Detection of chromosome-mediated tet(X4)-carrying Aeromonas caviae in a sewage sample from a chicken farm

Sir, Recently, novel plasmid-mediated tigecycline resistance mechanisms, Tet(X3) and Tet(X4), have been described in Enterobacteriaceae and Acinetobacter isolates from animals and humans.1 It raises a global antimicrobial resistance concern because these tetracycline-inactivating enzymes are able to inactivate the entire family of tetracycline antibiotics, including the newly FDA-approved eravacycline and omadacycline.1 The plasmid-borne tet(X3) and tet(X4) genes have been identified in >15 different Gram-negative species, with Escherichia coli being the most common.1,2 Here we report, to the best of our knowledge, the first identification of the tet(X4) gene on the chromosome of an Aeromonas caviae strain from sewage in China. Aeromonas species, including A. caviae, are important zoonotic pathogens of poikilotherms, but are now emerging as important human pathogens,3 and have been frequently found to carry antimicrobial resistance genes (e.g. blaNDM-1 and mcr-3 variants) on the chromosome.4–6 During a routine antimicrobial resistance surveillance study, a tet(X4)-positive strain, WCW1-2, was isolated on an LB agar plate containing tigecycline (2 mg/L) from a sewage sample from a chicken farm in 2018 in Guangdong, China. The 16S rRNA sequencing analysis further suggested that it belonged to A. caviae, which shared >99.9% nucleotide identity with the isolates from patients in USA and Brazil (accession numbers CP026055 and CP024198). Antimicrobial susceptibility testing was conducted by broth microdilution, with E. coli ATCC 25922 as the quality control strain, and interpreted according to the CLSI guideline.7 The tet(X4)-positive A. caviae WCW1-2 was resistant to tetracycline, amoxicillin/clavulanic acid, ciprofloxacin and trimethoprim/sulfamethoxazole, but remained susceptible to amikacin, cefotaxime, colistin, gentamicin and meropenem (Table S1, available as Supplementary data at JAC Online). In addition, WCW1-2 exhibited high MICs of tigecycline (16 mg/L), eravacycline (4 mg/L) and omadacycline (8 mg/L). The tigecycline resistance in A. caviae WCW1-2 failed to transfer to sodium azide-resistant E. coli J53 by filter mating, but further gene cloning of tet(X4) and its putative promoter into a pUC18 vector (primers in Table S2) confirmed the tet(X4)-mediated tigecycline resistance. Susceptibility testing results showed that the tet(X4) construct had 64- to 512-fold increases in MICs of tetracycline (128 mg/L), chlortetracycline (256 mg/L), oxytetracycline (128 mg/L), doxycycline (32 mg/L), minocycline (16 mg/L), tigecycline (8 mg/L), eravacycline (2 mg/L) and omadacycline (8 mg/L), which were consistent with the results for the parental strain WCW1-2 (Table S1). Genomic DNA of A. caviae WCW1-2 was then completely sequenced using a combination of the Nanopore GridION and Illumina HiSeq platforms (Nextomics, Wuhan, China), followed by assembling with Unicycler.8 The results of WGS revealed that WCW1-2 belonged to a novel ST, ST645, and harboured one chromosome of 4684096 bp (CP039832), but without plasmids. The tet(X4) gene was found to be on the chromosome of WCW1-2, which explained the failure of transfer of tigecycline resistance, and shared a homology region (namely upstream of the ΔmerR gene and downstream of the ucpA gene) with the chromosome of another A. caviae strain (Figure 1a). Moreover, WCW1-2 harboured an additional 15 antimicrobial resistance genes encoding resistance to β-lactams (blaMOX-5 and blaOXA-10), aminoglycosides [aadA1, aph(3')-Ia, aph(3'')-Ib and aph(6)-Id], fluoroquinolones [qnrVC4 and aac(6')-Ib-cr], phenicols (cmlA1, catB3 and floR), trimethoprim/sulfamethoxazole (sul1, dfrA14 and dfrB4) and tetracyclines [tet(A)]. (a) Comparison of the chromosomes of A. caviae strains. Arrows represent the positions and transcriptional directions of the ORFs. Regions of >99% homology are marked by shading. Δ indicates that the gene is truncated. (b) Genetic environments of tet(X4) genes. The putative replication initiation (oriIS; left-facing sticks in purple) and replication termination (terIS; right-facing sticks in purple) of the ISCR2 element are illustrated (5′ to 3′). The primers used for inverse PCR are also presented. (c) Gel picture of the PCR product generated by the inv-F and inv-R primers. DL5000 DNA marker is used as the size standard. (d) Schematic representation of the circular form obtained by sequencing the PCR product. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC. (a) Comparison of the chromosomes of A. caviae strains. Arrows represent the positions and transcriptional directions of the ORFs. Regions of >99% homology are marked by shading. Δ indicates that the gene is truncated. (b) Genetic environments of tet(X4) genes. The putative replication initiation (oriIS; left-facing sticks in purple) and replication termination (terIS; right-facing sticks in purple) of the ISCR2 element are illustrated (5′ to 3′). The primers used for inverse PCR are also presented. (c) Gel picture of the PCR product generated by the inv-F and inv-R primers. DL5000 DNA marker is used as the size standard. (d) Schematic representation of the circular form obtained by sequencing the PCR product. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC. A further BLASTn search for the tet(X4) gene against the NCBI database identified a series of Enterobacteriaceae carrying the same subtype from humans (e.g. NZ_NQAI01000053) and pigs (e.g. NZ_NQBP01000050), including the first described tet(X4)-harbouring plasmid, p47EC (MK134376) (Figure 1b). Analysis of their genetic environments revealed that the tet(X4) gene was usually located between two intact copies of ISCR2 (Figure 1b), and a 4608 bp circular form [namely catD-tet(X4)-ISCR2] was successfully identified from A. caviae WCW1-2 (Figure 1c and d). As previously reported for blaVEB, a single copy of ISCR2 could transpose adjacent DNA sequences by a rolling-circle transposition process and the circular form was generated between two copies of ISCR2.9 In this study, the ISCR2-mediated rolling-circle transposition was further confirmed for mobilization of tet(X4) at a frequency of (7.5±1.8) × 10−9 by cloning the catD-tet(X4)-ISCR2 unit with the suicide plasmid pSV03 using a ClonExpress Ultra One Step Cloning Kit (Vazyme, Nanjing, China) and filter mating with the recipient E. coli K-12 substrain MG1655 (recA-knockout), as described for mcr-1.10 In summary, we report for the first time, to the best of our knowledge, the emergence of tigecycline resistance mechanism Tet(X4) in A. caviae, a potential reservoir for the dissemination of tigecycline resistance into different environmental niches. The tet(X4)-carrying element analysis suggests that the catD-tet(X4)-ISCR2 gene cassette is highly active and may further spread into different bacterial species. Continuous monitoring of tet(X4) in humans, animals and environments should be considered, to improve understanding and address the spread of resistance to tetracyclines, including tigecycline. We thank Ke Cheng, Guo-Hui Deng, Jia-Qi Song and Bo Jiang (South China Agricultural University) for sample collecting. This work was jointly supported by the National Key Research and Development Program of China (2016YFD0501300), the Program for Innovative Research Team in the University of Ministry of Education of China (IRT_17R39) and the Foundation for Innovation and Strengthening School Project of Guangdong, China (2016KCXTD010). None to declare.