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Article

Concurrence of Inactivation Enzyme-Encoding Genes tet(X), blaEBR, and estT in Empedobacter Species from Chickens and Surrounding Environments

by
Chong Chen
1,2,3,
Yilin Lv
1,2,3,
Taotao Wu
1,2,3,
Jing Liu
1,2,3,
Yanan Guo
4 and
Jinlin Huang
1,2,3,*
1
Joint International Research Laboratory of Agriculture and Agri-Product Safety, Ministry of Education of China, Institutes of Agricultural Science and Technology Development, Yangzhou University, Yangzhou 225009, China
2
Jiangsu Key Laboratory of Zoonosis, Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou 225009, China
3
Key Laboratory of Prevention and Control of Biological Hazard Factors (Animal Origin) for Agrifood Safety and Quality, Ministry of Agriculture of China, Yangzhou University, Yangzhou 225009, China
4
Animal Science Institute, Ningxia Academy of Agriculture and Forestry Sciences, Yinchuan 750002, China
*
Author to whom correspondence should be addressed.
Foods 2024, 13(19), 3201; https://doi.org/10.3390/foods13193201
Submission received: 16 August 2024 / Revised: 18 September 2024 / Accepted: 7 October 2024 / Published: 9 October 2024
(This article belongs to the Special Issue Strategies for Controlling Pathogenic Microbial Contamination in Animal-Derived Food Products: An Antimicrobial Approach)
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Abstract

:
The emergence of inactivation enzyme-encoding genes tet(X), blaEBR, and estT challenges the effectiveness of tetracyclines, β-lactams, and macrolides. This study aims to explore the concurrence and polymorphism of their variants in Empedobacter sp. strains from food-producing animals and surrounding environments. A total of eight tet(X) variants, seven blaEBR variants, and seven estT variants were detected in tet(X)-positive Empedobacter sp. strains (6.7%) from chickens, sewage, and soil, including 31 Empedobacter stercoris and 6 novel species of Taxon 1. All of them were resistant to tigecycline, tetracycline, colistin, and ciprofloxacin, and 16.2% were resistant to meropenem, florfenicol, and cefotaxime. The MIC90 of tylosin, tilmicosin, and tildipirosin was 128 mg/L, 16 mg/L, and 8 mg/L, respectively. Cloning expression confirmed that tet(X6) and the novel variants tet(X23), tet(X24), tet(X25), tet(X26), and tet(X26.2) conferred high-level tigecycline resistance, while all of the others exhibited relatively low-level activities or were inactivated. The bacterial relationship was diverse, but the genetic environments of tet(X) and blaEBR were more conserved than estT. An ISCR2-mediated tet(X6) transposition structure, homologous to those of Acinetobacter sp., Proteus sp., and Providencia sp., was also identified in Taxon 1. Therefore, the tet(X)-positive Empedobacter sp. strains may be ignored and pose a serious threat to food safety and public health.

Graphical Abstract

1. Introduction

With the global spread of carbapenem resistance and colistin resistance, tigecycline has become one of the last-resorts for treating multidrug-resistant (MDR) bacterial infections [1]. Since approval by the United States Food and Drug Administration (USFDA) in 2005, tigecycline has been applied to treating acute bacterial skin and skin structure infections, complicated intra-abdominal infections, and community-acquired bacterial pneumonia worldwide [2]. On the other hand, a flavin-dependent monooxygenase Tet(X) can modify tigecycline through hydroxylation with NADPH, Mg2+, and molecular oxygen, leading to its low-level degradation [3]. Recently, the rapid emergence of high-level tigecycline resistance genes tet(X3) [4], tet(X4) [5], tet(X5) [6], tet(X6) [6,7], tet(X7) [8], and tet(X15) [9] in food-producing animals has compromised the clinical efficacy of tigecycline, especially in China. At present, the novel tet(X) variants and their transmission risks urgently need to be further evaluated.
The Weeksellaceae family is a complex group of Gram-negative, non-spore-forming, non-flagellated, aerobic, microaerobic, or facultatively anaerobic bacteria. It comprises the genera Empedobacter, Weeksella, Riemerella, Chryseobacterium, and Elizabethkingia, which formerly belonged to the Flavobacteriaceae family acting as the ancestral source of tet(X) genes [10,11,12]. Despite the few reports of Empedobacter sp. strains, Empedobacter stercoris was mainly recovered from animal fecal samples, while Empedobacter falsenii has become an opportunistic pathogen implicated in clinical blood, ear discharge, pleural fluid, pus, wound, vagina, respiratory tract, urinary tract, and stool samples [13,14,15,16,17,18]. Worrisomely, Empedobacter sp. strains exhibit resistance to multiple clinically important antibiotics, such as colistin, carbapenems, and tigecycline [16,17,18]. To the best of our knowledge, these strains are naturally resistant to colistin and carried a metallo-β-lactamase gene blaEBR [16,18,19,20]. The decreased tigecycline susceptibility mediated by tet(X2), tet(X3.2), tet(X14), and one unnamed tet(X) variant has also been sporadically detected in E. stercoris, E. falsenii, and Empedobacter brevis isolates from human, cattle, pig, shrimp, and chicken samples [10,17,21,22,23]. In 2023, an α/β-hydrolase EstT that inactivates 16-atom-containing macrolides was first reported in Sphingobacterium faecium [24]. However, the molecular polymorphism of inactivation enzyme-encoding genes tet(X), blaEBR, and estT in MDR Empedabacter sp. strains remains poorly understood.
Herein, we intend to explore the concurrence of multiple tet(X), blaEBR, and estT variants in Empedobacter sp. strains from chickens, sheep, sewage, and soil in Shandong, Jiangsu, and Ningxia provinces in China, followed by analyses of bacterial antimicrobial susceptibility, phylogenetic relationship, genetic diversity, clonal expression level, and antibiotic resistance gene transferability.

2. Materials and Methods

2.1. Bacterial Isolation and Identification

A total of 556 non-duplicate samples were collected from three chicken farms, one sheep farm, one live poultry market, and one chicken slaughterhouse between 2021 and 2023 in Shandong, Jiangsu, and Ningxia provinces of China (Table S1). These included fecal swabs of chickens (n = 317) and sheep (n = 95), carcass swabs of chickens (n = 60), and samples from the surrounding environment (soil, n = 48; sewage, n = 36). After dilution with 0.9% physiological saline at a weight/volume or volume/volume ratio of 1/5, 100 μL of them was spread evenly on Luria–Bertani agar (LBA, HuanKai, Guangzhou, China) containing tigecycline (2 mg/L). The tet(X)-positive strains were isolated via PCR detection of tet(X), and 16S rDNA sequencing was used for bacterial identification (Table S2).

2.2. Antimicrobial Susceptibility Testing

According to the Clinical and Laboratory Standards Institute (CLSI) guideline for Enterobacterales [25], minimum inhibitory concentrations (MICs) of 12 antibiotics against the tet(X)-positive Empedobacter sp. isolates were determined via two-fold agar dilution, including tetracycline (dilution range, 0.125–256 mg/L), amikacin (0.5–256 mg/L), gentamicin (0.25–256 mg/L), trimethoprim-sulfamethoxazole (10–160 mg/L), ciprofloxacin (0.004–64 mg/L), colistin (0.25–256 mg/L), cefotaxime (0.125–256 mg/L), meropenem (0.03125–64 mg/L), florfenicol (2–256 mg/L), tylosin (0.25–512 mg/L), tilmicosin (0.125–256 mg/L), and tildipirosin (0.125–256 mg/L). Additionally, the MIC of tigecycline (0.03125–64 mg/L) was determined via broth dilution and interpreted using the USFDA’s criteria for Enterobacterales [26]. Escherichia coli ATCC 25,922 was used as the quality control.

2.3. Whole-Genome Sequencing (WGS)

The genomic DNA of 37 tet(X)-positive Empedobacter sp. isolates was extracted using a TIANamp Bacteria DNA Kit (Tiangen, Beijing, China) and sequenced using an Illumina NovaSeq 6000 (ANOROAD, Beijing, China). The raw data were assembled using SPAdes version 3.12.0 and assessed using CheckM version 1.1.6 [27,28]. To obtain the complete sequences, E. stercoris YWS9-3 [tet(X2)- and tet(X26)-positive] was further subjected to Oxford Nanopore PromethION (BENAGEN, Wuhan, China), followed by assembling with Unicycler version 0.5.0 and correcting with Pilon version 1.12 [29,30]. The whole-genome sequences of tet(X)-positive Empedobacter sp. strains deposited in the National Center for Biotechnology Information (NCBI) database were also collected [31].

2.4. Bioinformatics Analyses

The Average Nucleotide Identity (ANI, >95%)-based bacterial species and Single Nucleotide Polymorphism (SNP)-based phylogenetic tree of tet(X)-positive Empedobacter sp. strains were analyzed using Integrated Prokaryotes Genome and pan-genome Analysis (IPGA) version 1.09 [32,33]. Antibiotic resistance genes (ARGs) were analyzed using ResFinder version 4.5.0, with a cutoff at 80% nucleotide identity and 60% nucleotide coverage, and a heatmap was generated with the phylogenetic tree using ggtreeExtra version 1.14.0 [34,35]. The allele numbers of the novel tet(X), blaEBR, and estT variants were assigned in the same manner as previously reported [36]. Multiple sequence alignment of Tet(X), EBR, and EstT variants was performed using ESPript version 3.0, respectively [37], with the secondary structure elements of Tet(X2) (Protein Data Bank accession number: 2XDO), EBR-4 (GenBank accession number: MN997121), and EstT-1 (GenBank accession number: CP094932) as their templates. Homology modeling of Tet(X) proteins was based on the published Tet(X2)-tigecycline complex (4A6N) with the online server SWISS-MODEL using default parameters [38]. Accurate structures of EBR and EstT proteins were not available, and therefore, they were predicted using the powerful server AlphaFold 3 [39]. A maximum likelihood tree of Tet(X) variants was constructed with 100 bootstraps using MEGA-X version 10.1.8 and visualized using FigTree version 1.4.4 [40]. Genome sequences of all tet(X)-positive Empedobacter sp. isolates were annotated using RAST version 2.0, and the genetic environments of tet(X), blaEBR, and estT genes were generated with Easyfig version 2.2.5 [41,42].

2.5. Cloning Expression

The novel tet(X), blaEBR, and estT variants were ligated with an L-arabinose-induced plasmid pBAD24 vis homologous recombination and then transformed into E. coli JM109 via heat shock as previously reported [11]. Except for the Nhe I- and Sal I-digested pBAD24 for estT-1.3, estT-1.4, estT-2, estT-3, estT-4, and estT-5 genes, all of the others were treated with EcoR I and Sal I. The putative transformants were selected on LBA containing ampicillin (100 mg/L) and confirmed using PCR and Sanger sequencing [43]. The amplification primers are listed in Table S2.
Thereafter, the MICs of tetracycline, tigecycline, doxycycline (0.125–256 mg/L), and minocycline (0.125–256 mg/L) against the novel tet(X) clones were determined via broth microdilution according to CLSI guidelines [25], with an addition of 0.1% L-arabinose. Our previously reported tet(X2), tet(X6), and empty clones were used as the control groups [44]. In addition, the MICs of meropenem and cefotaxime against the novel blaEBR clones and the reference blaEBR-4 clone were determined. The MICs of the 16-atom-containing tylosin, tilmicosin, and tildipirosin against the estT clones were also determined.

2.6. Conjugation Experiments

The transferability of tet(X)-mediated tigecycline resistance from Empedobacter sp. was determined via filter mating with the recipient Acinetobacter baylyi ADP1 (rifampicin-resistant) and E. coli C600 (streptomycin-resistant). The putative transconjugants were selected on LBA containing tigecycline (2 mg/L) supplemented with rifampin (100 mg/L) or streptomycin (1500 mg/L). Thereafter, all of them were further screened for tet(X) genes and confirmed using PCR fingerprints for A. baylyi and enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) for E. coli [45,46]. Transfer efficiencies were calculated based on the colony counts of the transconjugant and recipient cells in triplicate [47]. However, the conjugation experiments of blaEBR and estT genes were not conducted because of their weak inactivation activities.

3. Results

3.1. Sporadic Distribution of tet(X)-Positive MDR Empedobacter sp. Isolates

Among the 556 samples, a total of 37 tet(X)-positive Empedobacter sp. strains (6.7%) were isolated from chicken feces (7.9%, 25/317), sewage (30.6%, 11/36), and soil (2.1%, 1/48), but they were negative in chicken carcasses obtained after slaughter and sheep feces (Table S1). These strains consisted of 31 E. stercoris and 6 novel species Taxon 1, and the latter shared <95% ANI values with the reference Empedobacter species including E. falsenii 1681-1 (GCA_013488205.1), E. stercoris ES183 (GCA_014621655.1), E. brevis ATCC 43319 (GCA_000382425.1), Empedobacter tilapiae MRS2 (GCA_004785645.1), and Empedobacter sedimenti DT-LB-19 (GCA_030005555.1). By geography, they were mainly distributed in Shandong (11.7%, 34/291) but with few in Jiangsu (1.8%, 3/170) and Ningxia (0.0%, 0/95). The results of antimicrobial susceptibility testing showed that all tet(X)-positive E. stercoris and Taxon 1 strains were MDR. To be specific, 100% of them were resistant to tigecycline, tetracycline, colistin, and ciprofloxacin but remained susceptible to gentamicin, amikacin, and trimethoprim/sulfamethoxazole (Figure S1). Subsets were also resistant to meropenem (16.2%, 6/37), florfenicol (16.2%, 6/37), and cefotaxime (16.2%, 6/37). Meanwhile, the MIC90 of tylosin, tilmicosin, and tildipirosin was 128 mg/L, 16 mg/L, and 8 mg/L, respectively (Figure S2).
Based on the WGS data of 37 tet(X)-positive Empedobacteri sp. strains, in silico mining showed that they carried five classes of ARGs for tetracyclines [tet(X), n = 46; tet(36), n = 4], β-lactams (blaEBR, n = 37; blaXA-347, n = 2), macrolides [estT, n = 34; mef(C), n = 30; mph(G), n = 30; ere(D), n = 8; erm(F), n = 2], phenicols (floR, n = 36), and sulfonamides (sul2, n = 10) (Figure 1). For concurrence events, 24.3% of tet(X)-positive Empedobacter sp. isolates carried two tet(X) variants (e.g., B1-4) and 5.4% of them carried two estT variants (e.g., YWS11-3), while none of them carried more than two variants (Figure 1).

3.2. Polymorphism of tet(X), blaEBR, and estT Variants in Empedobacter Species

There were eight tet(X) variants identified in 37 tet(X)-positive Empedobacter sp. isolates, including tet(X2) (n = 14), tet(X6) (n = 2), and six novel variants (Figure 1). The results of multiple sequence alignment revealed that the novel tet(X) variants shared 66.5–99.7% amino acid sequence identities with previously reported ones (Figure S3; Table S3). According to the assignment rule of gene numbers, they were designated as tet(X2.2) (n = 1), tet(X23) (n = 1), tet(X24) (n = 1), tet(X25) (n = 3), tet(X26) (n = 22), and tet(X26.2) (n = 2), respectively (Figure 1). Homology modeling of their encoding proteins with Tet(X2) illustrated an analogous architecture, consisting of the substrate-binding domain, FAD-binding domain, and C-terminal α-helix (Figure 2). Except for Tet(X2.2) and Tet(X25), they shared similar amino acid substitutions (e.g., L282S, V329M, A339T, D340N, V350I, or K351E) as previously reported [44,48]. Functionally, the MICs of tigecycline against the tet(X23), tet(X24), tet(X25), tet(X26), and tet(X26.2) constructs increased by 64-fold when compared with that of the negative control E. coli JM109 carrying an empty pBAD24 vector (Table 1). In addition, the constructs exhibited a 32–128-fold increase for tetracycline, doxycycline, and minocycline, while the tet(X2.2) construct was only 2–8-fold (Table 1). By querying the NCBI database, tet(X2.3)-positive E. falsenii (n = 2), tet(X14)-positive E. stercoris (n = 2) as well as tet(X2)-positive E. falsenii (n = 5), E. stercoris (n = 5), and Taxon 1 (n = 2) were also detected, including one E. stercoris strain co-harboring tet(X2) and tet(X14) (Figure 1).
Besides, blaEBR-4 (n = 3) and six novel variants were randomly distributed in all 37 tet(X)-positive Empedobacter sp. strains. As per the rule mentioned above, the novel blaEBR variants were designated as blaEBR-5.2 (n = 6), blaEBR-5.3 (n = 7), blaEBR-6 (n = 3), blaEBR-7 (n = 1), blaEBR-7.2 (n = 7), and blaEBR-7.3 (n = 10), respectively, which shared > 81.6% amino acid sequence identities and three-dimensional structures with previously reported variants (Figure S4). The MIC results revealed that the blaEBR-5.2, blaEBR-6, and blaEBR-7.3 variants exhibited low-level activities for meropenem (2–4-fold increase) and cefotaxime (≥4-fold increase), but the blaEBR-5.3, blaEBR-7, and blaEBR-7.2 constructs exhibited no MIC changes (Table 1). Furthermore, the Empedobacter sp. strain carrying blaEBR-3 (n = 2), blaEBR-3.2 (n = 1), blaEBR-3.3 (n = 2), blaEBR-3.4 (n = 1), blaEBR-4 (n = 1), blaEBR-5 (n = 1), blaEBR-6.2 (n = 1), blaEBR-7.4 (n = 2), blaEBR-7.5 (n = 2), blaEBR-8 (n = 1), or blaEBR-9 (n = 1) was available in the NCBI database (Figure 1).
It is noted that 32 out of 37 tet(X)- and blaEBR-positive Empedobacter sp. strains carried seven novel estT variants, including estT-1.2 (n = 26), estT-1.3 (n = 1), estT-1.4 (n = 1), estT-2 (n = 2), estT-3 (n = 2), estT-4 (n = 1), and estT-5 (n = 1, Figure 1). Although they shared >82.2% amino acid sequence identities with the first reported estT-1 (CP094932), estT-1.2, estT-1.4, and estT-2 lacked an N-terminal random coil (Figure S5). The MICs indicated all exhibited low-level activities for 16-atom-containing tylosin (≥2-fold increase), tilmicosin (2–16-fold increase), and tildipirosin (4–16-fold increase, Table 1). Sporadically, E. falsenii strains carrying estT-1.2 (n = 2) and E. stercoris strains carrying estT-1.3 (n = 1), estT-1.6- (n = 1), both estT-1.2 and estT-1.7 (n = 1) were queried online (Figure 1).

3.3. Diverse Phylogeny of tet(X)-Positive Empedobacter sp. Strains

To explore the bacterial relationship between tet(X)-positive Empedobacter sp. strains, a WGS-based SNP analysis of 37 E. stercoris, 8 Taxon 1, and 7 E. falsenii from this study (n = 37) and the NCBI database (n = 15) was conducted. The phylogenetic tree revealed that E. stercoris, Taxon 1, and E. falsenii formed three separate clusters, with the maximal 21,822 SNPs (Figure 1). In part, eight pairs of strains exhibited a high similarity (SNPs ≤ 2), respectively, such as E. stercoris B32-4 and C25-4; E. stercoris C10-2 and C16-3; E. stercoris ES182 and ES183; E. stercoris YWS5-3 and YWS11-1; E. falsenii 1681-1 and EF1; E. falsenii N76-4 and N85-1; Taxon 1 LDH16-3 and LDH24-2; Taxon 1 B1-4 and B10-4. Significantly, there existed a potential evolutionary branch of Taxon 1 strains (SNPs ≤ 6694), which included tet(X2)-positive B4b2P8686 and Q1655 from humans, tet(X2)-positive C8-4 from chickens, and chicken-derived B1-4 and B10-4 co-harboring tet(X2) and tet(X6).

3.4. Genetic Characteristics of tet(X), blaEBR, and estT Genes

Although the resistance gene variants and bacterial phylogenetic relationship were diverse, the genetic environments of tet(X) and blaEBR were more conserved than estT. Briefly, all tet(X24), tet(X25), tet(X26), and tet(X26.2) genes shared a similar environment inserted between the MFS transporter gene and the homoserine O-acetyltransferase gene (e.g., E. stercoris YWS9-3; Figure 3). The blaEBR-4, blaEBR-5.2, blaEBR-5.3, blaEBR-6, blaEBR-7, blaEBR-7.2, and blaEBR-7.3 genes were also conserved between the hypothetical gene I and the agmatine deiminase gene (e.g., E. stercoris B32-4; Figure S6). By contrast, the genetic environments of estT genes were various and only estT-1.2 and estT-3 (e.g., E. stercoris YWS11-3) were similar to estT-1, which is adjacent to floR from environmental Sphingobacterium faecium WB1 (GCA_025340125.1; Figure S7).
According to the insertion sequences analyzed by ISfinder, an ISCR2-mediated transposon unit [namely ISCR2-hp-tet(X6)-estT-2-hp-ΔIS1595] was identified in chicken-derived Taxon 1 strains B1-4 and B10-4 (Figure 4). A series of Acinetobacter sp. (e.g., CP094557), Proteus sp. (e.g., CP047340), and Providencia sp. (e.g., CP084296) genomes carrying the tet(X6) transposon unit from food-producing animals were further identified in the NCBI database (Figure 4). Additionally, one IS1182-mediated transposon unit of tet(X26) and estT-1.2 genes were found in chicken-derived E. stercoris YF40-3 (Figure 3). The results of our conjugation experiments revealed that none of tet(X) genes could be transferred into the recipient A. baylyi ADP1 and E. coli C600. Nanopore sequencing of environmental E. stercoris YWS9-3 confirmed that tet(X2) and tet(X26) were distantly located on the chromosome cYWS9-3 (CP106831), which may lead to failure.

4. Discussion

The Empedobacter genus appears as a group of sporadically reported pathogens, such as E. stercoris, E. falsenii, E. brevis, E. sedimenti, and E. tilapiae. Since the emergence of tet(X2) and tet(X3.2) in E. brevis, tet(X2) and its N-truncated variant have been detected in E. falsenii as well as tet(X2) and tet(X14) in E. stercoris [17,21,22,23]. Except for tet(X3.2) (8 mg/L), the MICs of tigecycline against the E. coli clones with tet(X14) (2 mg/L), tet(X2) (0.25 mg/L), or its variant (1 mg/L) failed to reach the USFDA’s resistance breakpoint of 8 mg/L. Worrisomely, our study demonstrated a higher prevalence of tet(X)-mediated tigecycline-resistant E. stercoris and Taxon 1 strains in chickens and adjacent environments (especially sewage), indicating a potential transmission risk. As a major province of distribution, Shandong urgently needs to detect them. What is more, the tet(X)-positive E. stercoris and Taxon 1 strains were not detected in chicken carcasses obtained after slaughter, revealing a key process to prevent bacterial contamination.
To date, a total of 52 tet(X) variants have been reported from different bacterial hosts (Table S3, collected on 16 April 2024), of which 44 non-duplicate and non-frameshifted variants were confirmed (Figure S3). In this study, there were eight tet(X) variants identified, of which six were novel, and tet(X23), tet(X24), tet(X25), tet(X26), and tet(X26.2) were able to confer tigecycline resistance (8 mg/L). A series of key amino residue mutations such as L282S, V329M, A339T, D340N, V350I, and K351E have been reported as leading to enhanced Tet(X2) activity [44,48]. Homology modeling of Tet(X6), Tet(X23), Tet(X24), Tet(X26), and Tet(X26.2) with the template Tet(X2) using the online server SWISS-MODEL identified similar substitutions, but none were for Tet(X2.2) and Tet(X25) and requires further study (Figure 2B).
Moreover, there were 19 blaEBR variants as shown in Figure S4A, including five variants that have been previously reported in E. brevis (blaEBR-1), E. falsenii (blaEBR-2, blaEBR-3, blaEBR-4), and E. stercoris (blaEBR-5) [16,18,19,20]. We also reported six novel blaEBR variants in E. stercoris and Taxon 1, of which blaEBR-5.2, blaEBR-6, and blaEBR-7.3 exhibited 2–4-fold higher MICs for one of the last-resort antibiotic meropenem than blaEBR-1 but 2–4-fold lower than blaEBR-4 (Table 1) [20]. Apart from the tet(X) and blaEBR genes, a total of 11 estT variants were collected (Figure S5A), including seven novel variants in this study capable of inactivating 16-atom-containing macrolides like the first reported estT [24]. One another estT variant (designated estT-1.5 by us) was identified on the chromosome of Pasteurella multocida 17BRD-035 (CP082272), but its hydrolyzation activity needs to be confirmed [49]. The available data indicate that Empedobacter sp. strains appear as a reservoir of tet(X), blaEBR, and estT genes.
It is noted that the genetic environments of tet(X) and blaEBR variants were more conservative than estT variants on Empedobacter sp. genomes. However, their evolutionary routes remain to be explored. Our previous studies reported the ISCR2-mediated transposition events of tet(X3), tet(X4), and tet(X5), which probably originated from the Weeksellaceae (formerly Flavobacteriaceae) genomes but lack direct evidence [6,11,50]. Herein, we identified an ISCR2-mediated transposon unit in Taxon 1 B1-4 and B10-4 belonging to the Weeksellaceae family and supporting the evolutionary hypothesis. Although failing to be transferred by conjugation, a similar transposition structure was detected by querying in the NCBI database in Acinetobacter sp., Providencia sp., and Proteus sp. bacteria of chicken, pig, and duck origin, indicating a risk of cross-species dissemination in food-producing animals (Figure 4).

5. Conclusions

Taken together, our study demonstrates a diversity of tet(X)-, blaEBR-, and estT-positive MDR Empedobacter sp. strains in chickens and surrounding environments (especially sewage), and the slaughter process in slaughterhouses may be an effective means to clear the contamination. Particularly, there were eight tet(X) variants together with seven blaEBR variants and seven estT variants identified in this study, and the genetic environments of tet(X) and blaEBR are more conserved than estT. Given the emergence of ISCR2-mediated tet(X6) in the novel bacterial species Taxon 1, future efforts are needed to improve the surveillance of Empedobacter sp. strains from all related sectors and to evaluate their clinical impact.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/foods13193201/s1. Figure S1: Resistance rates of ten antibiotics against 37 tet(X)-positive Empedobacter sp. strains; Figure S2: MICs of tylosin (A), tilmicosin (B), and tildipirosin (C) against 37 tet(X)-positive Empedobacter sp. strains; Figure S3: Maximum-likelihood phylogenetic tree of the Tet(X) variants; Figure S4: Structural characteristics of the EBR proteins; Figure S5: Structural characteristics of the EstT proteins; Figure S6: Conserved genetic backgrounds of the blaEBR genes; Figure S7: Diverse genetic backgrounds of estT genes; Table S1: Distribution of tet(X)-positive Empedobacter sp. isolates of different sources; Table S2: Primers used in this study; Table S3: Summary of the tet(X) variants (n = 52, accessed on 16 April 2024). Refs. [4,7,9,11,17,21,23,51,52,53,54,55,56,57,58,59,60,61,62,63] are cited in Supplementary Materials.

Author Contributions

Conceptualization, C.C. and J.H.; methodology, C.C. and Y.L.; formal analysis, Y.L. and T.W.; investigation, J.L. and Y.G.; data curation, C.C., Y.L. and T.W.; writing—original draft preparation, C.C. and Y.L.; writing—review and editing, C.C. and J.H.; project administration, J.H.; funding acquisition, C.C. and J.H. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Natural Science Foundation of China (grant numbers 32402890 and 32172939), the China Postdoctoral Science Foundation (grant number 2023M732993), the Natural Science Foundation of Jiangsu Province of China (grant number BK20210803), the Yangzhou University Interdisciplinary Research Foundation for Veterinary Medicine Discipline of Targeted Support (grant number yzuxk202003), and the Key Research and Development Program of Yangzhou City (Social Development) (grant number YZ2022059).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

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

Acknowledgments

We acknowledge Hangning Ying and Suxing Zhang from Yangzhou University for sample collection.

Conflicts of Interest

The authors declare no conflicts of interest.

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Foods 13 03201 g001
Figure 1. Phylogenetic relationship and heatmap of tet(X)-positive Empedobacter sp. isolates. The bacterial strains belonging to E. falsenii, E. stercoris, and Taxon 1 are marked in green, purple, and pink, respectively. Their accession numbers and antibiotic resistance genes (solid circles) are also present. Bar, 0.3 nucleotide substitutions per site.
Figure 1. Phylogenetic relationship and heatmap of tet(X)-positive Empedobacter sp. isolates. The bacterial strains belonging to E. falsenii, E. stercoris, and Taxon 1 are marked in green, purple, and pink, respectively. Their accession numbers and antibiotic resistance genes (solid circles) are also present. Bar, 0.3 nucleotide substitutions per site.
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Foods 13 03201 g002aFoods 13 03201 g002b
Figure 2. Structural characteristics of Tet(X) variants. (A) Multiple sequence alignment of Tet(X) variants. Secondary structure elements of Tet(X2) (2XDO) are present on top, with triangles indicating the reported key amino acid sites. Identical residues are boxed in red. Similar residues in a group or across groups are marked with red characters and blue frames, respectively; (B) homology modelling of Tet(X) variants with Tet(X2) (4A6N). The substrate-binding domain (green), FAD-binding domain (pink), and C-terminal helix (blue) are marked, and the reported key amino acid sites are also displayed.
Figure 2. Structural characteristics of Tet(X) variants. (A) Multiple sequence alignment of Tet(X) variants. Secondary structure elements of Tet(X2) (2XDO) are present on top, with triangles indicating the reported key amino acid sites. Identical residues are boxed in red. Similar residues in a group or across groups are marked with red characters and blue frames, respectively; (B) homology modelling of Tet(X) variants with Tet(X2) (4A6N). The substrate-binding domain (green), FAD-binding domain (pink), and C-terminal helix (blue) are marked, and the reported key amino acid sites are also displayed.
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Figure 3. Conserved genetic backgrounds of tet(X24), tet(X25), tet(X26), and tet(X26.2) genes in Empedobacter sp. isolates. Regions of >76% nucleotide identity are marked by shading.
Figure 3. Conserved genetic backgrounds of tet(X24), tet(X25), tet(X26), and tet(X26.2) genes in Empedobacter sp. isolates. Regions of >76% nucleotide identity are marked by shading.
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Figure 4. ISCR2-related transposition events across different bacterial species. Regions of >79% nucleotide identity are marked by shading. The Δ symbol indicates that the gene is truncated.
Figure 4. ISCR2-related transposition events across different bacterial species. Regions of >79% nucleotide identity are marked by shading. The Δ symbol indicates that the gene is truncated.
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Table 1. MICs of E. coli JM109 + pBAD24 and its clones.
Table 1. MICs of E. coli JM109 + pBAD24 and its clones.
CloneMIC (mg/L) †
TCDOXMINTGCMEMCTXTYLTILTIP
Empty20.50.250.1250.25≤0.12512880.25
tet(X2)1640.50.25-----
tet(X2.2)1640.50.25-----
tet(X6)6416168-----
tet(X23)6416168-----
tet(X24)12832328-----
tet(X25)12832168-----
tet(X26)12832168-----
tet(X26.2)12832328-----
blaEBR-4----22---
blaEBR-5.2----0.51---
blaEBR-5.3----0.25≤0.125---
blaEBR-6----10.5---
blaEBR-7----0.25≤0.125---
blaEBR-7.2----0.25≤0.125---
blaEBR-7.3----0.51---
estT-1.2------512641
estT-1.3------>512321
estT-1.4------512161
estT-2------256641
estT-3------>5121284
estT-4------>5121284
estT-5------>512321
† TC, tetracycline; DOX, doxycycline; MIN, minocycline; TGC, tigecycline; MEM, meropenem; CTX, cefotaxime; TYL, tylosin; TIL, tilmicosin; TIP, tildipirosin.
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Chen, C.; Lv, Y.; Wu, T.; Liu, J.; Guo, Y.; Huang, J. Concurrence of Inactivation Enzyme-Encoding Genes tet(X), blaEBR, and estT in Empedobacter Species from Chickens and Surrounding Environments. Foods 2024, 13, 3201. https://doi.org/10.3390/foods13193201

AMA Style

Chen C, Lv Y, Wu T, Liu J, Guo Y, Huang J. Concurrence of Inactivation Enzyme-Encoding Genes tet(X), blaEBR, and estT in Empedobacter Species from Chickens and Surrounding Environments. Foods. 2024; 13(19):3201. https://doi.org/10.3390/foods13193201

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Chen, Chong, Yilin Lv, Taotao Wu, Jing Liu, Yanan Guo, and Jinlin Huang. 2024. "Concurrence of Inactivation Enzyme-Encoding Genes tet(X), blaEBR, and estT in Empedobacter Species from Chickens and Surrounding Environments" Foods 13, no. 19: 3201. https://doi.org/10.3390/foods13193201

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