Multiple Copies of Mobile Tigecycline Resistance Efflux Pump Gene Cluster tmexC2D2.2-toprJ2 Identified in Chromosome of Aeromonas spp.

ABSTRACT

The appearance and prevalence of novel plasmid-encoded tigecycline resistance efflux pump gene clusters tmexC1D1-toprJ1 and tmexC2D2-toprJ2 in Enterobacteriaceae have raised a threat to public health. Here, another tigecycline resistance gene cluster, tmexC2D2.2-toprJ2, was identified in two Aeromonas isolates recovered from fish meat and vegetables. Cloning confirmed the expression of tmexC2D2.2-toprJ2 mediated the resistance to tigecycline and decreased susceptibility to tetracyclines and cephalosporins in both Escherichia coli and Aeromonas. In an Aeromonas veronii strain, four copies of tmexC2D2.2-toprJ2 were located on the chromosome. Further analysis revealed that tmexC2D2.2-toprJ2 has been detected in the chromosomes of A. veronii, Aeromonas hydrophila, and Aeromonas caviae with one to four copies due to the insertion of a potential integrative transferable unit. The occurrence of multiple copies of chromosomal tmexC2D2.2-toprJ2 may act as a sink for this tigecycline resistance gene cluster, which requires continuous monitoring.

IMPORTANCE Tigecycline is regarded as one of the few effective drugs against multidrug-resistant bacterial infection. However, mobile tigecycline resistance efflux pump gene clusters such as tmexC1D1-toprJ1 and its variants have been identified in both animal- and human-origin Enterobacteriaceae. In this study, we first found another efflux pump gene cluster, tmexC2D2.2-toprJ2, in the Aeromonas chromosome. This gene cluster could mediate tigecycline resistance and decrease susceptibility to tetracyclines and cephalosporins in the Aeromonas host strain. Meanwhile, tmexC2D2.2-toprJ2 was detected with multiple copies in Aeromonas spp. This multidrug resistance efflux pump gene cluster with multiple copy numbers might stably exist in Aeromonas and serve as a reservoir for tmexCD2-toprJ2, facilitating its persistent presence and spread.

OBSERVATION

Antimicrobial resistance (AMR) is increasingly threatening public health, leaving tigecycline as one of the few effective drugs against multidrug-resistant bacteria (1, 2). The emergence of two types of novel mobile tigecycline resistance mechanisms, including plasmid-encoded efflux pump gene clusters (tmexCD1-toprJ1, tmexCD2-toprJ2, tmexCD3-toprJ1b, and tmexCD4-toprJ4) and the tigecycline-modifying enzyme Tet(X4) and its variants, has severely compromised the efficacy of this last-resort antibiotic (3–7). In addition to tigecycline, plasmid-carried tmexCD-toprJ-like efflux pump gene clusters can also confer resistance to other antimicrobials (3–6). The tmexCD-toprJ-like gene clusters have been identified mainly in China, but have disseminated to other countries (6, 8). tmexCD1-toprJ1 is the most widely disseminated gene cluster and has been identified in isolates from various sources, with chickens and Klebsiella pneumoniae being the major carrier and host bacterium, respectively (4, 9). In contrast, tmexCD2-toprJ2 is commonly found in patient-derived samples (6, 10, 11). Recent investigations have found tmexCD2-toprJ2 to be frequently accompanied by carbapenem resistance genes in Raoultella and Klebsiella strains, leading to resistance to both tigecycline and carbapenem (6, 10, 11). Although tmexCD2-toprJ2 was primarily identified in plasmids of Klebsiella and Raoultella spp., whether it has spread and conferred tigecycline resistance in other bacterial species remains unknown. In the present study, we characterized two foodborne Aeromonas strains carrying multiply copies of chromosomal tmexCD2-toprJ2 for the first time.

In December 2021, 45 retail fish meat and 125 fresh vegetable samples were collected from 20 farmers’ markets in Guangzhou, China. Strains GD21SC2284TT and GD21SC2322TT were isolated from fish meat and vegetables, respectively, using MacConkey agar supplemented with 4 mg/L tigecycline. The two isolates were identified as Aeromonas hydrophila and Aeromonas veronii using matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) (Bruker Daltonics, Bremen, Germany). Further PCR and Sanger sequencing confirmed that these two strains were positive for the tmexCD2-toprJ2 efflux pump gene cluster.

Antimicrobial susceptibility testing profiles of the two Aeromonas strains were determined using the broth or agar dilution method recommended by the Clinical and Laboratory Standards Institute (CLSI) with Escherichia coli ATCC 25922 as the quality control strain. The results were interpreted according to CLSI documents (12, 13). Both strains exhibited resistance to tigecycline (8 mg/L), tetracycline (128 mg/L), minocycline, doxycycline, gentamicin, cefquinome, ciprofloxacin (64 mg/L), and florfenicol (Table 1 and see Table S1 in the supplemental material). The two strains were susceptible to ceftazidime and imipenem (both 0.06 mg/L), as well as colistin (2 mg/L). To determine the profile of antibiotic resistance genes (ARGs) and the location of tmexCD2-toprJ2, the two Aeromonas strains were further subjected to whole-genome sequencing using the Illumina HiSeq and Nanopore MinION platforms. The complete genome data were assembled using Unicycler v.0.4.8 (14). Strain GD21SC2322TT contained a 4.70-Mb chromosome and a plasmid. The strain harbored 10 ARGs, including bla_TEM-1B, aac(6′)-Ib-cr, tet(A), and qnrS2 (Table S1), and tmexCD2-toprJ2 was located on the chromosome. GD21SC2284TT harbored a 5.01-Mb chromosome and two plasmids, wherein chromosomal tmexCD2-toprJ2 and β-lactamase genes bla_CTX-M-3 and bla_TEM-1B, together with other 14 ARGs, were identified (Table S1).

TABLE 1.

MICs of various antimicrobial agents against tmexC2D2.2-toprJ2-carrying strains and transformants^a

Strain	Strain information	MIC (mg/L) ofa:
		TIG	MIN	DOX	TET	CTX	CAZ	FEP	CQM	CIP
Aeromonas veronii GD21SC2322TT	Vegetable	8	16	128	128	0.125	0.5	1	2	64
Aeromonas hydrophila GD21SC2284TT	Fish meat	8	32	128	128	16	2	8	>128	64
E. coli
DH5α	E. coli recipient strain	0.25	1	1	1	0.015	0.06	0.03	0.03	0.001
DH5α/pHSG575	Transformant with low-copy-number vector pHSG575	0.25	1	1	1	0.015	0.06	0.03	0.03	0.001
DH5α/pHSG575-tmexC2D2-toprJ2	Transformants expressing tmexC2-tmexD2-toprJ2	2	4	4	4	0.125	0.25	0.125	0.25	0.008
DH5α/pHSG575-tmexC2D2.2-toprJ2	Transformants expressing tmexC2-tmexD2.2-toprJ2	2	4	4	4	0.125	0.25	0.125	0.25	0.008
DH5α/pHGR	Transformant with medium-copy-number vector pHGR	0.25	1	1	1	0.015	0.06	0.03	0.03	0.001
DH5α/pHGR-tmexC2D2.2-toprJ2	Transformants expressing tmexC2-tmexD2.2-toprJ2	4	8	8	8	0.25	0.5	0.5	1	0.008
A. veroniii
1AV	A. veroniii recipient strain	0.25	0.5	0.25	0.25	0.015	0.125	0.03	0.06	0.004
1AV/pHGR	A. veroniii strain expressing empty vector	0.25	0.5	0.25	0.25	0.015	0.125	0.03	0.06	0.004
1AV/pHGR-tmexC2D2.2-toprJ2	A. veroniii strain expressing tmexC2-tmexD2.2-toprJ2	4	4	1	1	0.06	0.25	0.25	0.5	0.008

^aTIG, tigecycline; MIN, minocycline; DOX, doxycycline; TET, tetracycline; CTX, cefotaxime; CAZ, ceftazidime; FEP, cefepime; CQM, cefquinome; CIP, ciprofloxacin.

The nucleotide sequences of the tmexCD2-toprJ2 gene cluster identified in the two Aeromonas strains were identical. Moreover, compared to the earliest reported tmexCD2-toprJ2 cluster (6), the efflux pump gene cluster found in this study had one nucleotide difference (T167A) in tmexD2, resulting in an amino acid change (Val56Glu) in TMexD2. This gene cluster was designated tmexC2-tmexD2.2-toprJ2. To assess the effect of this amino acid difference in TMexD2.2 on the function of TMexCD2-TOprJ2, the DNA fragment of tmexC2D2.2-toprJ2 was amplified and cloned into the low-copy-number vector pHSG575 with the primers listed in Table S2 and then transformed into E. coli DH5α. Compared with E. coli DH5α carrying empty vector, an 8-fold increase in tigecycline MIC and a 4- to 8-fold increase in MICs of other tetracyclines and cephalosporins were observed in E. coli host strain carrying pHSG575-tmexC2D2.2-toprJ2. Relative to the previously reported antibiotic resistance phenotype mediated by tmexCD2-toprJ2 (6), the same MIC values against various drugs were observed in the E. coli host strain expressing tmexC2D2.2-toprJ2. The findings indicated that this amino acid substitution (Val56Glu) in TMexD2 does not affect the function of the TMexCD2-TOprJ2 efflux system. To further investigate the function of TMexC2D2.2-TOprJ2 in Aeromonas spp., tmexC2D2.2-toprJ2 was cloned into the plasmid pHGR, a broad-host-range vector with medium copy number, and transformed into E. coli DH5α cells. The recombinant plasmid pHGR-tmexC2D2.2-toprJ2 with the correct sequences was further electroporated into environmental A. veronii strain 1AV. The MIC values of tigecycline and cephalosporins in E. coli DH5α bearing pHGR-tmexC2D2.2-toprJ2 were 2-fold higher than those of the same host strain harboring pHSG575-tmexC2D2.2-toprJ2 and pHSG575-tmexC2D2-toprJ2, which was due to the 5-fold to 14-fold-higher expression of the tmexC2D2.2-toprJ2 gene cluster in the pHGR vector than that in pHSG575 (Fig. 1A). Furthermore, the expression of tmexC2D2.2-toprJ2 in Aeromonas host strain 1AV resulted in a 16-fold increase in tigecycline MIC relative to that of the empty vector, indicating that tmexC2D2.2-toprJ2 could also confer tigecycline resistance in Aeromonas. The recombinant strain A. veronii 1AV harboring pHGR-tmexC2D2.2-toprJ2 exhibited 2- to 8-fold-higher MICs of other tetracyclines, cephalosporins, and ciprofloxacin than strains carrying empty plasmids. These results revealed that TMexC2D2.2-TOprJ2 could mediate tigecycline resistance and confer decreased susceptibility to multiple drugs in Aeromonas.

FIG 1.

Transcription level of tmexC2D2.2-toprJ2 in two Escherichia coli recombinant strains (A) and two wild-type Aeromonas strains (B). (A) Transcriptional expression level of tmexC2D2.2-toprJ2 in DH5α carrying pHGR-tmexC2D2.2-toprJ2 compared with that in DH5α carrying pHSG575-tmexC2D2.2-toprJ2; (B) expression level of tmexC2D2.2-toprJ2 in Aeromonas veronii GD21SC2322TT compared with that of Aeromonas hydrophila GD21SC2284TT. The data were analyzed by quantitative reverse transcription-PCR (qRT-PCR) and are shown as the relative mRNA fold changes normalized to 16S rRNA. Significant differences were analyzed by the unpaired Student's t test and are shown as follows: ***, P < 0.001; **, P < 0.01; *, P < 0.05. (C) Comparison of the genetic environment of tmexC2D2.2-toprJ2 in Aeromonas strains with those of closely similar sequences. Arrows with different colors indicate the extents and directions of different genes. The shaded region represents the 99% and 100% homology areas. The tmexC2D2.2-toprJ2-bearing IME regions are shown in the box, and the insertion sites are directly indicated. The truncated gene is indicated by the symbol Δ.

While GD21SC2284TT contained one copy of tmexC2D2.2-toprJ2 on the chromosome, intriguingly, four copies of tmexC2D2.2-toprJ2 were found on the chromosome of GD21SC2322TT, with genetic locations far from each other, two of which were relatively close (distance of 51 kb). Notably, although the same level of tigecycline resistance was observed between the two tmexC2D2.2-toprJ2-harboring Aeromonas strains (Table 1), GD21SC2322TT exhibited a higher tmexC2D2.2-toprJ2 transcriptional expression level than that of GD21SC2284TT (Fig. 1B). To find out how the multiply copies of tmexC2D2.2-toprJ2 appeared in strain GD21SC2322TT, the genetic context of tmexC2D2.2-toprJ2 was analyzed. Similarly to tmexCD1-toprJ1, tmexCD2-toprJ2, and tmexCD3-toprJ1b, chromosomal tmexC2D2.2-toprJ2 gene clusters in GD21SC2284TT and GD21SC2322TT were surrounded by the same upstream genetic structures, containing two integrase genes (int1 and int2) and two hypothetical genes (hp1 and hp2) (4–6), which formed an int1-like–int2-like–hp1–hp2–tnfxB2–ISBvi2–tmexC2–tmexD2.2–toprJ2 structure. The structure was assumed to be a novel integrative and mobilizable element (IME) (15). Furthermore, similarly to the IME units bearing tmexCD2-toprJ2 and tmexCD3-toprJ1b, the five tmexC2D2.2-toprJ2 IME units in GD21SC2322TT and GD21SC2284TT were all specifically inserted into umuC-like genes. Although the sequences of these umuC genes were not completely identical, relatively conserved insertion sites of these IME units were observed (Fig. 1C). BLAST analysis revealed multiple copies of tmexC2D2.2-toprJ2 in other Aeromonas species. Aeromonas caviae strain NUITM-VA2 from Vietnam (AP025280.1) carried four copies of the chromosome-located tmexC2D2.2-toprJ2, whereas two copies of tmexC2D2.2-toprJ2-like gene cluster, with one nucleotide difference compared with tmexD2.2, were found on the chromosome of the environmental A. hydrophila strain WCHAH045096 from China (CP028568.2). A single copy of chromosomal tmexC2D2.2-toprJ2 was identified in the human-derived A. caviae strain K433 from China (CP084031.1). The IME units carrying tmexC2D2.2-toprJ2 in these three Aeromonas strains were also inserted in umuC-like genes with similar insertion sites (Fig. 1C). In addition, our previously reported tmexCD2-toprJ2 IME identified in Raoultella ornithinolytica had two copies, of which one was inserted into the chromosome and another in the plasmid (6), illustrating the high transferability of the tmexCD-toprJ-like-carrying IME integrative units.

In addition to the multiple copies of tmexC2D2.2-toprJ2, two or more copies of other resistance genes, including tet(X) variants and β-lactamase genes, have been increasingly found (16–18).

These events are linked to intracellular transposition or recombination events generated by mobile genetic elements (MGEs) (19). For example, tet(X4) was observed in the form of tandem repeats in plasmids mediated by ISCR2 or IS26 (16). bla_NDM and bla_KPC were also observed for their increased copy numbers in clinical isolates after exposure to novel β-lactams, including cefiderocol and ceftazidime-avibactam (17, 18), indicating that selective pressure from antibiotics could facilitate the occurrence of multiple copy numbers of these AMR determinants (20). Consequently, the increased copy number of tmexC2D2.2-toprJ2 might be due to multiple insertion events mediated by the integrative unit occurring under drug stress, which needs further investigation. Despite the limited widespread transfer risk of chromosomal tmexC2D2.2-toprJ2, the multidrug resistance efflux pump gene cluster with multiple copy numbers might stably exist in Aeromonas and serve as a reservoir for tmexC2D2.2-toprJ2, facilitating the persistent presence of this novel ARG.

In conclusion, two multidrug-resistant Aeromonas strains carrying multiple copies of chromosomal tmexC2D2.2-toprJ2 were reported for the first time. TMexC2D2.2-TOprJ2 confers tigecycline resistance in both E. coli and Aeromonas spp. Given the wide distribution of Aeromonas species in aquatic environments and humans, the existence of tmexC2D2.2-toprJ2 with multiple copy numbers on the Aeromonas chromosome may act as a sink for this ARG, thus, requiring continuous monitoring.

Data availability.

The complete genome data of the Aeromonas strains GD21SC2322TT and GD21SC2284TT have been deposited in GenBank under accession no. CP102108, CP102109, CP102110, CP102111, and CP102112, respectively.