Pseudomonas Acts as a Reservoir of Novel Tigecycline Resistance Efflux Pump tmexC6D6-toprJ1b and tmexCD-toprJ Variants

ABSTRACT Several variants of the plasmid-carried tigecycline resistance gene cluster, tmexCD-toprJ, have been identified. This study characterized another novel variant, tmexC6D6-toprJ1b, located on the chromosome of environmental-origin Pseudomonas mendocina. TMexC6D6-TOprJ1 mediates resistance to multiple drugs, including tigecycline. The promoter activity of tmexC6D6-toprJ1b and negative transcriptional repression by the upstream regulator tnfxB6 are crucial for the expression of tmexC6D6-toprJ1b. tmexC6D6-toprJ1b was found in the plasmids or chromosomes of different Pseudomonas species from six countries. Two genetic backgrounds, class 1 integrons and int-carrying integrase units, were found adjacent to the tmexC6D6-toprJ1b gene cluster and might mediate the transfer of this novel efflux pump gene cluster in Pseudomonas. Further phylogenetic analysis revealed Pseudomonas as the major reservoir of tmexCD-toprJ variants, warranting closer monitoring in the future. IMPORTANCE Tigecycline is one of the treatment options for serious infections caused by multidrug-resistant bacteria, and tigecycline resistance has gained extensive attention. The emergence of a transferable tigecycline resistance efflux pump gene cluster, tmexCD-toprJ, severely challenged the efficiency of tigecycline. In this study, we identified another novel tmexCD-toprJ variant, tmexC6D6-toprJ1b, which could confer resistance to multiple classes of antibiotics, including tigecycline. Although tmexC6D6-toprJ1b was found only in Pseudomonas species, tmexC6D6-toprJ1b might spread to Enterobacteriaceae hosts via mobile genetic elements resembling those of other tmexCD-toprJ variants, compromising the therapeutic strategies. Meanwhile, novel transferable tmexCD-toprJ variants are constantly emerging and mostly exist in Pseudomonas spp., indicating Pseudomonas as the important hidden reservoir and origin of tmexCD-toprJ variants. Continuous monitoring and investigations of tmexCD-toprJ are urgent to control its spread.

In June 2022, isolate GD22SC3150TT was collected from an environmental sample of a farmers' market in Guangdong province of China using MacConkey agar plates supplemented with 4 mg/L tigecycline. GD22SC3150TT was classified as P. mendocina via matrixassisted laser desorption ionization-time of flight mass spectrometry (Bruker Daltonics, Bremen, Germany). Moreover, a PCR screening assay confirmed that a tmexCD-toprJ-like gene cluster was carried by this strain. Antimicrobial susceptibility testing was performed using the broth or agar dilution method (testing for tigecycline and colistin), according to the Clinical and Laboratory Standards Institute guidelines (16). GD22SC3150TT was found to be resistant to tigecycline (MIC = 8 mg/L), ceftazidime (MIC = 8 mg/L), cefotaxime (MIC = 16 mg/L), cefepime (MIC = 8 mg/L), and ciprofloxacin (MIC . 64 mg/L) but susceptible to colistin and imipenem (Table 1).
To determine the genetic location of this tmexCD-toprJ-like gene cluster, the complete genomic data of GD22SC3150TT were generated using the Illumina platform and Nanopore MinION, together with Unicycler version 0.4.7 for genome assemblies (17). GD22SC3150TT consisted of a 4,398,051-bp chromosome and a 35,410-bp plasmid. Six known ARGs, namely, determinants conferring resistance to aminoglycosides [aac(69)-IIa and aadA8], chloramphenicol (cmlA1), quinolone (qnrVC1), sulfonamide (sul1), and tetracycline [tet(G)], were found on the chromosome. Intriguingly, the tmexCD-toprJ-like gene cluster was also detected on the chromosome, accompanied by an adjacent tnfxB-like regulator gene, which  Table S1 in the supplemental material). In GD22SC3150TT, TMexC shared 96.90 to 98.45% amino acid identity with TMexC1 to TMexC4, and TMexD shared 97.61 to 99.90% amino acid identity with TMexD1 to TMexD5 (Table S1). The nucleotide sequence of the toprJ gene in GD22SC3150TT was the same as that of toprJ1b in tnfxB3-tmexCD3-toprJ1b (Table S1). Therefore, this gene cluster was designated tnfxB6-tmexC6D6-toprJ1b with the corresponding proteins being TNfxB6, TMexC6, TMexD6, and TOprJ1, respectively. To fully determine the evolutionary relationship between tmexC6D6-toprJ1b and various tmexCD-toprJ members, a phylogenetic tree was constructed based on the three protein sequences encoded by tmexCD-toprJ using MEGA X and iTOL (Fig. 1) (18, 19). A total of 46 representative nonredundant nucleotide sequences of tmexCD1-toprJ1-like gene clusters from FIG 1 Phylogenetic tree of tmexC6D6-toprJ1b gene clusters and its homologs. A total of 46 representative nonredundant nucleotide sequences of tmexCD1-toprJ1like gene clusters from GenBank were selected. This tree was constructed by MEGA-X using the neighbor-joining method with 2,000 replicates. Basic information on these tmexCD-toprJ-carrying bacteria, including their GenBank accession numbers, is shown in the corresponding branch. The six background colors indicate the six types of tmexCD-toprJ gene clusters.
To investigate the function of tmexC6D6-toprJ1b, we constructed plasmids containing tmexC6D6-toprJ1b expressed with its native promoter (pJN105-tmexC6D6-toprJ1b-P6) using the primers listed in Table S3 and transformed them in Escherichia coli DH5a. Recombinant strains carrying tmexC6D6-toprJ1b showed an 8-fold increase in tigecycline MIC compared to that of the same bacteria carrying an empty vector (Table 1). Notably, the potential promoter region of tmexC6D6-toprJ1b (P tmexC6 ) showed three nucleotide differences from the promoters of tmexC1 (P tmexC1 ) and tmexC3 (P tmexC3 ). To investigate whether these promoter region differences influenced the function of tmexC6D6-toprJ1b, we replaced the promoter region of tmexC6D6-toprJ1b with P tmexC1 and P tmexC3 , thereby generating two vectors, pJN105-tmexC6D6-toprJ1b-P1 and pJN105-tmexC6D6-toprJ1b-P3, respectively. The expression of tmexC6D6-toprJ1b controlled by P tmexC1 or P tmexC3 increased the MICs of tetracyclines (4-to 32-fold), cefquinome (8-fold), streptomycin (4-fold), and ciprofloxacin (4-fold) relative to the same host strain carrying pJN105, while there was only a 2-to 8-fold increase of these agents with P tmexC6 (Table 1). Meanwhile, different transcriptional expression levels of the tmexC6D6-toprJ1b gene cluster were observed in the three promoters, where strains with P tmexC1 and P tmexC3 exhibited significantly higher mRNA levels of efflux pump genes than those with P tmexC6 (Fig. 2A). These results indicate that the promoter activity is critical for the efflux function of TMexC6D6-TOprJ1. To verify the potential regulatory effects of TNfxB6, tnfxB6 was expressed accompanied by tmexC6D6-toprJ1b in the same pJN105 vector. A 4-fold decrease in tigecycline level was observed in the tnfxB6-tmexC6D6-toprJ1b-carrying strain relative to that in a strain without tnfxB6 (Table 1). Moreover, the presence of TNfxB6 downregulated the transcriptional expression of the tmexC6D6-toprJ1b gene cluster (Fig. 2B), indicating the repressive effect of TNfxB6 on the expression of TMexC6D6-TOprJ1.
In summary, we identified a novel multidrug resistance gene cluster, tmexC6D6-toprJ1b, which mediates tigecycline resistance, on the chromosome of Pseudomonas. Several tmexCD-toprJ variants are emerging in Pseudomonas, suggesting that Pseudomonas may be a major reservoir and ancestor of tmexCD-toprJ. Therefore, strong surveillance of these variants is necessary to prevent their uncontrolled spread.
Data availability. The complete sequence of the strain GD22SC3150TT chromosome was deposited in GenBank with the accession number CP115817.

SUPPLEMENTAL MATERIAL
Supplemental material is available online only. SUPPLEMENTAL FILE 1, PDF file, 0.2 MB.