key: cord-0431285-13jxwxqi authors: Maehana, Shotaro; Eda, Ryotaro; Niida, Nagi; Hirabayashi, Aki; Sakai, Kouji; Furukawa, Takashi; Sei, Kazunari; Kitasato, Hidero; Suzuki, Masato title: A novel mobile RND-type efflux pump gene cluster, tmexC3D2-toprJ3, confers tigecycline resistance in Pseudomonas alcaligenes date: 2021-08-03 journal: bioRxiv DOI: 10.1101/2021.07.26.453812 sha: 128dc387f6769eb747ccbb30a53cd5a5132c2718 doc_id: 431285 cord_uid: 13jxwxqi Tigecycline exhibits promising activity against multidrug-resistant gram-negative bacteria (MDR-GNB), whose infections are difficult to treat with antimicrobials. However, mobile tigecycline resistance genes, such as tmexCD-toprJ, have emerged in Enterobacterales isolated in China, Vietnam, and possibly other countries in the world. TMexCD-TOprJ complexes function as RND efflux pumps for multiple antimicrobials including tigecycline. To date, tmexCD1-toprJ1, tmexCD2-toprJ2, and tmexCD3-toprJ3 have been reported as variants of tmexCD-toprJ genes. Here, we identified a novel hybrid type of tmexCD-toprJ gene cluster, named tmexC3D2-toprJ3, in tigecycline- and carbapenem nonsusceptible isolates of Pseudomonas alcaligenes, a rare opportunistic human pathogen, obtained from medical sewage in Japan in 2020. Complete genomic analysis revealed that the P. alcaligenes isolate KAM426 co-harbors tmexC3D2-toprJ3 consisting of tmexC3, tmexD2, and toprJ3, along with two copies of the carbapenemase gene blaIMP-1 on the chromosome. tmexC3D2-toprJ3 in KAM426 was flanked by the IS5/IS1182 family transposase gene, suggesting that the gene cluster was acquired by horizontal gene transfer (HGT). Comparative genomic analysis using the public genome database showed that tmexC3D2-toprJ3 has spread to other Pseudomonas species such as Pseudomonas aeruginosa via HGT mediated by mobile gene elements such as a plasmid. This study suggests that diverse tmexCD-toprJ-like tigecycline resistance genes have spread among MDR-GNB worldwide and further epidemiological genomic studies are needed. Illumina reads were assembled de novo using Shovill v1. 1 Pseudomonas species (1). Interestingly, however, tmexCD1-toprJ1 has also 100 been shown to spread to Pseudomonas putida by a megaplasmid (7) . 101 Here, we report Pseudomonas alcaligenes isolates harboring a novel variant 102 of tmexCD-toprJ along with two copies of a metallo-β-lactamase (MBL) gene, gene), suggesting that these isolates were clonally disseminated (Fig. S1 ). One of the P. alcaligenes isolates, KAM426, was further sequenced using ONT The nfxB-like gene, which has been suggested to be involved in the expression 159 of the tmexCD-toprJ-like gene (1), was found upstream tmexC3D2-toprJ3 in P. (Fig. 1B) . ANI analysis confirmed that BJP69 is 98.0% 183 identical to Pseudomonas juntendi strain BML3 T (type strain, accession no. A0T30_05350 in accession no. CP014784) could be a hot spot for HGT ( Fig. 2A) , 203 but there have been no reports to suggest this possibility to date. The other copy of blaIMP-1 was contained within a partial structure of the integron 205 gene cassette consisting of two copies of aac(6')-Ib-cr followed by blaIMP-1, as 206 described previously herein ( Fig. 2A) , in a different location in the chromosome 207 of P. alcaligenes KAM426 (Fig. 2B) . Interestingly, a comparison between the (Fig. 2B) . The super-integron, which was first identified in Vibrio cholerae, is Emergence 334 of a Plasmid-Encoded Resistance-Nodulation-Division Efflux Pump Conferring 335 Resistance to Multiple Drugs, Including Tigecycline, in Klebsiella pneumoniae. 336 mBio Emerging 338 Tigecycline Resistance: Novel Tetracycline Destructases Spread via 339 the Mobile Tet(X) Tetracycline-inactivating enzymes from environmental, human commensal, and 343 pathogenic bacteria cause broad-spectrum tetracycline resistance Identification of novel tetracycline resistance gene tet(X14) and 347 its co-occurrence with tet(X2) in a tigecycline-resistant and colistin-resistant 348 Empedobacter stercoris Characterization of novel ISAba1-350 bounded tet(X15)-bearing composite transposon Tn6866 in Acinetobacter 351 variabilis Emergence of a plasmid-borne tigecycline resistance in Klebsiella pneumoniae 354 in Vietnam Emergence of a multidrug 356 resistance efflux pump with carbapenem resistance gene blaVIM-2 in a 357 Pseudomonas putida megaplasmid of migratory bird origin Transferable Resistance-Nodulation-Division Pump Gene Cluster Tigecycline Resistance in Raoultella ornithinolytica TOprJ3, an RND-Type Efflux System Conferring Resistance to Tigecycline in 365 Proteus mirabilis, and Its Associated Integrative Conjugative Element A transferable IncC-IncX3 hybrid plasmid 369 co-carrying blaNDM-4, tet(X), and tmexCD3-toprJ3 confers resistance to 370 carbapenem and tigecycline Genome 372 Sequence of a Strain of the Human Pathogenic Bacterium Pseudomonas 373 alcaligenes That Caused Bloodstream Infection The 377 lower respiratory tract microbiome of critically ill patients with COVID-19 Novel m4C modification in type I restriction-modification systems Plasmids of novel incompatibility group 384 IncpRBL16 from Pseudomonas species Integrons: past, present, and future Discovery and distribution of 389 super-integrons among pseudomonads A distinctive class of integron in 391 the Vibrio cholerae genome The evolutionary history of chromosomal super-integrons provides an ancestry 394 for multiresistant integrons Bacterial resistance evolution by 396 recruitment of super-integron gene cassettes