Studies on the Transmission of a Tigecycline Resistance-Mediating tet(A) Gene Variant from Enterobacter hormaechei via a Two-Step Recombination Process.

To investigate the contribution of a tet(A) variant to tigecycline resistance in Enterobacter hormaechei and the recombination events that occurred during transmission of this variant. MICs were determined by broth microdilution. E. hormaechei G17 was characterized by PCR, transfer assay, S1-PFGE, Southern blot hybridization, and WGS analysis. A tet(A) variant conferring resistance to tigecycline was present in E. hormaechei G17. This strain harbored two resistance plasmids (pG17-1, 264,084 bp and pG17-2, 68,610 bp) and its E. coli transformant Tm-G17TGC one resistance plasmid (pTm-G17, 93,013 bp). The comparative analysis of pG17-1, pG17-2, and pTm-G17 showed that a tet(A) variant-carrying multiresistance gene cluster (~23 kb) originating from pG17-1 had integrated into pG17-2, forming the novel plasmid pTm-G17. In a first step, this multiresistance gene cluster was excised from pG17-1 by recombination of homologous sequences, including △TnAs1 at both termini, thereby generating an unconventional circularizable structure (UCS). In a second step, this UCS integrated into pG17-2 via recombination between homologous sequences, including IS26 present on both, the UCS and pG17-2, thereby giving rise to the new plasmid pTm-G17. In summary, a tet(A) variant conferring resistance to tigecycline was reported in E. hormaechei. Transfer of a tet(A) variant-carrying multiresistance gene cluster between plasmids occurred in a two-step recombination process, in which homologous sequences, including either △TnAs1 or IS26, were involved. IMPORTANCE Tigecycline is an important last-resort broad spectrum antimicrobial agent. This study describes the two-step recombination processes resulting in the transfer of the tet(A) variant gene between different plasmids in E. hormaechei, which depicts the role of recombination processes in the generation of UCSs and new plasmids, both carrying a tet(A) variant conferring resistance to tigecycline. Such processes enhance the dissemination of resistance genes, which is of particular relevance for resistance genes, such as the tet(A) variant. The presence and transmission of a tet(A) variant in E. hormaechei will compromise the efficacy of tigecycline treatment for E. hormaechei associated infection.

INTRODUCTION

Enterobacter cloacae complex (ECC) is versatile, and the taxonomic status is constantly updated. There are more than 20 species and clades (without taxonomy terms) classified into it (1). It has been reported that Enterobacter hormaechei and E. kobei are the predominant ESBL-positive species accounting for community-acquired ECC strains collected from northern China (2). Nowadays the widespread existence of carbapenem-producing Enterobacteriaceae (CPE), has greatly limited the use of carbapenems in clinical treatment (3). However, the use of the alternative antimicrobial agents colistin and tigecycline is also restricted by newly emerged plasmid-mediated resistance genes, such as mcr, tet(A) and tet(X) variants (4–7).

Tigecycline belongs to the glycylcyclines, a new class of tetracycline derivatives. It is a broad-spectrum agent and exhibits bacteriostatic activity by binding to the 30S ribosomal subunit of bacteria (8). The mechanisms of resistance to tigecycline mainly include overexpression of efflux systems and enzyme modification. The AcrAB mutant efflux pump was the major mechanism of tigecycline resistance among Gram-negative bacteria, including Klebsiella pneumoniae and Enterobacter cloacae (9, 10). In recent years, it was reported that in Klebsiella pneumoniae, Enterobacter cloacae and Salmonella Typhimurium, the mutation of the ramR gene leaded to the increased expression of AcrAB efflux pump, which enhanced the resistance to various drugs, including tigecycline (11–13). In 2010, it was discovered in Salmonella that a variant of the efflux gene tet(A) can lead to low-level tigecycline resistance (14). The TetX enzyme, which modifies tigecycline, was first reported in 2005 (15). Two plasmid-mediated high-level tigecycline resistance genes tet(X3) and tet(X4) were identified in 2019 (6, 7). The latest tet(X) variants reported in 2021 include 27 new variants, from tet(X18) to tet(X44), which were identified in Riemerella anatipestifer (16), and tet(X15) in Acinetobacter variabilis (17).

Insertion sequences (ISs) seem to play an important role in the dissemination of antimicrobial resistance genes in both Gram-positive and Gram-negative bacteria. If two identical ISs, located in the same orientation, bracket DNA sequences, these two ISs can recombine and form translocatable units (TUs), which contain one copy of the IS and the region between the two ISs, which often comprises resistance genes (18, 19). These TUs may then integrate into plasmids or chromosomal sites, thereby fostering the dissemination of resistance genes (19). More rarely, it has been reported that circular structures can also be formed through recombination of homologous sequences at both termini that lack recombinase genes (20–25). These structures were tentatively referred to as unconventional circularizable structures (UCSs) in 2013 (26). The existence of these structures broadens the way for the spread of resistance genes.

In this study, the contribution of a tet(A) variant to tigecycline resistance in E. hormaechei was investigated. In addition, the transmission of this variant was analyzed with regard to the formation of an UCS and the integration of this UCS into a new plasmid background.

RESULTS AND DISCUSSION

Two multidrug resistance plasmids harboring UCS in E. hormaechei G17.

E. hormaechei strain G17 contained two plasmids, designated pG17-1 and pG17-2, which were 264,084 bp and 68,610 bp in size, respectively (Fig. 1). Both of them were hybrid plasmids, with pG17-1 belonging to the incompatibility group IncHI2/IncHI2A, and pG17-2 to IncFIA(HI1)/IncR. pG17-1 harbored a tigecycline resistance-mediating tet(A) gene variant, the rifamycin resistance gene arr-3, the phenicol resistance gene floR, the aminoglycoside resistance genes aph(6)-Id, aac(3)-IId, aac(3′)-Ia, and aadA22, the sulfonamide resistance gene sul3, the trimethoprim resistance gene dfrA14, the lincomycin resistance gene lnu(F), the quinolone resistance gene qnrS1, the β-lactam resistance genes blaCTX-M-55, blaLAP-2, and blaTEM-1B, as well as the macrolide resistance genes mph(A) and mef(B) (Fig. 1a). As shown in Fig. 2, the tet(A) variant was flanked by copies of homologous sequences, including △TnAS1, which can recombine and form an UCS, designated UCS1. This was also evidenced by PCR using the primers listed in Table S1 and Sanger sequencing (Fig. S1).

FIG 1

The structures of plasmids pG17-1 (a) and pG17-2 (b) from E. hormaechei G17. The circles in (a) and (b) depict (from the outside to inside): (i) the size scale in bp; (ii) the positions of predicted coding sequences transcribed in the clockwise orientation; (iii) the positions of predicted coding sequences transcribed in the counterclockwise orientation; (iv) the GC content plotted against 50%, with blue indicating >50% and green indicating <50%; and (v) GC skew [(G- C)/(G+C)] in a 10,000 bp window. Genes are color-coded, depending on functional annotations: red, antimicrobial resistance; gray, transposition; black, other genes and plasmid replication.

FIG 2

Transfer of a tet(A) variant-carrying multiresistance gene cluster between the plasmids via a two-step recombination process revealed by the comparative analysis of the relevant regions of plasmids pG17-1, pG17-2, and pTm-G17. UCS1 was formed by the recombination between a pair of similar repeated sequences (blue dashed boxes), covered by a horizontal straight line. UCS1 integrated into pG17-2 via another pair of homologous sequences (red dashed boxes). The integration process was shown in the dotted line. The genes are shown as arrows, with the arrowhead indicating the direction of transcription. Antimicrobial resistance genes are shown in red, the transposition-related genes in gray and other genes in black. The gray-shaded area indicates regions of >99% nucleotide sequence identity.

pG17-2 carried the aforementioned resistance genes arr-3 and floR, but also the tetracycline resistance gene tet(D), the aminoglycoside resistance genes aadA16 and aac(6’)-Ib-cr, the sulfonamide resistance gene sul1, the trimethoprim resistance gene dfrA27, and the quinolone resistance gene qnrB6 (Fig. 1b). The quinolone resistance gene qnrB6 located downstream of the class I integron was flanked by two copies of homologous sequences, including qacEΔ1 and sul1 genes on pG17-2. Each of these copies was 1,181 bp in size and the upstream and downstream copies were identical in their nucleotide sequences. PCR revealed that UCS2 consisting of the qnrB6 gene and one copy of hybrid sequence derived from recombination between the homologous sequences, including qacEΔ1 and sul1 genes was formed (Fig. S1), suggesting that the UCS2 containing qnrB6 is active.

tet(A) variant on pG17-1 in E. hormaechei was identified to confer resistance to tigecycline.

In order to verify whether the tet(A) variant in E. hormaechei G17 confers resistance to tigecycline, E. hormaechei G17 was used as the donor in conjugation and transformation experiments. No transconjugant was obtained in multiple attempts when using 0.25 mg/L tigecycline for screening. However, transformants were obtained on screening media supplemented with 0.25 mg/L tigecycline or 4 mg/L tetracycline, respectively. Two different transformants, designated E. coli Tm-G17TGC (selected from media supplemented with 0.25 mg/L tigecycline) and E. coli Tm-G17TET (selected from media supplemented with 4 mg/L tetracycline), respectively, were chosen for further analysis. The transformant E. coli Tm-G17TGC displayed resistance to both tigecycline (8-fold MIC increase) and tetracycline (>128-fold MIC increase) compared with the recipient E. coli DH5α. However, the transformant E. coli Tm-G17TET displayed no increase in the MIC of tigecycline, but a >64-fold increase in the MIC of tetracycline, compared with the recipient E. coli DH5α (Table 1).

TABLE 1

MICs of E. hormaechei G17, transformant Tm-G17TET, transformant Tm-G17TGC and E. coli DH5α

Strains	MICs (mg/L)a
	TET	TGC	CHL	FEP	TMP	CAZ	RIF
E. hormaechei G17	256	8	>512	128	2	512	>512
Tm-G17TGC	128	1	256	<1	2	<1	>512
Tm-G17TET	64	0.125	128	<1	2	<1	512
E. coli DH5α	<1	0.125	2	<1	<1	<1	8

TET, tetracycline; TGC, tigecycline; CHL, chloramphenicol; FEP, cefepime; TMP, trimethoprim; CAZ, ceftazidime; RIF, rifampin.

PCR analysis revealed that the transformant E. coli Tm-G17TGC was not only positive for the conserved backbone sequence on pG17-2, but also positive for both, the tet(A) variant and tet(D), whereas the transformant E. coli Tm-G17TET was only positive for the conserved backbone sequence on pG17-2 and tet(D). S1-PFGE revealed that E. hormaechei G17 contained two plasmids (pG17-1, ~264 kb and pG17-2, ~68 kb), E. coli Tm-G17TGC and E. coli Tm-G17TET contained an ~93 kb plasmid (pTm-G17) and an ~68 kb plasmid (pG17-2), respectively (Fig. S2). Southern blot hybridization showed that the tet(A) variant was originally located on the ~264 kb pG17-1 in E. hormaechei G17, but appeared on the ~93 kb plasmid pTm-G17 in E. coli Tm-G17TGC after transformation, suggesting the tet(A) variant can be transferred between the plasmids. Protein sequence analysis with the software DNAMAN8.0 indicated that the Tet(A) in E. hormaechei G17 in this study had the same amino acid sequence as the previously reported Tet(A) variant in Klebsiella pneumoniae KP267 (Fig. S3), which had been proved to confer resistance to tigecycline (4). There are seven amino acid substitutions occurring in the deduced amino acid sequence of the Tet(A) from E. hormaechei G17 (Identity, 99.42%), compared with the reference RP1 Tet(A) from E. coli plasmid RP1 (27, 28). Of them, the key amino acid substitutions 201-SFV-203 in Tet(A) from RP1 to 201-ASF-203 in Tet(A) from pGF17 and K. pneumoniae KP267 had been considered to be involved in tigecycline resistance (28).

The tet(A) variant was transferred between the plasmids via a two-step recombination process.

The comparative analysis of plasmids pG17-1, pG17-2, and pTm-G17 showed that a tet(A) variant-carrying multiresistance gene cluster (~23 kb), originating from pG17-1 had integrated into pG17-2, forming the novel plasmid pTm-G17 (~93 kb, Fig. 2). The specific integration process is as follows: in a first step, the tet(A) variant-carrying multiresistance gene cluster was looped out from plasmid pG17-1 by recombination between homologous sequences of 849 bp, which contained a truncated copy of transposon TnAs1, designated △TnAs1.These homologous sequences were present at both termini of the cluster (Fig. 2), and recombination between them formed UCS1. In a second step, the tet(A) variant-carrying UCS1 integrated into pG17-2 via another pair of homologous sequences which were present on both the UCS1 and plasmid pG17-2. These homologous sequences had a size of 2,470 bp and contained a copy of IS26 (Fig. 2). In pTm-G17, the pG17-1-derived multiresistance gene cluster was bracketed by copies of the homologous sequences containing IS26. Where and when plasmid pTm-G17 was generated is unknown. However, since both partner plasmids, pG17-1 and pG17-2, are required for the formation of pTm-G17, it is likely that pTm-G17 was already formed in the donor strain and was present there at a very low copy number, too low to be detected by PFGE and subsequent hybridization. When transformed into E. coli, this plasmid became the only plasmid in the new host and was now detectable at the regular copy number. The two-step recombination processes detailed above resulted in the transfer of the tet(A) variant gene between different plasmids. Such processes enhance the dissemination of resistance genes, which is of particular relevance for resistance genes, such as the tet(A) variant gene which mediates resistance not only to tetracycline but also to tigecycline.

MATERIALS AND METHODS

Bacterial strains and antimicrobial susceptibility testing.

E. hormaechei strain G17 was obtained from the liver of a diseased duck in a traditional duck farm in Henan province, China, in 2019. It was identified and stored in our lab. Antimicrobial susceptibility testing (AST) was conducted by broth microdilution according to the recommendations of the Clinical and Laboratory Standards Institute (CLSI) for tetracycline, tigecycline, chloramphenicol, cefepime, trimethoprim, ceftazidime and rifampin (29). The tigecycline MICs were interpreted according to the recommendations of EUCAST (https://www.eucast.org/ast_of_bacteria/). E. coli ATCC 25922 served as the quality control strain in AST.

Transfer experiments.

Conjugation and transformation experiments were performed using E. hormaechei G17 as the donor, and E. coli DH5α as the recipient. For the screening of the transconjugants, LB agar was supplemented with 0.25 mg/L tigecycline. For the screening of the transformants, LB agar was supplemented with 0.25 mg/L tigecycline or 4 mg/L tetracycline, respectively. Colonies that grew on these selective plates at 37°C after incubation for 24 h were further confirmed by AST as well as PCR amplification followed by Sanger sequencing and analysis of the tet(A) gene.

PCR analysis.

The resistance gene tet(A) variant, the conserved backbone sequence on the plasmids pG17-1 or pG17-2, and the presence of UCSs were detected by PCR using the primers and conditions listed in Table S1 (available as supplemental material). All the PCR products were subjected to Sanger sequencing.

S1-PFGE and Southern blot hybridization.

The genomic DNA of the donor E. hormaechei G17, the recipient E. coli DH5α and two transformants E. coli Tm-G17TGC and E. coli Tm-G17TET in agarose gel plugs were digested with S1 endonuclease (TaKaRa, Dalian, China), separated by PFGE as previously described (30), transferred to Amersham Hybond-N+ membranes (GE Healthcare), and hybridized with a digoxigenin-labeled tet(A) variant probe (tet[A] [nt 7292–7676; X61367.1]). Detection was performed by using a DIG-DNA labeling and detection kit (Roche Diagnostics, Germany).

Sequencing and sequence analysis.

The whole-genome DNA of E. hormaechei strain G17 and the transformant E. coli Tm-G17TGC were sequenced by using the PacBio RS and Illumina MiSeq platforms (Shanghai Personal Biotechnology Co., Ltd., China). The PacBio sequence reads were assembled with HGAP4 and CANU (Version 1.6), corrected by Illumina MiSeq with pilon (Version 1.22). The prediction of ORFs and their annotations were performed using Glimmer 3.0. The blast software was used following the procedures at https://blast.ncbi.nlm.nih.gov. The Illumina short read sequences and PacBio long read sequences were mapped to the spliced sequences by BWA (Version: 0.7.17-r1188), and the chromosome and plasmid depths were calculated by mosdepth 0.3.3. In G17 strain, the read depths of chromosome and three plasmids by short sequence alignment were 2.71, 27.47, 16.01, and 140, respectively, and the read depths of long sequence alignment were 31.38, 13.94, 13.99, and 30.20, respectively. The read depths of chromosome and a plasmid in Tm-G17TGC by short sequence alignment were 258.85 and 470.75, respectively, and the read depths of long sequence alignment were 325.56 and 264.78, respectively.

Data availability.

The sequences of three plasmids pG17-1, pG17-2 and pTm-G17 and two chromosomes from G17 and Tm-G17TGC determined in this study have been deposited in GenBank under accession numbers CP079936.1, CP079937.1, CP080248.1, CP079939.1, and CP080247.1, respectively.