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Article

Multiple Copies of Tigecycline Gene Cluster tmexC6D6-toprJ1b in Pseudomonas mendocina in a Swine Farm

by
Renjie Wu
1,2,
Yongliang Che
1,2,
Longbai Wang
1,2,
Qiuyong Chen
1,2,
Bing He
1,2,
Jingli Qiu
1,2,
Xuemin Wu
1,2,
Rujing Chen
1,2,
Yutao Liu
1,2,* and
Lunjiang Zhou
1,2,*
1
Institute of Animal Husbandry and Veterinary Medicine, Fujian Academy of Agricultural Sciences, Fuzhou 350013, China
2
Fujian Animal Diseases Control Technology Development Center, Fuzhou 350013, China
*
Authors to whom correspondence should be addressed.
Antibiotics 2025, 14(5), 500; https://doi.org/10.3390/antibiotics14050500
Submission received: 28 February 2025 / Revised: 11 April 2025 / Accepted: 6 May 2025 / Published: 13 May 2025
(This article belongs to the Special Issue Antimicrobial Susceptibility of Veterinary Origin Bacteria)
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Abstract

:
Background/Objectives: The emergence and transmission of the tigecycline resistance efflux pump gene cluster tmexCD-toprJ among humans, animals and the environment have posed a serious threat to public health. The objective of this study was to characterize Pseudomonas strains carrying multiple copies of tmexC6D6-toprJ1b from a pig farm and illustrate the genetic context of tmexC6D6-toprJ1b in the NCBI database. Methods: The characterization of Pseudomonas strains FJFQ21PNM23 and FJFQ21PNM24 was determined by antimicrobial susceptibility testing, whole-genome sequencing, and RT-qPCR. Results: The tmexCD-toprJ-positive P. mendocina strains FJFQ21PNM23 and FJFQ21PNM24 were isolated from nasal swabs in a pig farm. Sequence analysis showed that the two P. mendocina strains harbored multiple antimicrobial resistance genes, including tigecycline resistance gene tmexC6D6-toprJ1b. WGS analysis indicated that tmexC6D6-toprJ1b gene was located on a classical transferable module (int1-int2-hp1-hp2-tnfxB-tmexCD-toprJ) and a multiresistance region in FJFQ21PNM24 and FJFQ21PNM23, respectively. Further analysis revealed that 39 additional tmexC6D6-toprJ1b genes in the NCBI database were all identified in Pseudomonas spp., and the genetic features of tmexC6D6-toprJ1b were summarized into three distinct structures. Conclusions: This study is the first to identify and report the tigecycline resistance gene tmexCD-toprJ in a swine farm. Our findings summarize the three structures in the genetic context of tmexC6D6-toprJ1b and reveal that Pseudomonas serves as the only known reservoir of tmexC6D6-toprJ1b.

1. Introduction

Tigecycline is a semisynthetic glycylcycline, belonging to the third-generation tetracyclines, with a broad spectrum of antimicrobial activity [1]. Notably, tigecycline is recognized as one of the few effective treatments for infections caused by multidrug-resistant (MDR) bacteria [2]. However, the rapid emergence and spread of tigecycline resistance, particularly the plasmid-borne tet(X)-encoding flavin-dependent monooxygenase gene and the resistance-nodulation-division (RND) family efflux pump gene cluster tmexCD-toprJ, pose a serious threat to public health [1,3]. Since tmexCD1-toprJ1 was first reported on the plasmid of Klebsiella pneumoniae, six tmexCD-toprJ variants have been identified in various bacterial species, including Klebsiella spp., Pseudomonas spp., and Aeromonas spp., among others [1,4,5,6]. Notably, Pseudomonas is speculated to be an ancestral host of tmexCD-toprJ and has played a significant role in the reservoir and transmission of tmexCD-toprJ [4,5,7]. Furthermore, TmexCD-TOprJ efflux pump not only mediates tigecycline resistance but also confers multidrug resistance by expelling multiple drugs, such as quinolones, cephalosporins, and aminoglycosides [1,5]. Alarmingly, the co-occurrence of colistin (mcr) or carbapenem (e.g., blaNDM, blaKPC, blaOXA) resistance genes in tmexCD-toprJ-bearing MDR strains threatens the clinical efficacy of multiple drugs in both human and livestock [8,9,10,11].
To date, six tmexCD-toprJ gene clusters have been found in humans, animals, food, and environmental samples [5,12,13,14,15,16]. Although tigecycline has been banned from use in the livestock industry, livestock is the main origin of tmexCD1-toprJ1 [4,7]. Tetracyclines are the most widely used veterinary antibiotics; they are frequently used to treat infections of the respiratory and digestive tracts in swine farms and serve as feed additives in broiler chicken farming [17,18]. Therefore, the accumulation of tetracycline-resistant pathogens is driven by the selective pressure of this drug, which results in partial tetracycline resistance bacteria showing cross-resistance against tigecycline [7]. Consequently, since tmexCD1-toprJ1 was first isolated from K. pneumoniae in chicken feces in a chicken farm, tmexCD-toprJ variants have spread extensively across the chicken food production chain with high positivity rates [1,9,16,19,20]. Moreover, tmexCD-toprJ has been sporadically detected in swine slaughterhouses and pork, while it has not been reported in pig farms [1,12,19,20,21]. Therefore, the emergence and dissemination of tmexCD-toprJ variants in the pig food production chain remain unclear.
Thus, this study aimed to screen the tigecycline resistance gene cluster tmexCD-toprJ among bacteria isolated from a swine farm in Fujian Province, China. In this study, we first detected tmexCD-toprJ gene clusters in a pig farm and characterized two tmexCD-toprJ-positive Pseudomonas mendocina strains. Additionally, this study also characterized the diverse genetic context of tmexCD6-toprJ1b in Pseudomonas spp.

2. Results

2.1. Identification of tmexCD-toprJ-Positive Strains

Two swine-origin isolates, FJFQ21PNM23 and FJFQ21PNM24, were identified as P. mendocina by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) (Table 1). Moreover, a PCR screening assay confirmed that both P. mendocina strains carried tmexCD-toprJ-like gene cluster. Further Sanger sequencing suggested that FJFQ21PNM23 and FJFQ21PNM24 carried a tmexC6D6-toprJ1b gene cluster (Table 1).

2.2. Antimicrobial Susceptibility of tmexC6D6-toprJ1b-Positive P. mendocina

Antimicrobial susceptibility testing suggested that both strains showed resistance to multiple antimicrobial agents, including tetracyclines (MIC ≥ 128 mg/L), doxycycline (MIC = 16 mg/L), florfenicol (MIC > 256 mg/L), chloramphenicol (MIC > 256 mg/L), and trimethoprim–sulfamethoxazole (MIC > 16/304). Additionally, FJFQ21PNM23 exhibited resistance to ciprofloxacin (MIC = 16 mg/L), and FJFQ21PNM24 exhibited resistance to gentamicin (MIC = 16 mg/L) and streptomycin (MIC > 256 mg/L). The two strains were susceptible to cefquinome, cefotaxime, and ceftazidime (Table 2). Notably, the MIC values of tigecycline for FJFQ21PNM23 and FJFQ21PNM24 were 1 and 2, respectively.

2.3. Characterization of tmexC6D6-toprJ1b-Positive P. mendocina

To further determine the genetic location of tmexC6D6-toprJ1b and the antibiotic resistance genes (ARGs) profiles of the two P. mendocina strains, the complete genomes of FJFQ21PNM23 and FJFQ21PNM24 were obtained using Illumina and Nanopore sequencing. The sequence analysis suggested that FJFQ21PNM23 harbored a 4.78 Mb chromosome and a 142,064 bp plasmid. Therein, six ARGs were identified on the chromosome, including genes conferring resistance to tetracyclines [tmexC6D6-toprJ1b and tet(G)], quinolone (qnrVC1), chloramphenicol (floR), sulfonamide (sul1), and aminoglycoside (aac(6′)-IIa), while the sulfonamide gene dfrA1 was located on the plasmid (Table 1). In contrast, FJFQ21PNM24 contained 10 ARGs, namely tmexC6D6-toprJ1b, tet(G), aph(6)-Id, dfrA1, aph(3″)-Ib, ant(2″)-Ia, dfrA1, sul1, sul2, qnrVC1, and floR, all of which were found on a 4.79 Mb chromosome (Table 1).

2.4. Genetic Context of tnfxB6-tmexC6D6-toprJ1b

The function of tmexC6D6-toprJ1b and the regulatory effect of TNfxB6 have been reported in a previous study [5]. Here, the genetic structure of chromosomal tmexC6D6-toprJ1b located in FJFQ21PNM23 and FJFQ21PNM24 was analyzed. FJFQ21PNM23 harbored one copy of tmexC6D6-toprJ1b, whereas FJFQ21PNM24 contained two copies, separated by a genetic distance of approximately 406 kb (Figure 1). As expected, the transcription level of tmexC6D6-toprJ1b gene in FJFQ21PNM24 was higher than that in FJFQ21PNM23 (Figure 2), which may account for FJFQ21PNM24 exhibiting two-fold to four-fold higher MICs for tetracyclines compared with FJFQ21PNM23.
Similarly to tnfxB1-tmexCD1-toprJ1, tnfxB2-tmexCD2-toprJ2, and tnfxB3-tmexCD3-toprJ1b gene clusters, two hypothetical protein genes (hp1-like and hp2-like) and two hypothetical integrase genes (int1 and int2) were located upstream of chromosomal tnfxB6-tmexC6D6-toprJ1b in FJFQ21PNM24 (Figure 1), forming a classical transferable module (int1-int2-hp1-hp2-tnfxB-tmexCD-toprJ). In addition to tnfxB1-tmexCD1-toprJ1, the transferable module was found to specifically insert into the umuC-like gene. An identical tnfxB6-tmexC6D6-toprJ1b-carrying module region was inserted into the umuC-like gene on the chromosome of Pseudomonas sp. strain 13,159,349 (CP045553.1). In addition, this module was also found on the plasmid of Pseudomonas aeruginosa S201409-209 (CP131786.1) and S201405-249 (CP131793.1), as well as on the chromosomes of Pseudomonas juntendi L4008hy (CP146690.1) and Pseudomonas alcaligenes KAM426 (AP024354.1) (Figure S1). Unlike the genetic context of tnfxB-tmexCD-toprJ in the classical transferable module, tnfxB6-tmexC6D6-toprJ1b was inserted into a 28,302 bp multiresistance region (MRR) upstream of the umuC and umuD genes in FJFQ21PNM23 (Figure 1) [15]. The MRR carried qnrVC1, aac(6′)-IIa, floR, tet(G), tmexC6D6-toprJ1b, and two copies of sul1 resistance genes. Interestingly, all chromosomal resistance genes in FJFQ21PNM23 were located within this MRR. Further genomic analysis showed that the 10,848 bp structure qacE∆1-sul1-orf5-∆hp2-like-tnfxB6-tmexC6D6-toprJ1b in FJFQ21PNM23 was identical to the corresponding region of the first reported tnfxB6-tmexC6D6-toprJ1b-positive P. mendocina GD22SC3150TT chromosome (CP115817.1) and Pseudomonas stutzeri ZDHY95 chromosome (CP063358.1) (Figure S2).
The Nucleotide Basic Local Alignment Search Tool (BLASTn) analysis results revealed that 39 additional tmexC6D6-toprJ1b or tmexC6D6-toprJ1b-like gene clusters (>99.60% nucleotide identity) originating from different Pseudomonas species exhibited diverse genetic environments of tmexC6D6-toprJ1b (Table S2). The genomic context analysis suggested that the highly homologous structure aph(3″)-Ib-aph(6)-Id-∆hp2-like-tnfxB6-tmexC6D6-toprJ1b was located on the chromosomal tnfxB6-tmexC6D6-toprJ1b-carrying MRRs of Pseudomonas kurunegalensis NY4817 (CP131921.1), P. aeruginosa ZM21 (CP141683.1), Pseudomonas sp. BJP69 (CP041933), Pseudomonas monteilii L2757hy (CP146841.1), P. monteilii 170620603RE (CP043396.1), and P. monteilii 170918607 (CP043395.1) (Figure S2). Of note, the ∆hp-2-like gene in the NY4817-like strains showed 99.91–100% identity but only 41–59% coverage compared to the corresponding gene in FJFQ21PNM24 and FJFQ21PNM23 (Table S2). In addition to the classical structure (int1-int2-hp1-hp2) and a part of the hp2 gene, tnfxB6-tmexC6D6-toprJ1b was adjacent to IS1182 (Figure S1), where two copies of IS1182 flanked the tnfxB6-tmexC6D6-toprJ1b gene cluster on the plasmid (CP084322.1) or chromosome (CP061777.1 and CP061779.1) of P. aeruginosa. Notably, the structures sul1-qacE∆1-drfA47-arr-2-qnrVC1 and IS1182-tnfxB6-tmexC6D6-toprJ1b formed an MRR that was found on the plasmids of P. aeruginosa (CP132998.1, CP073081.1, and CP095921.1) and Pseudomonas putida (CP134603.1) (Figure S1).

3. Discussion

Unlike the prevalence of tmexCD1-toprJ1, tmexCD2-toprJ2, and tmexCD3-toprJ1b among tmexCD-toprJ gene clusters, tmexC6D6-toprJ1b has rarely been reported [4]. Here, we identified two P. mendocina strains carrying chromosomal tmexC6D6-toprJ1b, which originated from a pig farm in China. The tmexCD-toprJ gene cluster has been reported in humans, environmental samples (e.g., sewage from urban areas, hospitals, and food markets, farm markets, and river water), food products (e.g., vegetables, fish meat, chicken meat), and animals (e.g., chickens, migratory birds, fish, ducks, flies) [1,5,6,20,22,23,24,25,26,27]. Despite the widespread dissemination of the tmexCD-toprJ gene cluster among various sources in China, it has been sporadically reported in swine and has only been found in swine feces from slaughterhouses and retail pork (Table S3) [1,12,19,20]. It is noteworthy that this study is the first to report the tmexCD-toprJ gene cluster isolated from nasal swabs from swine in a pig farm.
The tmexC6D6-toprJ1b was first detected in P. mendocina of environmental origin, and its variant tmexC6D6.2-toprJ1b was reported in P. aeruginosa from a retail chicken sample [5,11]. Subsequently, this study first identified multiple copies of tmexC6D6-toprJ1b in Pseudomonas spp. However, we did not detect multiple copies of tmexC6D6-toprJ1b in 39 additional tmexC6D6-toprJ1b-like-positive strains from the NCBI database (Table S2). In contrast, multiple copies of tmexCD2-toprJ2-like gene were found on the chromosome of Aeromonas spp. [26]. Unlike the first reported tmexC6D6-toprJ1b-carrying strain P. mendocina (MIC = 8), the MICs of tigecycline in FJFQ21PNM23 (MIC = 1 mg/L) and FJFQ21PNM24 (MIC = 2 mg/L) were lower. P. mendocina GD22SC3150TT (MIC = 8) was isolated using an agar plate supplemented with 4 mg/L tigecycline, whereas P. mendocina FJFQ21PNM23 (MIC = 1 mg/L) and FJFQ21PNM24 (MIC = 2 mg/L) were randomly obtained using agar plates without antibiotics, which may have resulted in the differences in the MICs of tigecycline of P. mendocina between the two studies [5]. In addition to the intrinsic resistance of P. aeruginosa to tigecycline, the MICs of tigecycline for other Pseudomonas strains carrying tmexCD-toprJ ranged from 1 to 16 mg/L [5,7,24,28].
To date, K. pneumoniae and Pseudomonas spp. have been the predominant tmexCD-toprJ-bearing strains, while tmexCD-toprJ gene was also found in Aeromonas spp., Raoultella ornithinolytica, Oceanimonas sp., Proteus spp., Citrobacter youngae, and other Enterobacteriaceae [4,10,13,14,15,26]. Although Escherichia coli carrying tmexCD-toprJ was found in the NCBI database using bioinformatics analysis [4,8], we supposed that E. coli may harbor tmexCD-toprJ spread among the intestinal tract. The BLASTn analysis revealed that tmexC6D6-toprJ1b or tmexC6D6-toprJ1b-like gene clusters in our study and the NCBI database were all located on the plasmids and chromosomes of Pseudomonas spp. (Table S2), indicating that Pseudomonas is the only known repository of tmexC6D6-toprJ1b. Of these 41 Pseudomonas spp., tmexC6D6-toprJ1b or tmexC6D6-toprJ1b-like genes were predominantly identified in P. aeruginosa (n = 16), while P. stutzeri (n = 5), P. putida (n = 4), and P. monteilii (n = 3) also acted as reservoirs of tmexC6D6-toprJ1b (Table S2). Previous studies have showed that chromosomal mexCD-oprJ in P. aeruginosa serves as a potential ancestral source of tigecycline efflux pump tmexCD-toprJ clusters [4,29]. In addition to tmexCD1-toprJ1 and tmexCD1-toprJ2, other tmexCD-toprJ were mostly found in Pseudomonas [4,5,7,13,28]. Consequently, we surmise that Pseudomonas spp. play a vital role in the reservoir and transmission of tmexCD-toprJ gene clusters.
The tmexCD1-toprJ1 was first found in 2020 in China; since then, tmexCD-toprJ gene clusters have been globally reported, mainly in China and some Asian countries [1,4]. The BLASTn analysis revealed that tmexC6D6-toprJ1b or tmexC6D6-toprJ1b-like genes were isolated from seven countries, including China, Japan, Lebanon, Pakistan, Spain, Poland, and Brazil (Table S2). The chromosomal tmexC6D6-toprJ1b of FJFQ21PNM24 was identified on a similar genetic module (int1-int2-hp1-hp2-tnfxB-tmexCD-toprJ) to those found in the tmexCD1-toprJ1, tmexCD2-toprJ2, and tmexCD3-toprJ1b gene clusters (Figure 1). This genetic structure carrying tmexC6D6-toprJ1b was found in P. mendocina, P. juntendi, P. alcaligenes, P. aeruginosa, and Pseudomonas sp. originating from humans and animals, revealing that it can promote the transfer of tmexC6D6-toprJ1b (Figure S1). Although a previous study summarized seven types of genomic environments of tmexCD-toprJ in Pseudomonas spp. [29], the genetic context of tmexC6D6-toprJ1b can be simplified into three structures. In addition to the classical transfer module structure, the other two structures are characterized by an insertion sequence (IS) or ∆hp2-like gene combined with a gene cassette carrying multiple antibiotic genes, located upstream of tmexC6D6-toprJ1b to form an MRR. Further analysis illustrated that the structure (resistances-intI1-IS-tnfxB6-tmexC6D6-toprJ1b) was predominantly found in P. aeruginosa, while the structure (intI1-resistances-∆hp2-like-tnfxB6-tmexC6D6-toprJ1b) was identified in various Pseudomonas spp. (Figures S1 and S2). Notably, tmexC6D6-toprJ1b-bearing Pseudomonas strains carried not only multidrug resistance efflux pump TmexC6D6-TOprJ1 but also multiple antibiotic resistance genes, such as carbapenem (blaOXA-246, blaIMP-1, blaVIM-2, blaIMP-15), aminoglycoside [aac(6′)-IIa, aph(3″)-Ib, aph(6)-Id], sulfonamide (dfrA47, sul1, dfrB1), and other resistance genes. This highlights the need for vigilance regarding the emergence and spread of tmexC6D6-toprJ1b among Pseudomonas. Given the limited reports on tmexC6D6-toprJ1b [5,11], future studies should also prioritize monitoring tmexC1D1-toprJ1, tmexC2D2-toprJ2, and tmexC3D3-toprJ1b in swine-origin bacteria.

4. Materials and Methods

4.1. Bacterial Strains

A total of 77 isolates were collected from one lung sample and 19 nasal swabs from a swine farm in Fujian Province, China, in September 2021. Among them, the strains FJFQ21PNM23 and FJFQ21PNM24 were isolated from nasal swabs. These isolated strains were collected with tryptic soy agar (TSA) (OXOID, Basingstoke, UK) supplemented with 5% sterile defibrillated equine blood (Zhengzhou Jiulong Biotechnology, Zhengzhou, China).
Bacterial species identification was performed using MALDI-TOF MS (Bruker Daltonics, Bremen, Germany). An isolated colony was selected as a sample, which was transferred onto the target plate. A matrix was added to the sample on the plate, and the MALDI-TOF spectrum was automatically generated by the software.
Subsequently, PCR was performed to screen for tmexCD-toprJ-like gene clusters using previously described primers (Table S1) [1]. PCR was performed using the GoTaq® Green Master Mix (Promega, Beijing, China) on the VeritiProTM thermal cycle (Thermo Fisher Scientific, Waltham, MA, USA). The amplification was performed under the following conditions: 95 °C for 2 min; 30 cycles of 95 °C, 30 s; 60 °C, 30 s; 72 °C, 1 min, followed by the final extension of 72 °C at 5 min. The positive products were subjected to Sanger sequencing (TsingKe Biological Technology, Beijing, China), and the obtained sequences were verified by the National Center for Biotechnology Information (NCBI) BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 12 June 2024).

4.2. Antimicrobial Susceptibility Testing

The minimum inhibitory concentrations (MICs) of tmexCD-toprJ-positive strains against 13 antimicrobial agents were determined using broth microdilution (for tigecycline) and agar dilution methods (for minocycline, doxycycline, tetracycline, cefquinome, cefotaxime, ceftazidime, florfenicol, chloramphenicol, gentamicin, streptomycin, ciprofloxacin, and trimethoprim–sulfamethoxazole) based on the Clinical and Laboratory Standard Institute guidelines (CLSI, M100-S31) [30]. Mueller–Hinton (MH) agar (Hope Biotechnology, Qingdao, China) was used in the agar dilution method. The MH broth (Hope Biotechnology, Qingdao, China) and a 96-well cell culture plate (Beijing Labgic Technology, Beijing, China) were used in the broth microdilution method. Escherichia coli ATCC 25922 served as the quality control strain. The MICs were interpreted in accordance with the CLSI and European Committee on Antimicrobial Susceptibility Testing (EUCAST, version 9.0) [31] guidelines (for tigecycline).

4.3. Whole-Genome Sequencing (WGS) Analysis

The genomic DNA of tmexC6D6-toprJ1b-positive strains was extracted using the Hipure Bacterial DNA Kit (Magen, Guangzhou, China), according to the product manual. The whole-genome sequencing of two P. mendocina strains, FJFQ21PNM23 and FJFQ21PNM24, was performed using the Illumina NovaSeq and Oxford Nanopore MinION platforms (Beijing Novogene Technology, Beijing, China). The de novo assembly was obtained using SPAdes, and the complete sequences were generated through hybrid assembly with Unicycler [32,33]. Antimicrobial resistance genes (ARGs) were analyzed using Resfinder from the Center for Genomic Epidemiology server (http://genepi.food.dtu.dk/resfinder, accessed on 12 June 2024) [34]. The genomic environmental analysis of tmexC6D6-toprJ1b was performed by Easyfig [35]. To compare the genetic contexts of tmexC6D6-toprJ1b, our study collected and summarized the characteristics of an additional 39 strains carrying tmexC6D6-toprJ1b from the NCBI database (Table S2).

4.4. RNA Extraction, cDNA Synthesis, and RT-qPCR

The total RNA of tmexC6D6-toprJ1b-positive strains was extracted using the TransZol Up Plus RNA Kit (TransGen Biotech, Beijing, China), according to the product manual. The quality of the RNA was assessed by agarose gel electrophoresis and quantified by Nanodrop (Thermo Fisher Scientific, Waltham, MA, USA) [36]. Subsequently, cDNA was synthesized using TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China). qPCR was performed using the PerfectStart Green qPCR SuperMix (TransGen Biotech, Beijing, China) on the LightCycle® 96 real-time PCR detection system (Roche, Basel, Switzerland). The cq value was analyzed by the software LightCycle® 96 SW 1.1, according to the product manual. The relative transcriptional expression levels of tmexC6, tmexD6, and toprJ1b were determined using the 2−ΔΔCt method, with the 16S rRNA gene used as a reference gene [37]. The data on the transcriptional expression levels of tmexC6D6-toprJ1b gene passed the normality test, as analyzed by the Shapiro–Wilk test. The statistical significance of the data was further analyzed by Student’s t-test. The qPCR primers are listed in Table S1.

4.5. Nucleotide Sequence Accession Numbers

The complete nucleotide sequences of the chromosomes of strains FJFQ21PNM23 and FJFQ21PNM24 were deposited in GenBank under the accession numbers CP176751.1 and CP176620.1, respectively.

5. Conclusions

In summary, we report, for the first time, the tigecycline resistance gene cluster tmexCD-toprJ in nasal samples from a pig farm. Our study identified multiple copies of tmexC6D6-toprJ1b and summarized the three genetic contexts of tmexC6D6-toprJ1b in Pseudomonas. As the only known reservoir of tmexC6D6-toprJ1b and a potential ancestral origin of tmexCD-toprJ, greater attention and surveillance should be directed toward the emergence and transmission of tmexCD-toprJ in Pseudomonas spp.

Supplementary Materials

The following supporting information can be download at: https://www.mdpi.com/article/10.3390/antibiotics14050500/s1. Figure S1: Comparison of the genetic context of tmexC6D6-toprJ1b in Pseudomonas mendocina FJFQ21PNM24 with those of similar sequences carrying tmexC6D6-toprJ1b. The tmexCD-toprJ genes are shown as orange arrows, while other ARGs are depicted by red arrows. Other different genes are labeled with corresponding colors. The truncated genes are denoted by the symbol ∆. The plasmid backbone or chromosome is represented by a horizontal black line. The gray shade indicates the nearly 100% homologous region. Figure S2: Comparison of the genetic context of tmexC6D6-toprJ1b in Pseudomonas mendocina FJFQ21PNM23 with those of similar sequences carrying tmexC6D6-toprJ1b. The tmexCD-toprJ genes are shown as orange arrows, and other ARGs are depicted by red arrows. Other different genes are labeled with corresponding colors. The truncated genes are denoted by the symbol ∆. The plasmid backbone or chromosome is represented by a horizontal black line. The gray shade indicates the nearly 100% homologous region. Table S1: Primers used in this study; Table S2: tmexC6D6-toprJ1b and tmexC6D6-toprJ1b-like gene clusters found in GenBank that all originated from Pseudomonas spp.; Table S3: The information for tmexC6D6-toprJ1b and tmexC6D6-toprJ1b-like gene clusters of pig origin. References [1,12,19,20,21,26] are cited in the supplementary materials.

Author Contributions

Conceptualization, R.W., L.Z. and Y.L.; Methodology, R.W., Y.C. and B.H.; Software, R.W., Q.C. and B.H.; Validation, R.W., Y.C. and L.W.; Formal Analysis, R.W., Q.C. and B.H.; Investigation, R.W., Y.C., Q.C. and X.W.; Resources, L.Z. and L.W.; Data Curation, R.W., Y.C. and J.Q.; Writing—Original Draft Preparation, R.W.; Writing—Review and Editing, R.W., L.Z. and Y.L.; Visualization, R.W., Q.C., B.H. and R.C.; Supervision, L.Z., Y.L. and Y.C.; Project Administration, L.Z., Y.L. and L.W.; Funding Acquisition, L.Z. and Y.C. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Fujian Academy of Agriculture Science Research Project (No. XTCXGC2021008, CXTD2021007-2, YDXM2023007, and ZYTS202422).

Institutional Review Board Statement

The collection of animal samples was approved by the Animal Welfare and Ethics Committee of the Institute of Animal Husbandry and Veterinary Medicine, Fujian Academy of Agriculture Sciences (IAHV-AEC-2021-070 and 10 June 2021).

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Materials. Further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors report no conflicts of interest regarding this work.

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Antibiotics 14 00500 g001
Figure 1. Comparison of the genetic context of tmexC6D6-toprJ1b in Pseudomonas mendocina FJFQ21PNM23 and FJFQ21PNM24 with those of its similar sequences. The extents and directions of the genes are shown by arrows labeled with the corresponding gene names. The tmexCD-toprJ genes are shown as orange arrows, and other ARGs are depicted by red arrows. Hypothetical protein genes (hp1-like and hp2-like) and hypothetical integrase genes (int1 and int2) are labeled with dark yellow and light yellow arrows, respectively. The two copies of the classical transferable module (int1-int2-hp1-hp2-tnfxB-tmexCD-toprJ) in FJFQ21PNM24 are shown within a box. The MRR in FJFQ21PNM23 is labeled with a dotted line. Other different genes are labeled with corresponding colors. The truncated genes are denoted by the symbol ∆. The plasmid backbone or chromosome is represented by a horizontal black line. The gray shade indicates a nearly 100% homologous region.
Figure 1. Comparison of the genetic context of tmexC6D6-toprJ1b in Pseudomonas mendocina FJFQ21PNM23 and FJFQ21PNM24 with those of its similar sequences. The extents and directions of the genes are shown by arrows labeled with the corresponding gene names. The tmexCD-toprJ genes are shown as orange arrows, and other ARGs are depicted by red arrows. Hypothetical protein genes (hp1-like and hp2-like) and hypothetical integrase genes (int1 and int2) are labeled with dark yellow and light yellow arrows, respectively. The two copies of the classical transferable module (int1-int2-hp1-hp2-tnfxB-tmexCD-toprJ) in FJFQ21PNM24 are shown within a box. The MRR in FJFQ21PNM23 is labeled with a dotted line. Other different genes are labeled with corresponding colors. The truncated genes are denoted by the symbol ∆. The plasmid backbone or chromosome is represented by a horizontal black line. The gray shade indicates a nearly 100% homologous region.
Antibiotics 14 00500 g001
Antibiotics 14 00500 g002
Figure 2. Comparison of the transcriptional expression levels of tmexC6D6-toprJ1b gene between Pseudomonas mendocina FJFQ21PNM23 and FJFQ21PNM24. The statistical significance of the data was analyzed by Student’s t-test.
Figure 2. Comparison of the transcriptional expression levels of tmexC6D6-toprJ1b gene between Pseudomonas mendocina FJFQ21PNM23 and FJFQ21PNM24. The statistical significance of the data was analyzed by Student’s t-test.
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Table 1. Characterization of the tmexC6D6-toprJ1b-positive P. mendocina strains in this study.
Table 1. Characterization of the tmexC6D6-toprJ1b-positive P. mendocina strains in this study.
StrainSpeciesSourcePlasmid or ChromosomeSize (bp)Resistance Genes
FJFQ21PNM23Pseudomonas mendocinaNasal swabs of swine Chromosome4,779,589aac(6′)-IIa, qnrVC1, floR, sul1, tet(G),
tmexC6D6-toprJ1b
Plasmid pFJFQ21PNM23 a142,064dfrA1
FJFQ21PNM24Pseudomonas mendocinaNasal swabs of swineChromosome4,785,273aph(6)-Id, dfrA1, aph(3″)-Ib,
ant(2″)-Ia, qnrVC1, floR, sul1, sul2, tet(G),
tmexC6D6-toprJ1b
a The replicon type of pFJFQ21PNM23 was unknown according to WGS analysis.
Table 2. MIC (mg/L) values of 13 different antimicrobials for two P. mendocina isolates FJFQ23PNM23 and FJFQ23PNM24 carrying tmexC6D6-toprJ1b.
Table 2. MIC (mg/L) values of 13 different antimicrobials for two P. mendocina isolates FJFQ23PNM23 and FJFQ23PNM24 carrying tmexC6D6-toprJ1b.
MIC of Drug a
StrainTIGMINDOXTETCQMCTXCAZFFCCHLGENSTRCIPSXT
FJFQ21PNM231116128421>256>256148>16/304
FJFQ21PNM242416256424>256>25616>2562>16/304
a Abbreviations: TIG, tigecycline; MIN, minocycline; DOX, doxycycline; TET, tetracycline; CQM, cefquinome; CTX, cefotaxime; CAZ, ceftazidime; FFC, florfenicol; CHL, chloramphenicol; GEN, gentamicin; STR, streptomycin; CIP, ciprofloxacin; SXT, trimethoprim–sulfamethoxazole.
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Wu, R.; Che, Y.; Wang, L.; Chen, Q.; He, B.; Qiu, J.; Wu, X.; Chen, R.; Liu, Y.; Zhou, L. Multiple Copies of Tigecycline Gene Cluster tmexC6D6-toprJ1b in Pseudomonas mendocina in a Swine Farm. Antibiotics 2025, 14, 500. https://doi.org/10.3390/antibiotics14050500

AMA Style

Wu R, Che Y, Wang L, Chen Q, He B, Qiu J, Wu X, Chen R, Liu Y, Zhou L. Multiple Copies of Tigecycline Gene Cluster tmexC6D6-toprJ1b in Pseudomonas mendocina in a Swine Farm. Antibiotics. 2025; 14(5):500. https://doi.org/10.3390/antibiotics14050500

Chicago/Turabian Style

Wu, Renjie, Yongliang Che, Longbai Wang, Qiuyong Chen, Bing He, Jingli Qiu, Xuemin Wu, Rujing Chen, Yutao Liu, and Lunjiang Zhou. 2025. "Multiple Copies of Tigecycline Gene Cluster tmexC6D6-toprJ1b in Pseudomonas mendocina in a Swine Farm" Antibiotics 14, no. 5: 500. https://doi.org/10.3390/antibiotics14050500

APA Style

Wu, R., Che, Y., Wang, L., Chen, Q., He, B., Qiu, J., Wu, X., Chen, R., Liu, Y., & Zhou, L. (2025). Multiple Copies of Tigecycline Gene Cluster tmexC6D6-toprJ1b in Pseudomonas mendocina in a Swine Farm. Antibiotics, 14(5), 500. https://doi.org/10.3390/antibiotics14050500

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