Identification of a novel tetracycline resistance gene, tet(63), located on a multiresistance plasmid from Staphylococcus aureus

Abstract

Objectives

To identify and characterize a novel tetracycline resistance gene on a multiresistance plasmid from Staphylococcus aureus SA01 of chicken origin.

Methods

MICs were determined by broth microdilution according to CLSI recommendations. The whole genome sequence of S. aureus SA01 was determined via Illumina HiSeq and Oxford Nanopore platforms followed by a hybrid assembly. The new tet gene was cloned and expressed in S. aureus. The functionality of the corresponding protein as an efflux pump was tested by efflux pump inhibition assays.

Results

A novel tetracycline resistance gene, tet(63), was identified on a plasmid in S. aureus SA01. The cloned tet(63) gene was functionally expressed in S. aureus and shown to confer resistance to tetracycline and doxycycline, and a slightly elevated MIC of minocycline. The tet(63) gene encodes a 459 amino acid efflux protein of the major facilitator superfamily that consists of 14 predicted transmembrane helices. The results of efflux pump inhibitor assays confirmed the function of Tet(63) as an efflux protein. The deduced amino acid sequence of the Tet(63) protein exhibited 73.0% identity to the tetracycline efflux protein Tet(K). The plasmid pSA01-tet, on which tet(63) was located, had a size of 25664 bp and also carried the resistance genes aadD, aacA-aphD and erm(C).

Conclusions

A novel tetracycline resistance gene, tet(63), was identified in S. aureus. Its location on a multiresistance plasmid might support the co-selection of tet(63) under the selective pressure imposed by the use of macrolides, lincosamides and aminoglycosides.

Introduction

Tetracyclines are broad-spectrum antimicrobial agents that are effective against a wide range of bacteria.¹ During recent decades, tetracycline resistance has increased rapidly as a result of continuous selective pressure conferred by the extensive use of tetracyclines in human and veterinary medicine, but also in agriculture, horticulture, beekeeping and aquaculture.² Bacterial resistance to tetracyclines is usually due to acquired tetracycline resistance genes.³ Until now, 62 different tetracycline resistance genes have been identified (http://faculty.washington.edu/marilynr/; last updated February 2020), which represent mainly three distinct mechanisms: active efflux, ribosomal protection and enzymatic inactivation.¹ Active efflux is the most common resistance mechanism and 35 distinct genes encoding efflux proteins have been described so far. Most of the corresponding proteins were assigned to the major facilitator superfamily of transporters, which are able to effectively decrease the intracellular tetracycline concentration by exchanging a tetracycline molecule against a proton.⁴ Efflux genes have been widely reported in both Gram-positive and Gram-negative bacteria.⁵ In addition, many of the tetracycline efflux genes are associated with mobile genetic elements, such as plasmids or transposons, which supports their wide dissemination across species or genus boundaries.³ According to the tetracycline nomenclature database (http://faculty.washington.edu/marilynr/tetweb3.pdf), 12 different tet genes have been identified in staphylococci, including tet(K, L, M, O, S, U, W, 38, 42, 43, 44 and 45). Among them, the genes tet(K), tet(L), tet(38), tet(42), tet(43) and tet(45) encode efflux proteins.

In this study, we report a new multiresistance plasmid from S. aureus of chicken origin, which harbours a novel plasmid-borne tetracycline efflux gene, designated tet(63).

Materials and methods

Bacterial strain and plasmids

S. aureus SA01 was isolated from a faecal sample of a diseased chicken from a chicken farm in Heilongjiang province, China, in 2019. Bacterial species identification was conducted by 16S rRNA gene sequencing and biochemical profiling using API 20E (bioMérieux, Shanghai, China). MLST and spa typing were carried out as described previously.⁶ Plasmids of S. aureus SA01 were extracted using the Qiagen Plasmid Midi Kit (Qiagen, Germany) and transferred into S. aureus RN4220 via electrotransformation.⁷ The transformants were selected on brain heart infusion (BHI) agar plates containing 1 mg/L tetracycline.

Functional cloning of tet(63)

To confirm the drug resistance phenotypes conferred by the tet(63) gene, a 2582 bp DNA segment was amplified using a pair of primers [tet-F (5′-CGGGGATCCTCTAGAGTCGACTCTGATAACGACCTATTATG-3′) and tet-R (5′-CTTGCATGCCTGCAGGTCGACAATACACATAATAAAACACC-3′)] and an annealing temperature of 54°C. These primers consist of the partial sequences of the vector and the sequences that belong to the SA01 sequence (single underlined). In addition, these primers contain a SalI restriction site (double underlined) for subsequent cloning experiments. This amplified segment comprised the tet(63) gene as well as 218 bp of its upstream region and 132 bp of its downstream region. The PCR amplicon was ligated into the ampicillin- and chloramphenicol-resistant Escherichia coli–S. aureus shuttle plasmid pLI50 within the SalI site and the resulting recombinant plasmid was first transformed into E. coli DH5α and then transformed into S. aureus RN4220 by electrotransformation.⁷E. coli and S. aureus transformants were selected on LB plates containing 50 mg/L ampicillin and 1 mg/L tetracycline as well as BHI plates containing 25 mg/L chloramphenicol and 1 mg/L tetracycline, respectively.

WGS and bioinformatic analysis

Genomic DNA was prepared using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). S. aureus SA01 was subjected to WGS using a combination of Nanopore MinION (Oxford Nanopore Technologies, Shanghai, China) and Illumina HiSeq (Genewiz, Nanjing, China) platforms. The de novo hybrid assembly of both short- and long-read data was performed using Unicycler v.0.4.3 and error correction was conducted using Pilon v.1.22. Genome sequences were annotated using the RAST online tool (http://rast.theseed.org/FIG/rast.cgi) and corrected via BLASTn (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Antimicrobial resistance genes were detected by the ResFinder tool on the CGE website (https://cge.cbs.dtu.dk/services/). The BLAST Ring Image Generator tool was used to draw the circular map of plasmid pSA01-tet. The presence of transmembrane domains (TMDs) in Tet(63) was predicted using the TMHMM Server v.2.0 web service. A multisequence alignment of the amino acid sequence of Tet(63) along with those of other tetracycline efflux proteins was conducted using ClustalX 2.0 and the phylogenetic tree was generated using the MEGA 6.0.6 software package using the maximum likelihood method.

Antimicrobial susceptibility testing

Antimicrobial susceptibility testing was performed by broth microdilution. Resulting MICs were interpreted according to the recommendations given in CLSI documents VET08⁸ and M100.⁹S. aureus ATCC 29213 served as a quality control strain.

Efflux pump inhibition assay

Subinhibitory concentrations of two known efflux pump inhibitors (EPIs), CCCP and reserpine, for S. aureus SA01 and RN4220 were used to test efflux pump inhibition as previously described.¹⁰ The aforementioned EPIs were added at subinhibitory concentrations of 0.4 mg/L CCCP or 0.2 mg/L reserpine to the broth medium in order to evaluate efflux pump inhibition by MIC reduction.

Nucleotide sequence accession numbers

The complete nucleotide sequences of the chromosomal DNA and the three plasmids of strain SA01 have been deposited in GenBank under accession numbers CP053075–CP053078.

Results and discussion

Characteristics of S. aureus SA01 and detection of the novel tet gene

Susceptibility testing showed that S. aureus SA01 was resistant to erythromycin, clindamycin, chloramphenicol, gentamicin, tetracycline and doxycycline, and intermediate to tedizolid and minocycline, and showed relatively high MICs of lincomycin, florfenicol and spectinomycin compared with the recipient strain S. aureus RN4220 (Table S1, available as Supplementary data at JAC Online). MLST results revealed that strain SA01 represented a new single-locus variant of ST5, ST6324, in which glpF allele 1 was replaced by new glpF allele 839. spa typing showed that strain SA01 belonged to t002. The assembled whole genome sequence revealed that S. aureus SA01 harboured a circular chromosomal DNA (2882539 bp) and three plasmids [pSA01-tet (25664 bp), pSA01-03 (17257 bp) and pSA01-04 (2992 bp)]. The ResFinder results showed that three antimicrobial resistance genes [the oxazolidinone resistance gene optrA, the phenicol resistance gene fexA and the tetracycline resistance gene tet(M)] were present in the chromosomal DNA of S. aureus SA01. Plasmid pSA01-tet was an MDR plasmid containing the aadD gene encoding an aminoglycoside adenyltransferase (conferring resistance to kanamycin, neomycin, paromomycin and tobramycin), the erm(C) gene encoding a 23S rRNA methylase (mediating combined resistance to macrolides, lincosamides and streptogramin B antibiotics) and the aacA-aphD gene encoding a bifunctional aminoglycoside-modifying enzyme (mediating resistance to gentamicin, kanamycin and tobramycin). Plasmids pSA01-03 and pSA01-04 did not carry any resistance genes.

In addition to the three known resistance genes aadD, aacA-aphD and erm(C), the annotation of plasmid pSA01-tet indicated an ORF of 1380 bp encoding a 459 amino acid protein. The deduced amino acid sequence of this protein exhibited 73.0% identity to the tetracycline efflux protein Tet(K). According to the current nomenclature for tetracycline resistance genes, a new gene can be defined when the corresponding protein shows <79% amino acid homology to all known determinants.¹¹ This putative tetracycline resistance gene received the designation tet(63) from Marilyn C. Roberts, the curator of the tetracycline resistance nomenclature centre (http://faculty.washington.edu/marilynr/).

Confirmation of tet(63) as a tetracycline resistance gene

To determine whether tet(63) confers resistance to tetracyclines, plasmid pSA01-tet was successfully transformed into S. aureus RN4220. Antimicrobial susceptibility testing of the transformants indicated that pSA01-tet conferred a ≥8-fold increase in the MICs of all tetracyclines tested, except tigecycline, compared with the ‘empty’ recipient strain RN4220 (Table S1).

To further identify the functionality of tet(63) and its involvement in tetracycline resistance, the tet(63) gene, together with some flanking regions, was cloned in the E. coli–S. aureus shuttle vector pLI50. In comparison with S. aureus RN4220 carrying the empty vector pLI50, S. aureus RN4220 carrying plasmid pLI50+tet(63) exhibited 256-, 64- and 8-fold increases in the MICs of tetracycline, doxycycline and minocycline, respectively, while the same low tigecycline MIC of 0.125 mg/L was observed for S. aureus RN4220/pLI50 and S. aureus RN4220/pLI50+tet(63) (Table 1). No differences in the susceptibility to other antimicrobial agents were detected following expression of tet(63) in the S. aureus host (data are shown in Table S1 for S. aureus RN4220/pSA01-tet).

Table 1.

MICs for S. aureus SA01, S. aureus RN4220, E. coli DH5α and their transformants

Bacterial isolate	MIC (mg/L)
	tetracycline	doxycycline	minocycline	tigecycline
S. aureus SA01	64	16	8	0.5
S. aureus RN4220	0.25	0.06	0.03	0.125
Transformant S. aureus RN4220/pSA01-tet	32	4	0.25	0.25
Transformant S. aureus RN4220/pLI50	0.25	0.06	0.03	0.125
Transformant S. aureus RN4220/pLI50+tet(63)	64	4	0.25	0.125
E. coli DH5α	1	1	1	0.125
Transformant E. coli DH5α/pLI50	1	1	1	0.125
Transformant E. coli DH5α/pLI50+tet(63)	4	2	1	0.25

Relatedness of Tet(63) with other tetracycline efflux proteins

A phylogenetic tree based on an amino acid sequence alignment indicated that Tet(63) clustered together with Tet(K), Tet(L), Tet(45) and Tet(58) (Figure 1). According to their amino acid sequences, tetracycline efflux proteins have been differentiated into seven groups,¹² with Tet(K), Tet(L) and Tet(45) belonging to group 2.^1,13 The group 2 Tet proteins have been identified primarily in clinical isolates of the Gram-positive genera Staphylococcus, Bacillus and Streptococcus¹⁴ and possess 14 predicted TMDs.¹ The TMHMM Server predicts that the Tet(63) protein also has 14 TMDs (Figure S1).

Figure 1.

Phylogeny of known tetracycline efflux pumps. Protein sequences were aligned using ClustalX 2.0 and the tree was generated using MEGA 6.0.6. The clade containing Tet(63) is highlighted. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.

Verification of Tet(63) as an efflux protein

To confirm the function of Tet(63) as an efflux protein, the effect of two known EPIs (CCCP and reserpine) on Tet(63) was tested. In the presence of either CCCP or reserpine, the MIC of tetracycline for both the transformants S. aureus RN4220/pSA01-tet and S. aureus RN4220/pLI50+tet(63) was lowered 4- to 8-fold (Table S2). However, no change in the tetracycline MIC for S. aureus SA01 was observed, probably due to the fact that S. aureus SA01 carried a chromosomal tet(M) gene encoding a ribosome protective protein, which was not affected by EPIs.

Regulation of tet(63) gene expression

The expression of tet genes can be either inducible or constitutive. Analysis of the upstream region of the tet(63) gene revealed the presence of a regulatory region, known as the translational attenuator. Translational attenuators have already been described upstream of the tet(K) and tet(L) genes.¹⁵ In addition to a small ORF, designated lp, encoding a short leader peptide of 14 amino acids, three pairs of inverted repeated (IR) sequences were found immediately upstream of tet(63) (Figure S2). These IR sequences can form different mRNA secondary structures in the absence or presence of tetracycline, thereby allowing or preventing the translation of the tet(63) transcripts.

Characterization of plasmid pSA01-tet

Plasmid pSA01-tet is 25664 bp in size, has a GC content of 30.58% and consists of 23 ORFs (Figure 2a). This plasmid harboured one truncated (ΔrepA) and two complete (repA and repU) replication genes as well as a 705 bp ORF encoding a replication-associated protein. The region between ΔrepA and repU includes reading frames for a 171 amino acid protein with homology to the MarR family of transcriptional regulators, a 119 amino acid rhodanese-like domain-containing protein and a type II toxin–antitoxin system consisting of an 88 amino acid toxin protein and an 85 amino acid antitoxin protein. In addition to tet(63), three known antimicrobial resistance genes [aadD, aacA-aphD and erm(C)] were detected downstream of the repU gene. The aacA-aphD gene was located on the non-conjugative composite transposon Tn4001, in which this resistance gene was flanked by two copies of the IS256 element in opposite orientations. An 8 bp DR (5′-ATTATTTG-3′) was found upstream and downstream of this transposon (Figure 2b). It has been demonstrated that the aacA-aphD gene in staphylococci of poultry origin is commonly associated with complete or truncated copies of Tn4001.¹⁶ In addition, the erm(C) gene was flanked by two copies of the IS431 element in the same orientation. Immediately downstream of tet(63), the gene for a 209 amino acid recombinase family protein was found. A complete type I restriction-modification system was detected upstream of the tet(63) gene, consisting of three host specificity determining genes (hsdS, hsdR and hsdM) encoding the S, M and R subunits, respectively. Both type I restriction-modification systems and the toxin–antitoxin system have been shown to have important functions for plasmid stabilization and gene regulation.^17,18 These characteristics provide additional advantages to host bacteria that carry pSA01-tet, allowing for adjustment to various environments. The result of BLASTn, based on the complete plasmid sequence, indicated that no other plasmid so far deposited in the GenBank database showed significant similarity to pSA01-tet. However, sequence comparison revealed that the resistance region of plasmid pSA01-tet ranging from the repU gene to the rec gene exhibited high homology to the corresponding region in the chromosomal DNA of the porcine MRSA isolate NX-T55 from China (accession no. CP031839) (Figure 2b).

Figure 2.

(a) Circular presentation of the map of the plasmid pSA01-tet. ORFs with different functions are presented in various colours. The backbone regions of the plasmid and some of the more important genes of pSA01-tet are indicated in the map. The circles show (from outside to inside): predicted coding sequences, GC skew, GC content and scale in kb. (b) Comparison of plasmid pSA01-tet identified in the present study with chromosomal corresponding regions of S. aureus NX-T55 from swine in China. The arrows indicate the extents and directions of transcription of the genes. ORFs with different functions are presented in various colours. ISs are shown as black boxes with a green arrow indicating the transposase gene. The regions with >99% homology between plasmid pSA01-tet and the chromosome of S. aureus NX-T55 are indicated by grey shading. The 8 bp DRs are shown in boxes. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.

Conclusions

A novel plasmid-borne tetracycline resistance gene, tet(63), was identified in multiresistant S. aureus SA01 of chicken origin, which encoded a tetracycline-specific efflux protein of the major facilitator superfamily. It should be noted that tet(63) was co-located with several other antimicrobial resistance genes on plasmid pSA01-tet. Thus, co-selection and co-transfer of tet(63) may also occur under the selection pressure imposed by the use of the respective non-tetracycline antimicrobial agents.

Acknowledgements

We thank Marilyn C. Roberts for the designation of the novel tet(63) gene.

Funding

This work was supported by the National Key Research and Development Program of China (grant no. 2016YFD0501304 and grant no. 2017YFD0500102), the Natural Science Foundation of Heilongjiang Province of China (YQ2019C031) and the German Federal Ministry of Education and Research (BMBF) under project number 01KI1727D as part of the Research Network Zoonotic Infectious Diseases.

Transparency declarations

None to declare.

Supplementary data

Tables S1 and S2 and Figures S1 and S2 are available as Supplementary data at JAC Online.

Author notes

Yao Zhu and Changzhen Wang authors contributed equally to the work.