to an overall limited clinical impact of the poxtA gene. However, the discovery of a novel transferable mechanism able to reduce the susceptibility to both linezolid and tedizolid among Gram-positive cocci is a matter of concern. In fact, the poxtA gene could also act synergistically with other oxazolidinone resistance mechanisms to further increase the level of resistance to these drugs in a stepwise manner. Indeed, the AOUC-0915 strain exhibited high-level resistance to linezolid (due also to the presence of the cfr gene and of a G152D substitution in the L3 ribosomal protein) and it will be interesting to ascertain the contribution of poxtA and cfr to this resistance phenotype by separate and sequential gene inactivation experiments. On the other hand, screening for the presence of the poxtA gene in collections of clinical and veterinary isolates would be of interest to assess the real prevalence of this gene. Funding This study was supported by internal funding. Transparency declarations None to declare. Supplementary data Figure S1 is available as Supplementary data at JAC Online. References 1 Kerr ID. Sequence analysis of twin ATP binding cassette proteins involved in translational control, antibiotic resistance, and ribonuclease L inhibition . Biochem Biophys Res Commun 2004 ; 315 : 166 – 73 . Google Scholar CrossRef Search ADS PubMed 2 Dean M , Rzhetsky A , Allikmets R. 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Plasmid DNA transformation in Escherichia coli: effect of heat shock temperature, duration, and cold incubation of CaCl2 treated cells . Int J Biotechnol Biochem 2010 ; 6 : 561 – 8 . 19 Clinical and Laboratory Standards Institute . Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically—Tenth Edition: Approved Standard M07-A10 . CLSI , Wayne, PA, USA , 2015 . 20 EUCAST . Breakpoint Tables for Interpretation of MICs and Zone Diameters, Version 8.0, 2018. http://www.eucast.org/clinical_breakpoints/. 21 Krogh A , Larsson B , von Heijne G et al. Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes . J Mol Biol 2001 ; 305 : 567 – 80 . Google Scholar CrossRef Search ADS PubMed 22 Petersen TN , Brunak S , von Heijne G et al. SignalP 4.0: discriminating signal peptides from transmembrane regions . Nat Methods 2011 ; 8 : 785 – 6 . Google Scholar CrossRef Search ADS PubMed 23 Tamura K , Stecher G , Peterson D et al. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 2013 ; 30 : 2725 – 9 . Google Scholar CrossRef Search ADS 24 Sullivan MJ , Petty NK , Beatson SA. Easyfig: a genome comparison visualizer . Bioinformatics 2011 ; 27 : 1009 – 10 . Google Scholar CrossRef Search ADS PubMed 25 Wilson DN. The ABC of ribosome-related antibiotic resistance . MBio 2016 ; 7 : e00598-16. Google Scholar CrossRef Search ADS PubMed 26 Hot C , Berthet N , Chesneau O. Characterization of sal(A), a novel gene responsible for lincosamide and streptogramin A resistance in Staphylococcus sciuri . Antimicrob Agents Chemother 2014 ; 58 : 3335 – 41 . Google Scholar CrossRef Search ADS PubMed 27 Seoane A , García Lobo JM. Identification of a streptogramin A acetyltransferase gene in the chromosome of Yersinia enterocolitica . Antimicrob Agents Chemother 2000 ; 44 : 905 – 9 . Google Scholar CrossRef Search ADS PubMed 28 Tennigkeit J , Matzura H. Nucleotide sequence analysis of a chloramphenicol-resistance determinant from Agrobacterium tumefaciens and identification of its gene product . Gene 1991 ; 98 : 113 – 6 . Google Scholar CrossRef Search ADS PubMed 29 Doublet B , Schwarz S , Kehrenberg C et al. Florfenicol resistance gene floR is part of a novel transposon . Antimicrob Agents Chemother 2005 ; 49 : 2106 – 8 . Google Scholar CrossRef Search ADS PubMed 30 Hao H , Sander P , Iqbal Z et al. The risk of some veterinary antimicrobial agents on public health associated with antimicrobial resistance and their molecular basis . Front Microbiol 2016 ; 7 : 1626. Google Scholar PubMed © The Author(s) 2018. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com. 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Journal of Antimicrobial ChemotherapyOxford University Press

Published: Apr 4, 2018

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