Emergence of Carbapenem- and Tigecycline-Resistant Proteus cibarius of Animal Origin

The emergence of tet(X) and carbapenemase genes in Enterobacterales pose significant challenges to the treatment of infectious diseases. Convergence of these two categories of genes in an individual pathogen would deteriorate the antimicrobial resistance (AMR) crisis furthermore. Here, tigecycline-resistant Enterobacterales strains were isolated and detected with carbapenemase genes, characterized by antimicrobial susceptibility testing, PCR, conjugation assay, whole genome sequencing, and bioinformatics analysis. Three tigecycline-resistant isolates consisting of one plasmid-mediated tet(X4)-bearing Escherichia fergusonii and two chromosomal tet(X6)-bearing Proteus cibarius were recovered from chicken feces. The tet(X4) was located on a conjugative IncX1 plasmid pHNCF11W-tetX4 encoding the identical structure as reported tet(X4)-bearing IncX1 plasmids in Escherichia coli. Among two P. cibarius strains, tet(X6) was located on two similar chromosomal MDR regions with genetic contexts IS26-aac(3)-IVa-aph(4)-Ia-ISEc59-tnpA-tet(X6)-orf-orf-ISCR2-virD2-floR-ISCR2-glmM-sul2 and IS26-aac(3)-IVa-aph(4)-Ia-ISEc59-tnpA-tet(X6)-orf-orf-ISCR2-glmM-sul2. Apart from tet(X6), P. cibarius HNCF44W harbored a novel transposon Tn6450b positive for blaNDM–1 on a conjugative plasmid. This study probed the genomic basis of three tet(X)-bearing, tigecycline-resistant strains, one of which coharbored blaNDM–1 and tet(X6), and identified P. cibarius as the important reservoir of tet(X6) variants. Emergence of P. cibarius encoding both blaNDM–1 and tet(X6) reveals a potential public health risk.


INTRODUCTION
Antimicrobial resistance (AMR) is a serious threat to public health globally. Carbapenem and tigecycline are regarded as vital antimicrobials reserved for clinical use due to their broad antibacterial spectrum. However, the increasing spread of carbapenemase-encoding genes has rendered carbapenem-resistant Enterobacteriaceae (CRE) infection a great threat (Du et al., 2018;Tacconelli et al., 2018). Plasmid-mediated resistance genes tet(X3) and tet(X4) conferring high-level resistance to tigecycline in Enterobacterales and Acinetobacter has been found to be ubiquitous in animals and food of animal origin (Bai et al., 2019;He et al., 2019;Sun et al., 2019;Li et al., 2020c). This undermines the efficacy of tigecycline as the last-resort drug in treating MDR bacterial infections, especially those caused by carbapenem-resistant, Gram-negative bacteria, such as CRE.
Initially, tet(X) genes were mainly found in Bacteroides species and bacteria derived from environmental microbiota (Forsberg et al., 2015;Tian et al., 2019). Alarmingly, novel tet(X) variants are being identified from the pathogens of animal, food, and human origins Wang et al., 2019;Liu et al., 2020;Peng et al., 2020), which implies that tet(X) is expanding from commensals to pathogens (Aminov, 2013). Thus, the convergence of mobile tigecycline-resistant tet(X) and carbapenem-resistant genes in Enterobacterales may be inevitable. Acinetobacter strains coharboring tet(X) and bla NDM−1 were identified from samples of waterfowl and dairy cows recently (Cui et al., 2020;He T. et al., 2020), highlighting the potential convergence risk of both critical resistance genes among bacteria. However, Enterobacterales carrying both tet(X) and bla NDM−1 is unknown, which constitutes a more severe concern. To cover this knowledge gap, we performed a tigecyclineresistant Enterobacterales screening pilot program and identified a Proteus cibarius coharboring both tet(X6) and bla NDM−1 , implying that wide surveillance of Enterobacterales conferring resistance to carbapenems and tigecycline among bacteria should be performed worldwide.

Bacterial Isolates
A total of 16 chicken fecal samples were collected from four chicken farms in Henan province, China, in 2019. Tigecyclineresistant Enterobacterales were selected on MacConkey agar plates containing tigecycline (4 mg/L). The species of purified tigecycline-resistant isolates was identified by 16S rRNA gene sequencing and further confirmed by analyzing WGS data. The tet(X) and carbapenem-resistant genes were determined by PCR with reported primers (Dallenne et al., 2010;He et al., 2019).

Antimicrobial Susceptibility Testing
The antimicrobial susceptibility testing (AST) of tet(X)-positive tigecycline-resistant strains against 13 antibiotics was performed based on the broth microdilution method with Escherichia coli ATCC 25922 as the quality control and interpreted according to CLSI guidelines (CLSI, 2018). The tigecycline non-susceptibility in Enterobacterales was interpreted with an MIC of ≥4 mg/L (Marchaim et al., 2014).

Conjugation Assay and S1-PFGE
A conjugation assay was conducted using rifampicin-resistant E. coli C600 (Rif r ) as the recipient to investigate the transferability of tet(X) and bla NDM−1 . The donor and recipient strains were mixed at a ratio of 1:4 on a filter, followed by culturing on LB agar plates at 30 and 37 • C, respectively, overnight and screening on MacConkey plates containing tigecycline (4 mg/L) or meropenem (2 mg/L) with rifampicin (300 mg/L). Transconjugants were confirmed with PCR. S1 nuclease pulsedfield gel electrophoresis (S1-PFGE) was utilized to characterize the plasmid profiles.

Whole Genome Sequencing and Bioinformatics Analysis
Genomid DNA (gDNA) was purified with the TIANamp Genomic DNA Kit and quantified by Qubit 4 Fluorometer. Shortread Illumina sequencing and long-read Nanopore sequencing were combined to generate complete genome sequences . The de novo assembled sequences were annotated by RAST 1 . Antibiotic resistance genes (ARGs) and plasmid replicon types were determined using the ResFinder and PlasmidFinder 2 . BRIG and Easyfig were used to compare the plasmid and MDR structures (Alikhan et al., 2011;Sullivan et al., 2011).

Functional Confirmation of tet(X6) and Phylogenetic Analysis
To confirm the resistance function of the identified tet(X6) variant, TA-cloning and AST were performed. Briefly, the new variant, together with its natural promoter sequence, was amplified by PCR using primers HNCF44W-F/AGCGAACAAGAATATGACTTTACT and HNCF44W-R/CGCCTTTCTGTTTTATAGATTCAT, cloned into a pMD19-T vector and transformed chemically into E. coli DH5α. The resistance phenotype of tet(X6) was tested by measuring the MICs of different tetracycline antibiotics. Phylogenetic analysis of Tet(X) amino acid sequences were conducted in MEGA X using the neighbor-joining method (Kumar et al., 2018).

Nucleotide Sequence Accession Numbers
The complete sequences obtained in this study have been deposited in the GenBank database under BioProject number PRJNA625637. The novel bla NDM−1 -bearing Tn6450b was also deposited in the NCBI database (MT701524).
Phylogenetic analysis of Tet(X) indicated that of all reported Tet(X6) variants clustered in a clade, most related to Tet(X5) (Figure 3). Although most tet(X6) variants were found in Proteus and Acinetobacter spp., analysis of the NCBI database shows that E. coli positive for tet(X6) existed as early as 2003 in Denmark (JWJM01000174), indicating the transmission of tet(X6) occurred previously. Furthermore, chromosomal tet(X6)bearing ICEs and MDRs are found in Proteus, Acinetobacter, and Myroides phaeus Liu et al., 2020;Peng et al., 2020). Plasmid-mediated tet(X6) in Proteus, Acinetobacter, and E. coli also emerged according to published reports (Cui et al., 2020;Li et al., 2020c;Xu et al., 2020), implying tet(X6) is at the stage of being expanded via different mobile elements. This highlights that tet(X6) variants would be the next possible prominent tet(X) allele, approaching the widespread situation of tet(X4) in E. coli (Bai et al., 2019;He et al., 2019;Sun et al., 2019). Nomenclature of tet(X) by different allele numbers is not recommended because many of the alleles are 85-100% aa identical 3 , but an identical tet(X) naming system may not benefit research communications and AMR surveillance, especially as different tet(X) alleles possess different resistance phenotypes, genetic contexts, hosts, and evolutionary routes as more research reveals. For example, tet(X4) was found to be more common in E. coli than tet(X6) owing to the fact that tet(X4) has merged into conjugative plasmids adapted in E. coli. Also, the plasmids coharboring both bla NDM−1 and tet(X) in Acinetobacter spp. (Cui et al., 2020) could be divided into tet(X3)-or tet(X6)bearing plasmids, which could make it difficult to decipher the accurate different genetic contexts if only tet(X) represented the two alleles simultaneously. To better investigate the molecular epidemiology, genomic evolution, and functional analysis and avoid confusion in tet(X) designations, a clear nomenclature scheme acceptable to researchers worldwide should be proposed as soon as possible. Because mcr genes exist in certain bacterial species as housekeeping genes and certain alleles leap over species boundary limits to Enterobacterales via mobile elements like Tn6330 (Li et al., 2016;Shen et al., 2020), tet(X) genes may behave similarly because they exist as core genes in certain bacteria and transfer to Enterobacterales by mobile elements like ISCR2. Thus, the mcr nomenclature scheme may be a good example when it comes to tet(X) designation (Partridge et al., 2018). More research should be performed to put forward a widely accepted tet(X) allele numbering system to facilitate research communications.

CONCLUSION
To conclude, we characterized three tet(X)-bearing Enterobacterales strains of chicken origin and found that one P. cibarius coharbored both bla NDM−1 and tet(X6). To the best of our knowledge, this is the first report of convergence of tet(X6) and bla NDM−1 genes in Enterobacterales, highlighting 3 http://faculty.washington.edu/marilynr/ the importance of continuous surveillance of tigecycline-and carbapenem-resistant Enterobacterales of animal origin, which may act as the potential reservoir of clinical Enterobacterales resistant to tigecycline and carbapenems.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/ Supplementary Material.