Emergence of plasmid-borne tet(X4) resistance gene in clinical isolate of eravacycline- and omadacycline-resistant Klebsiella pneumoniae ST485

ABSTRACT Omadacycline and eravacycline are gradually being used as new tetracycline antibiotics for the clinical treatment of Gram-negative pathogens. Affected by various tetracycline-inactivating enzymes, there have been reports of resistance to eravacycline and omadacycline in recent years. We isolated a strain carrying the mobile tigecycline resistance gene tet(X4) from the feces of a patient in Zhejiang Province, China. The strain belongs to the rare ST485 sequence type. The isolate was identified as Klebsiella pneumoniae by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The MICs of antimicrobial agents were determined using either the agar dilution method or the micro broth dilution method. The result showed that the isolate was resistant to eravacycline (MIC = 32 mg/L), omadacycline (MIC > 64 mg/L), and tigecycline (MIC > 32 mg/L). Whole-genome sequencing revealed that the tet(X4) resistance gene is located on the IncFII(pCRY) conjugative plasmid. tet(X4) is flanked by ISVsa3, and we hypothesize that this association contributes to the spread of the resistance gene. Plasmids were analyzed by S1-nuclease pulsed-field gel electrophoresis (S1-PFGE), Southern blotting, and electrotransformation experiment. We successfully transferred the plasmid carrying tet(X4) to the recipient bacteria by electrotransformation experiment. Compared with the DH-5α, the MICs of the transformant L3995-DH5α were increased by eight-fold for eravacycline and two-fold higher for omadacycline. Overall, the emergence of plasmid-borne tet(X4) resistance gene in a clinical isolate of K. pneumoniae ST485 underscores the essential requirement for the ongoing monitoring of tet(X4) to prevent and control its further dissemination in China. IMPORTANCE There are still limited reports on Klebsiella pneumoniae strains harboring tetracycline-resistant genes in China, and K. pneumoniae L3995hy adds a new example to those positive for the tet(X4) gene. Importantly, our study raises concerns that plasmid-mediated resistance to omadacycline and eravacycline may spread further to a variety of ecological and clinical pathogens, limiting the choice of medication for extensively drug-resistant bacterial infections. Therefore, it is important to continue to monitor the prevalence and spread of tet(X4) and other tetracyclines resistance genes in K. pneumoniae and diverse bacterial populations.

is rapidly increasing and, with the global spread of plasmid-mediated mobile colistin resistance (MCR), tigecycline is now one of the last options for the treatment of severe infections caused by K. pneumoniae (2,3).With the inevitable emergence of tigecycline resistance since its introduction to clinical treatment in 2005, this undoubt edly poses a significant challenge for the treatment of CRKP (4).In 2018, two novel tetracyclines were approved by the US Food and Drug Administration (FDA): eravacy cline and omadacycline.These two third-generation tetracyclines showed lower adverse effects and better antibacterial activity compared with tigecycline, respectively, and are therefore considered to be the most appropriate treatment for patients with severe community-sourced XDR bacterial infections in patients with severe community origin (5,6).
Chromosomal mutations, overexpression of the efflux pump, or mutations in the ribosome, have long been recognized as the primary mechanisms leading to resistance to tigecycline in K. pneumoniae (7,8).Mutations in acrR, ramR, plsC, rpsJ, trm, tet(A), and tet(M) were found to decrease tigecycline sensitivity (9,10).The plasmid-borne tet(X) resistance gene, encoding a flavin monooxygenase, represents a novel mechanism of resistance to a class of tigecycline (11).Indeed, tet(X) effectively degrades almost all tetracycline antibiotics in vitro, mediating high levels of resistance to tigecycline antibiotics (12,13).Various tet(X) gene variants mediate different levels of tigecycline resistance, with the plasmid-mediated expression product of the tet(X4) gene belonging to the core members of the degradative enzyme machinery (5,9,14).Except for the discovery of a strain of K. pneumoniae carrying the tet(X4) resistance gene by Zhai et al. in Beijing in 2019 (15), clinical carriage of the tet(X4) resistance gene is currently seen mainly in bacteria such as Escherichia coli.K. pneumoniae ST485 is a rare isolate.Previously, Kang screened 24/27 strains of ST-type K. pneumoniae in the ICU environ ment, suggesting that no major outbreak of ST485 transmission has developed in the country (16).
Given the emergence of clinical tet(X4)-carrying K. pneumoniae, it is urgent to investigate the genetic environment and transmission mechanisms of the tet(X4) resistance gene.This study reports the identification and comprehensive characteriza tion of multidrug-resistant K. pneumoniae isolated from patient feces.This strain carries the tet(X4) resistance gene on a transferable plasmid that confers resistance to omadacy cline and eravacycline.

Isolation and characterization of tet(X4)-carrying K. pneumoniae strain L3995hy
Following admission to the hospital in 2021 with a headache and fever, the male patient received treatment with ceftriaxone and meropenem.After being diagnosed with tuberculous meningitis (TBM) and receiving anti-tuberculosis treatment, he was later discharged from a Grade-A tertiary hospital in Zhejiang Province following hospitaliza tion for an intracranial infection.Strain L3995hy was isolated from the feces of this male patient and identified as K. pneumoniae.Clinical reports showed it was resistant to tigecycline, eravacycline, and omadacycline.PCR revealed the presence of a tet(X4) gene.

Bacterial electrotransformation
The transformant L3995-DH5α was selected and identified as E. coli by MALDI-TOF MS.They were confirmed to be tet(X4)-positive by PCR.Consequently, it was demonstrated that the plasmid carrying tet(X4) from the donor L3995hy could transfer into E. coli DH5α.

DISCUSSION
K. pneumoniae is one of the prevalent bacterial pathogens responsible for nosocomial and serious community-acquired infections.The escalating antimicrobial drug resistance it exhibits presents a formidable therapeutic challenge (17,18).Eravacycline has higher clinical efficacy and better tolerability than tigecycline for abdominal infections caused by common pathogens such as K. pneumoniae.Previous studies have found that the minimum inhibitory concentration (mg/L) inhibiting 90% of isolates (MIC 90 ) of eravacy cline is generally lower than that of tigecycline and omadacycline (19).The gene tet(X4), which is one of the most prevalent tigecycline resistance genes, has been detected in K. pneumoniae from the environment, animals, and edible meats (20)(21)(22).However, clinical isolates of K. pneumoniae carrying tet(X4) have rarely been reported.In this article, we isolated a strain of K. pneumoniae carrying tet(X4) from a clinical fecal sample, which is highly resistant to all tetracycline antibiotics.We successfully transferred the plasmid carrying tet(X4) to the recipient bacteria by electrotransformation experiment.Whole-genome sequencing (WGS) analysis revealed that insertion sequences ISVsa3 both upstream and downstream of tet(X4) contributed to the transmission of drug-resist ant genes.This differs from the common ISCR2-tet(X4)-ISCR2 sequence associated with tet(X4).
Previously, the tet(X4) gene has been identified on various plasmid types, such as ColE2-like, IncQ, IncX1, IncA/C2, IncFII, IncFIB, among others.Notably, the IncX1-type plasmid emerges as the predominant vector for the tet(X4) gene.The type of replicon carrying tet(X4) in isolate L3995hy is identified as IncFII(pCRY).The STs of K. pneumoniae carrying tet(X4)-resistant genes are diverse, with the dominant clone type isolated from a Chinese pig, ST414-1LV (23).It is worth noting that up to now, we have not detected tet(X4) resistance genes in the more popular K. pneumoniae STs, such as ST11, ST15, and ST258.In contrast to the more prevalent K. pneumoniae STs, L3995hy represents a rare ST485 isolate.Importantly, this marks the initial identification of the tet(X4) resistance gene in K. pneumoniae ST485.
pL3995-Tet(X4) carries both tet(X4) and tet(A) genes.In contrast to the majority of plasmids carrying the tet(X4) resistance gene, there is an absence of ISCR2 both upstream and downstream of tet(X4).Instead, there are mobile genetic elements (MGEs) (TnAs3 and ISVsa3) (24).ISCR2 is commonly located both upstream and downstream of tet(X4) and mediates the horizontal transfer of tet(X4) resistance gene through roll-over replication.Moreover, it often forms a complex genetic structure with Tn3 that facili tates the transmission of tet(X4) resistance gene.Both ISVsa3 and ISCR2 are IS91-like transposases capable of mobilizing resistance genes through rolling circle replication.Our observation implies that the involvement of an expanding array of IS elements is involved in the mobilization of the tet(X4) gene.Likewise, we believe that the tet(X4) gene can be incorporated into a new plasmid with the assistance of TnAs3 and ISVsa3 insertion sequences.In addition to the downstream of tet(X4), the insertion of the IS26 element is observed in the plasmid housing tet(X4).This presence of IS26 is significant, as IS26-mediated translocation has been documented to play a pivotal role in mobilizing antimicrobial resistance genes (15,24).
Historically, tigecycline resistance in K. pneumoniae was attributed mainly to the overexpression of genes encoding the AcrAB-TolC efflux pump, which is controlled by the local repressor acrR and global transcriptional activators (25).Various efflux mechanisms are associated with low levels of tigecycline resistance (26), and in the case of L3995hy, it carries the acrR and tet(A) efflux pump gene on chromosome and plasmid, respectively, contributing to low-level resistance to tigecycline.Antimicrobial resistance sensitivity demonstrated that L3995hy exhibited high resistance to all tetracyclines, including the recently FDA-approved eravacycline and omadacycline.Compared with the DH-5α, MICs of the transformant L3995-DH5α were increased by eight-fold for eravacycline, two-fold higher for omadacycline, and ranged from four-to 64-fold higher for tetracyclines such as tigecycline.This suggests that a combination of efflux mecha nisms and drug-resistance genes may exert a synergistic effect on resistance (27).
Biofilms play a key role in expressing resistance and virulence phenotypes.Biofilm formation assays showed that ATCC 700603 had moderate biofilm formation ability (2*ODc <ODs ≤ 4*ODc) (28), and K. pneumoniae L3995hy had strong biofilm formation ability (ODs > 4*ODc) (Fig. 4), suggesting that it has some ability to adhere and colonize.This may mean that this K. pneumoniae ST485 strain has the ability to cause clinical challenges.OriTfnder results indicated the absence of the oriT, Relaxase, T4CP, and T4SS in Plasmid3, Plasmid4, Plasmid5, and Plasmid6.The lack of conjugated transfer gene regions in these plasmid backbones is consistent with the fact that in vitro conjugation experiments were not successful (29).In conclusion, given the previous experience of rapid dissemination of carbapenem-resistant plasmids (bla KPC-2 or bla NDM-1 plasmids) and colistin-resistant plasmids (mcr plasmids), there is a strong suspicion that the emergence of a transmissible tigecycline-resistant plasmid (tet[X4]) will significantly contribute to the development of global pan-drug resistance.

Sample collection and bacterial culture
In 2021, a tigecycline-resistant strain of K. pneumoniae (strain L3995hy) was isolated from a fecal sample of a male inpatient at a tertiary care hospital in Zhejiang Province, China.The sample was incubated on MacConkey agar plates at 37°C for 18-24 hours.Subse quently, the strain and resistance genes in the isolate were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry and PCR amplification.

Location of tet(X4) gene and transferability of plasmids carrying tet(X4)
The number and size of the plasmid of K. pneumoniae L3995hy were determined with the S1 nuclease pulsed-field gel cataphoresis (S1-PFGE) method, as described previously (30).In addition, the location of the tet(X4) gene was determined according to Southern blotting and hybridization with a digoxigenin-labeled tet(X4) specific probe.Salmonella strain H9812 was used as a control strain and size marker (31).The recombinant vector was transferred into E. coli DH5α by the electrotransformation method as described previously (5).Plasmid extraction was performed using the QIAGEN Large-Contruct Kit, and the extracted plasmid was electrotransformed into receptive E. coli DH5α by voltage shock, after incubation in SOC medium, it was uniformly applied to a drug-sensitive plate containing tigecycline.Finally, tet(X4) was verified by PCR and MALDI-TOF/MS performed strain identification.

Antimicrobial susceptibility testing
Antimicrobial susceptibility testing (AST) was performed for tetracycline, β-lactam, aminoglycoside, and quinolone antibiotics using either the agar dilution method or the micro-broth dilution method.Polymyxins were interpreted according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (https://www.eucast.org/),FIG 4 The biofilm biomass of K. pneumoniae L3995hy was compared with that of the control strains.
ATCC 700603 served as a reference control, while LB broth was employed as the negative control.The critical value, ODc, represents the mean optical density (OD) of the negative control.and tigecycline, omadacycline, and eravacycline were interpreted according to FDA definitions (Tigecycline-Injection products | FDA; Omadacycline Injection and Oral Products | FDA; Eravacycline -Injection Products | FDA).The remaining antibiotics were interpreted according to Clinical and Laboratory Standards Institute (CLSI) standards, and E. coli ATCC 25922 was used as a quality control.

Biofilm formation experiment
According to the tests described in earlier studies, the formation of biofilm was assessed (32).First, the bacteria were inoculated in LB broth for overnight incubation, 200 μL of 0.5 McFarland standard turbidity suspension was added to a 96-well plate, and three well replicates were done for each sample.After standing overnight incubation at 37°C, the plate was washed three times with PBS to eliminate all non-adherent bacteria, then fixed with methanol and stained by adding 150 μL of 0.1% crystal violet solution to each well.After washing three times with PBS and discarding the washing solution, 100 µL of DMSO was added to dissolve the crystal violet attached to the biofilm, followed by incubation for 5 minutes.The OD (optical density) was measured at 590 nm.Three replicate experiments were performed.The standard strain ATCC 700603 was used as a control.LB broth was used as a negative control.ODs ≤ ODc, 2*ODc < ODs ≤ 4*ODc, and ODs > 4*ODc, indicating no biofilm formation ability, moderate biofilm formation ability, and strong biofilm formation ability of bacterium, respectively.

Whole-genome sequencing and bioinformatics analysis
Genomic DNA was extracted by using a Bacterial DNA Kit (QIAGEN, Hilden, Germany).Following that, the DNA was sequenced to acquire data using both the Illumina NovaSeq 6000 (Illumina, San Diego, CA, USA) and Oxford Nanopore platforms (Oxford Nanopore Technologies, Oxford, United Kingdom) to obtain the strain's final, whole genome sequencing, sequenced segments were assembled using Unicycler v0.4.7.RAST 2.0 (http://rast.nmpdr.org)was used to annotate it after that.By using the ISfinder database, insertion elements (ISs) were found.Acquired antibiotic resistance genes (ARGs) and plasmid incompatibility types were identified using the ResFinder (https://cge.cbs.dtu.dk/services/ResFinder/) and Plasmid Finder (https://cge.cbs.dtu.dk/services/PlasmidFinder/)databases.Multilocus sequence typing (MLST) was performed on the tigecycline-resistant K. pneumoniae isolates by amplifying and sequencing seven housekeeping genes (gapA, infB, mdh, pgi, phoE, rpoB, and tonB) according to a previously described protocol.Sequence types (STs) were assigned using the online database (http://pubmlst.org/ecloacae).Using oriTfinder, the source of transfers in the DNA sequences of bacterial mobile genetic elements was found (https://tool-mml.sjtu.edu.cn/oriTfinder/oriTfinder.html).VFDB.1 was used to find the virulence factors.BLASTN was used to compare plas mid sequences with the GenBank database (https://blast.ncbi.nlm.nih.gov/blast.cgi).Finally, the circular image of plasmid comparison and the comparative map of the genetic environment surrounding the tet(X4) gene were plotted by BLAST Ring Image Generator (BRIG) and Easyfig, respectively.

FIG 1
FIG 1The plasmid size of K. pneumoniae L3995hy was determined by S1-PFGE, with Salmonella enterica serotype Braenderup H9812 as the size marker.Southern blotting hybridization with a tet(X4)-specific probe.

FIG 3
FIG 3 Genomic analyses of plasmid pL3995-tet(X4).Open reading frames (ORFs) are indicated by arrows and are denoted according to their presumed function.Blue indicates resistance genes, pink indicates removable element-related genes, and orange indicates other functional genes.Regions with a high degree of homology are shaded in blue.

TABLE 1
Susceptibility of K. pneumoniae L3995hy and its transformant to commonly used antibiotics a a R, resistant; S, susceptible; I, intermediate.