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. 2023 Feb 10;102(5):102579. doi: 10.1016/j.psj.2023.102579

Molecular epidemiology and transmission of rmtB-positive Escherichia coli among ducks and environment

Guihua Li *,†,‡,1, Xiaoshen Li *,†,‡,1, Jianxin Hu *,†,, Yu Pan *,†,, Zhenbao Ma §, Lingxuan Zhang *,†,, Wenguang Xiong *,†,, Dongping Zeng *,†,, Zhenling Zeng *,†,‡,2
PMCID: PMC10023955  PMID: 36913759

Abstract

This study aimed to investigate the transmission and molecular epidemiological characteristics of the rmtB gene in Escherichia coli (E. coli) strains isolated from duck farms in Guangdong Province of China from 2018 to 2021. A total of 164 (19.4%, 164/844) rmtB-positive E. coli strains were recovered from feces, viscera, and environment. We performed antibiotic susceptibility tests, pulsed-field gel electrophoresis (PFGE), and conjugation experiments. We obtained the genetic context of 46 rmtB-carrying E. coli isolates and constructed a phylogenetic tree via whole genome sequencing (WGS) and bioinformatic analysis. The isolation rate of rmtB-carrying E. coli isolates in duck farms increased yearly from 2018 to 2020 but decreased in 2021. All rmtB-harboring E. coli strains were multidrug resistant (MDR), and 99.4% of the strains were resistant to more than 10 drugs. Surprisingly, duck- and environment-associated strains similarly showed high MDR. Conjugation experiments revealed that the rmtB gene horizontally cocarried blaCTX-M and blaTEM gene dissemination via IncFII plasmids. Insertion sequences IS26, ISCR1, and ISCR3 were closely associated with the spread of rmtB-harboring E. coli isolates. WGS analysis indicated that ST48 was the most prevalent sequence type. The results of single nucleotide polymorphism (SNP) differences revealed potential clonal transmission between ducks and the environment. Based on One Health principles, we need to strictly use veterinary antibiotics, monitor the distribution of MDR strains, and evaluate the impact of plasmid-mediated rmtB gene on human, animal, and environmental health.

Key words: duck, Escherichia coli, rmtB, multidrug resistance, whole genome sequencing

INTRODUCTION

Aminoglycosides antibiotics (AAs) are bactericidal antimicrobial drugs that have a broad spectrum of extent in gram-negative strains and some gram-positive strains (Eljaaly et al., 2019). They have strong concentration-dependent bactericidal power, postantibiotic effects, and are usually used in combination with penicillins, cephalosporins, or other extended-spectrum β-lactamase (ESBL) antibiotics (Castanheira et al., 2018). AAs are used not only to treat severe infections caused by multidrug-resistant (MDR) bacteria but also to boost animal growth (Fritsche et al., 2008). Given that AAs have been abused in human clinics and animal husbandry for a long time, bacteria have extensive resistance to many antibacterial drugs, such as streptomycin, kanamycin, gentamycin, and neomycin (Deng et al., 2013).

AAs resistance mechanisms include modified structure by aminoglycoside-modifying enzymes, mutated and altered ribosomal mutations, and augmented expression of efflux genes (Castanheira et al., 2018). In recent years, 16S rRNA methylases (RMTases) reportedly showed high-level resistance to many kinds of AAs (Castanheira et al., 2018). 16S RMTases can methylate the 16S rRNA in the 30S subunit of the bacterial ribosome, so that AAs cannot bind to it and play a part in antibiosis, resulting in bacteria resistant to this kind of antibiotics (Fournier et al., 2022). Since the first report on 16S RMTase-encoding gene armA in 2003, rmtA, rmtB, rmtC, rmtD, rmtE, rmtF, rmtG, rmtH, armA, and npmA have been found in different countries or areas (Yamane et al., 2005; Tada et al., 2019).

RmtB genes are the most frequently discovered in the whole world (Ma et al., 2009; Xia et al., 2016). They are closely related to ESBL-encoding genes (Ma et al., 2009; Wangkheimayum et al., 2017). For example, a study found that the rmtB-positive Escherichia coli (E. coli) strains isolated from a hospital in Bulgaria contained the carbapenemase-encoding gene blaNDM-1 and the ESBL-encoding gene blaCTX-M-15 in 2014 (Poirel et al., 2014). In 2016, another report found that rmtB-positive Klebsiella pneumoniae (K. pneumoniae) strains isolated from Brazil also carry blaCTX-M gene (Longo et al., 2019). A combination of factors has facilitated the rapid transmission of 16S RMTase-encoding genes among different bacteria, such as coselection, which is mainly because of conjugative plasmids and other mobile genetic elements (Taylor et al., 2021).

RmtB is mainly found on IncFII plasmid, but it is also present in IncN and IncI1 plasmids (Xia et al., 2016). IncFII plasmid is a narrow host range type of plasmid, which is mainly detected in Enterobacteriaceae, such as E. coli, Enterobacter cloacae, K. pneumoniae, and Salmonella enterica serovar Indiana (Hou et al., 2012; Habeeb et al., 2013; Yang et al., 2015a). For example, a study reported that the isolate of K. pneumoniae ST11 cocarries blaKPC-2, blaCTX-M, and rmtB genes on the same IncFII plasmid (Uchida et al., 2019). Another report found that MDR genes blaTEM, fosA3, and rmtB coexist on the IncFII plasmid, such as F33:A-:B-, F2:A-:B-, and F18:A-:B plasmids (Cao et al., 2017). In addition, the rmtB, qepA, and blaCTX-M genes are colocated in F2:A:B and F33: A: B plasmids in Enterobacteriaceae isolated from pets (Deng et al., 2011a). The insertion sequence and mobile plasmids play important roles in the acquisition of resistance and virulence genes. The spread of rmtB is associated with IS26, Tn3, Tn2, ISCR1, and ISCR3 (Camelena et al., 2020; Wang et al., 2020). The insertion sequence IS26 is often inserted into the integron Tn2/Tn3 of the plasmid carrying the rmtB gene, and the tnpA or tnpR gene of the integron Tn2/Tn3 is truncated (Fang et al., 2019).

Antibiotics have been unreasonably used in food animals for a long time, leading to the emergence of antimicrobial resistance (AMR) (Pholwat et al., 2020). E. coli is a kind of symbiotic bacterium, which causes serious infection in human and animal clinics (Kim et al., 2022). The emergence of MDR E. coli has become a serious global medical problem, and MDR E. coli can be transmitted to humans through edible animals and the environment (Liu et al., 2022). To date, rmtB-positive E. coli has been reported in diverse food-producing animals, pets, and humans in China (Deng et al., 2011b; Xia et al., 2016; Camelena et al., 2020). Most rmtB-positive E. coli are MDR strains and cocarry other antibiotic resistance genes. Ducks are an important vector to transmit MDR strains to animals and humans (Wang et al., 2021). Ducks are widely raised in China, but few studies have reported the specific transmission and prevalence of rmtB-bearing E. coli isolates in duck farms in Guangdong Province.

Therefore, we focused on rmtB-carrying E. coli isolates recovered from duck farms in Guangdong Province, China, including the cities of Zhaoqing, Foshan, and Qingyuan, from October 2018 to December 2021. The purpose of this study was to describe the MDR and phylogenetic relationship of the rmtB-bearing E. coli strains. We also aimed to determine the epidemiology and transmission characteristics of rmtB-harboring E. coli strains isolated from ducks and the environment.

MATERIALS AND METHODS

Samples Collection

From October 2018 to December 2021, a total of 1,209 samples including 582 feces, 200 dead duck viscera samples, 427 environmental samples collected from 24 large-scale duck farms from Guangdong province (including the cities of Zhaoqing, Foshan, and Qingyuan), China. The sick duck viscera samples included heart, liver, spleen, lungs, and intestine. The environmental samples included pool water, pool mud, air, feed, and soil.

Isolation of E. Coli Strain and Detection of Antibiotic Resistance Genes

All samples were placed in 2 mL of Luria-Bertani (LB) liquid broth and grown with 180 rpm/min at 37°C for 12 h. According to the typical morphological features of colonies, the E. coli strain was selected and purified using MacConkey agar plates and eosin methylene blue (EMB) agar plates. Each single isolated colony was confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and 16S rRNA sequencing (Deng et al., 2011a). The identified E. coli strain's DNA was extracted by a simple boiling method. All 16S-RMTase genes were detected by a multiplex-PCR method previously studied (Zhou et al., 2010). Finally, all 16S-RMTase-produced E. coli strains were further amplified into ESBL-encoding genes blaCTX-M and blaTEM, colistin-resistant gene mcr-1, and carbapenemase-encoding gene blaNDM by specific polymerase chain reaction (PCR) (Yang et al., 2017; Long et al., 2019).

Antibiotic Susceptibility Testing

Antimicrobial susceptibilities to 14 antibiotics were tested using the agar dilution method and to apramycin, colistin, and tigecycline using the broth microdilution method. Escherichia coli ATCC 25922 was used as the quality control strain. The following 17 antimicrobials were used for susceptibility testing: amoxicillin (AMO), ceftiofur (CEF), gentamicin (GEN), amikacin (AMI), doxycycline (DOX), tigecycline (TIG), ciprofloxacin (CIP), enrofloxacin (ENR), neomycin (NEO), ampicillin (AMP), cefotaxime (CTX), florfenicol (FLR), ceftazidime (CAZ), apramycin (APR), colistin (CL), meropenem (MEM), and sulfamethoxazole-trimethoprim (SXT). Minimum inhibitory concentrations (MICs) were interpreted according to Clinical and Laboratory Standards Institute (CLSI) guidelines (CLSI, 2018). The breakpoints of apramycin were based on the National Antibiotic Resistance Monitoring System (NARMS) (https://www.cdc.gov/narms/annual/2001/2001.pdf), and the breakpoints of tigecycline for Enterobacteriaceae were interpreted according to EUCAST Version 6.0 (EUCAST, 2016).

Pulsed-Field Gel Electrophoresis

Depending on the different sources, hosts, years, and cities, 84 rmtB-bearing E. coli strains randomly selected for pulsed-field gel electrophoresis (PFGE). PFGE analyzed DNA fingerprint of the strains that digest genomic DNA through XbaI enzymes. It was performed using the CHEF-MAPPER System (Bio-Rad Laboratories, Hercules, CA) with 1% agarose in 0.5 × Tris-borate-EDTA (TBE) at 150 V. It was run at the condition of temperature 14°C, voltage 6 V/cm, pulse angle 120°, and pulse duration of 2.2 to 54.3 s for 20.3 h (Murase et al., 1996). Salmonella enterica H9812 served as a standardized DNA size marker in this study. The PFGE patterns were analyzed using BioNumerics software 7.6 (Applied-Maths, Kortrijk, Belgium). We used the unweighted pair-group approach with arithmetic mean (UPGMA) algorithm according to the Jaccard resemblance size. It used a cut-off value of 85% of the similarity as Criteria for determining genetic relatedness (Magistrali et al., 2015).

Conjugation Experiments and PBRT

To describe the plasmids cocarrying 16S RMTase genes and other important drug-resistant genes, we performed mating-out assays. Conjugation experiments were assessed by LB mating test with E. coli C600 (resistant to streptomycin) as the recipient strain and 164 rmtB-harboring E. coli isolates as the donor strains. Donor strains and recipient strain were respectively grown in LB liquid broth with 180 rpm for 5 h at 37°C. They were sufficiently mixed (ratio of 1:4) in LB broth and incubated for 12 h at 37°C. Transconjugants grown on LB agar plates including streptomycin (3,000 mg/L) and amikacin (64 mg/L) were screened (Xiang et al., 2015). PCR was performed with the related rmtB-specific primers to evaluate the success of transconjugants. All rmtB-positive transconjugants were subjected to antimicrobial susceptibility test, PCR-based replicon typing (PBRT), and PCR to determine other important drug-resistant genes (Amin et al., 2019). PBRT was achieved by PCR different plasmid replicon types such as IncFII, IncN, IncI, IncFIA, IncFIB, IncX, IncR, and IncHI2 (Saha et al., 2006). To further describe FII, FIA, and FIB groups, all transconjugants carrying the IncF plasmid were subjected to replicon sequencing typing (RST). Alleles were obtained by submitting the amplicon sequence to the plasmid multilocus sequence typing (MLST) website as described in a previous study (Yang et al., 2015b).

Whole Genome Sequencing and Analysis

In view of the result of PFGE, PBRT, and MDR, 46 characteristic rmtB-positive E. coli isolates were selected for WGS. Genomic DNA was extracted using the HiPure Bacterial DNA Kit (TIANGEN China, Beijing, China). WGS sequenced by the Illumina NOVAseq 6000 sequencing platform (Novogene Company, Beijing, China) (Chin et al., 2013). Illumina Novaseq sequences were assembled using CLC Genomics Workbench 10 (CLC Bio, Aarhus, Denmark). All assembled genomes were annotated using RAST and IS finder web (Overbeek et al., 2014). The data of MLST, plasmid incompatibility (Inc) groups, antibiotic resistance genes, and virulence genes were identified using MLST, PlasmidFinder, ResFinder, and VirulenceFind from Center for Genomic Epidemiology. A phylogenetic tree was constructed based on core-genome single nucleotide polymorphism (SNP). It was formed by using default parameters with CSI Phylogeny in Center for Genomic Epidemiology (http://www.genomicepidemiology.org) (Treangen et al., 2014; Guneri et al., 2022). The assembled genome of the GDB20D1 strain was served as a reference genome. RmtB-flanking region genetic context comparison was performed using Easyfig 2.1 (Sullivan et al., 2011). The rmtB sequence homology was completed via BLAST from the National Center for Biotechnology Information (NCBI) website.

RESULTS

Prevalence of Antibiotic Resistance Genes

In this study, a total of 164 (19.4%, 164/844) 16S-RMTase-produced E. coli strains were recovered from 469 feces, 239 environment and 136 viscera in duck farms in Guangdong province (including the cities of Zhaoqing, Foshan, and Qingyuan) of China from October 2018 to December 2021. All 16S-RMTase-produced E. coli strains carried the rmtB gene, and other 16S-RMTase-encoding genes were not found. Compared with the detection rate of rmtB among E. coli isolates from different sources, viscera had the highest detection rate (33.1%), followed by the environment (19.3%) and feces (15.6%). Viscera samples included samples from the intestine (8.54%), heart (5.49%), liver (5.49%), lung (4.27%), and spleen (3.66%). Environmental samples included pond water (17.68%), feed (4.27%), air (3.66%), pond mud (1.22%), and soil (1.22%) (Supplementary Table 3). The isolation rate of rmtB in E. coli increased every year from 2018 to 2020 (7.4% in 2018, 10.7% in 2019, and 41.3% in 2020) but decreased in 2021 (17%) (Figure 1A and Supplementary Table 1). Furthermore, the ESBL-encoding genes blaCTX-M (89.63%) and blaTEM (86.58%) appeared at a high percentage in most of the rmtB-bearing E. coli strains. However, the detection rates of the blaNDM and mcr-1 genes were at a very low proportion (<3.05%) (Supplementary Table 4).

Figure 1.

Figure 1

The detection rate and antimicrobial resistance rate of rmtB-positive E. coli strains. (A) The positive detection rate of the rmtB-positive E. coli strains from feces, environment, and viscera in duck farms in Guangdong Province of China from 2018 to 2021. The detection rate of maximum and minimum were respectively shown in blue and white. (B) The bar graph indicates the total number of drugs with resistance in each duck and environment-associated isolates. Duck-associated isolates include feces and viscera. (C) Heat map denotes the antimicrobial resistance rate of 164 rmtB-positive E. coli strains isolated from feces, viscera, and environment to 17 drugs. The figures were performed in Graphpad Prism 8.0 (Graphpad Software, La Jolla, CA).

Antimicrobial Susceptibility

The antimicrobial susceptibility of 164 rmtB-carrying E. coli strains was tested by 17 different antibiotics. Notably, all rmtB-harboring E. coli strains were considered MDR strains on the basis of the MIC results, and these strains were resistant to at least 6 antimicrobial drugs. Some rmtB-carrying E. coli strains (37.19%, 61/164) were resistant to 13 antimicrobial drugs, and the main pattern (54.09%, 33/61) of MDR was AMP-AMO-CEF-CTX-CAZ-GEN-AMI-NEO-DOX-FLR-ENR-CIP-SXT. Strains resistant to 14 or 12 antimicrobial drugs comprised 20.12 and 14.02%, respectively, of the sample population. Worryingly, 99.4% of the strains were resistant to more than 10 drugs (Supplementary Table 3). Duck- and environment-associated isolates were similarly resistant to a large amount of antimicrobial drugs (median = 13; Figure 1B). All strains isolated from feces, viscera, and environment emerged as highly resistant to AAs, including gentamicin, neomycin, and amikacin; ESBL antibiotics containing ampicillin, amoxicillin, and cefotaxime; sulfonamide antibiotic sulfamethoxazole-trimethoprim; tetracycline antibiotics doxycycline, and florfenicol; and quinolone antibiotics such as enrofloxacin and ciprofloxacin (>82%). However, they were poorly resistant to meropenem, tigecycline, and colistin (<12.80%). In addition, 129 (77.71%) rmtB-positive E. coli strains remained resistant to ceftazidime, whereas 81 (48.80%) were resistant to apramycin (Figure 1C and Supplementary Table 3).

Pulsed-Field Gel Electrophoresis

A total of 84 rmtB-positive E. coli isolates from different hosts and sources in duck farms were successfully studied for their genetic relatedness by XbaI–PFGE (Figure 2). The results showed multiple types of PFGE patterns. PFGE analysis indicated that the genetic relatedness of most rmtB-carrying E. coli strains was distant, and those strains were epidemiologically not closely connected. However, small amounts of rmtB-bearing E. coli strains recovered from the same duck farm presented 100% clonal semblance. For example, GDQ20D85 and GDQ20D90 strains (100% clonal semblance) both isolated from pond water were clonal strains. Sick duck intestine- and lungs-associated isolates GDQ20D115 and GDQ20D112 with 100% clonal semblance were also clonal strains. Thus, vertical clonal transmission occurred among some rmtB-harboring E. coli strains in an identical duck farm.

Figure 2.

Figure 2

PFGE patterns and features of 84 rmtB-positive E. coli strains isolated from different sources in duck farms in Guangdong Province of China from 2018 to 2021.

Conjugation Experiments and Plasmid Analysis

A total of 110 (67.07%) rmtB genes were successfully and horizontally transferred to E. coli C600 through conjugation experiments. All rmtB-positive transconjugants underwent drug sensitivity tests, and all transconjugants were MDR strains and resistant to more than 4 drugs. The most frequently found pattern of MDR was AMP-AMO-CEF-CTX-GEN-AMI-NEO, which was resistant to 7 antimicrobial drugs (25.45%). Transconjugants resistant to more than 6 antimicrobial drugs accounted for 91.8%. Most rmtB-positive transconjugants were highly resistant to AAs, including gentamicin, amikacin, ampicillin, and ESBL antibiotics containing amoxicillin, ceftiofur, and cefotaxime (>79.1%). However, they were poorly resistant to ceftazidime, enrofloxacin, and ciprofloxacin (<9.1%). All transconjugants were susceptible to meropenem, tigecycline, and colistin. Furthermore, some transconjugants remained resistant to neomycin, apramycin, doxycycline, florfenicol, sulfamethoxazole-trimethoprim (from 27.3 to 53.6%) (Supplementary Table 5).

The PBRT of 110 transconjugants revealed that the most prevalent incompatible plasmid in rmtB was IncFII plasmid (87.27%), but small amounts of IncN and IncI1 IncY, IncFIB, and IncHI2 plasmids also coexisted in the transconjugants. RST revealed that the F33:A-:B- plasmid (56.25%, 54/96) was the most popular IncFII plasmid. Other IncFII plasmids F16:A-:B- (19.79%), F29:A-:B- (11.46%), F18:A-:B1 (4.17%), and F40:A-:B- (3.13%) were also detected. Five transconjugants carried F14:A-:B1, F16:A-:B1, F18:A-:B-, F33:A-:B1, and F63:A-:B- plasmids. PCR demonstrated that most rmtB-harboring transconjugants cocarried ESBL-encoding genes, including blaCTX-M (83.64%), blaTEM (90.00%), and blaOXA (2.73%) (Supplementary Table 6).

RmtB Flanking Region Genetic Contexts

On the basis of rmtB gene locus contig annotation and blast comparison of the relative flank environment, the rmtB genetic context of 46 sequenced E. coli strains were classified into 5 types (Types I, II, III, IV, and V) (Figure 3). Other antimicrobial genes were also present, including qnrs1, qepA1, blaTEM-1, blaCTX-M, and sul1 surrounding rmtB. Insertion sequences containing IS3, IS6, IS26, IS1380, ISCfr1, ISCR1, and ISCR3 and integron Tn3 were also found. Most of the rmtB contig sequences were very short in this study at only about 1,200 bp. Therefore, the genetic structure type II (n = 35) blaTEM-1-rmtB-ΔIS26 was the most prevalent. In type I (n = 2), the genetic background was rmtB-hp-hp-ISCR3-hp-qepA1. This structure was highly similar to a rmtB-qepA-blaCTX-M-27-carrying IncFII plasmid pGDD25-16 (GenBank accession number MH316135) from a Salmonella enterica serotype Indiana strain of duck origin isolated from Guangdong Province, China. Type III structure (n = 3) included blaTEM, sul1, ISCfr1, and ISCR1. ISCfr1 was located upstream of the blaTEM gene, whereas ISCR1 was located downstream of rmtB. The genetic context was ISCfr1-blaTEM-1-rmtB-hp-ISCR1-hp-sul1. It showed a high level of similarity to a plasmid pSCKLB138-1 (accession number MH161192) from a Chinese K. pneumoniae strain. In the type IV (n = 2) structure, ΔIS6 and ΔTn3 were located upstream of blaTEM. The genetic context of the rmtB flanking region was ΔIS6-ΔTn3-hp-blaTEM-1-rmtB-ΔIS26. In type V (n = 2), the rmtB contig sequence was long at about 14,695 bp, and the genetic background was relatively more complex and complete. Horizontal mobile gene elements containing IS3, IS1380, and Tn3 were located upstream of blaTEM, whereas the insertion sequence ΔIS26 was located downstream of rmtB. This long genetic background was qnrs1-IS3-Tn3-hp-blaCTX-M-IS1380-hp-hp-blaTEM-1-rmtB-ΔIS26. This was almost identical to a plasmid pSD21 (accession number CP093915) from an Enterobacter cloacae strain of human origin isolated from Shandong Province in China. Thus, the rmtB gene was disseminated to other Enterobacteriaceae of diverse origins.

Figure 3.

Figure 3

Schematic representation and comparison of 5 different types (Types I, II, III, IV, V) of rmtB-flanking region genetic contexts in 46 rmtB-positive E. coli strains. Comparing the corresponding regions with 3 strains from the NCBI database. Arrows denote the direction of transcription of each of the genes. The rmtB gene is shown as a red arrow, green arrows indicate other antimicrobial resistance genes and blue arrows indicate horizontal transfer element. Hypothetical proteins or other functions are depicted in pink arrows. Regions of >98% nucleotide sequence identity are shaded in gray. “Δ” indicates a truncated gene.

The Phylogenetic Tree and MLST

The maximum likelihood phylogenetic tree, MLST, sources, Inc.type plasmids, antibiotic resistance genes, and virulence genes of 46 sequenced rmtB-positive E. coli strains are shown in Figure 4. The sources of strains were diverse, including feces, pond water, pond mud, feed, air, soil, liver, intestine, spleen, and lungs. A total of 23 various sequence types (STs) were found among them. The most prevalent ST was ST48 (19.5%), followed by ST4383 (10.8%), ST224 (8.6%), and ST1011 (8.6%). The remaining 19 isolates represented individual STs. ST48 strains were mainly recovered from feces, soil, liver, and pond water in the same duck farm. Most strains carried IncFII plasmid (72.3%). IncX, IncN, and IncI1 plasmids were also found. Most of the sequenced strains carried aminoglycosides resistance gene APH(6)-Id, ESBL-encoding genes containing blaCTX-M, blaOXA, and blaTEM; sulfonamide resistance genes such as dfrA14 and sul2; florfenicol resistance gene floR; tetracycline resistance gene tet(A); quinolone resistance gene qnrS1 and fosfomycin resistance gene fosA3 (>61%). The most populous virulence genes were terC and traT (89%). Other virulence genes gad, ompT, and papC were also detected.

Figure 4.

Figure 4

Maximum likelihood phylogenetic tree and MLST of 46 rmtB-positive E. coli strains isolated from feces, environment, and viscera from 2018 to 2021. Inc.type plasmids, resistance genes, and virulence genes were respectively shown in green, red, and blue, empty squares indicate nonexistence.

All sequenced rmtB-positive E. coli strains in the phylogeny shared a total of 37,158 core genome SNPs. A strain ST48 E. coli GDQ8D193 isolated from soil shared only 9 SNPs with the ST48 GDQ8D93 strain isolated from feces and shared 0 SNP with ST48 GDQ8D78 isolated from feces in Zhaoqing City in this study. Sick duck intestine-associated isolate GDS21D219 was closely related to GS21D142 isolated from pond mud (with a difference of 2 SNPs). The ST48 GFQ9D161 strain isolated from pond water was intimately correlated with feces-associated isolate ST48 GFQ9D86 (0 SNP difference). The GDS21D14 strain recovered from feces differed by only 18 SNPs from the pond water isolate GDS21D156 isolated in this study. At the same time, GDS21D14 was closely related to feed-associated isolate GDQ20D92 (1 SNP difference). Feces-associated isolate GDS21D110 was closely related to intestine-associated isolate GDS21D222 (7 SNP differences). Potential clonal transmission was noted between ducks and the environment. Thus, the environment and food chain probably play an important part in the dissemination of rmtB-harboring E. coli strains.

DISCUSSION

16S-RMTase-encoding genes are highly resistant to AAs, leading to drug treatment failure in most gram-negative bacteria. We found that the rmtB gene was the most prevalent in 16S-RMTase-encoding genes, which was identical to other results about the prevalence of 16S-RMTase-encoding genes (Xia et al., 2016; Fang et al., 2019). Notably, the isolation rate of rmtB in E. coli strains increased yearly from 2018 to 2020. The isolation rate in 2020 (41.3%) was higher than that in most studies (Yang et al., 2015a; Xia et al., 2016; Fang et al., 2019; Long et al., 2019), and this difference may be attributed to the extensive dissemination of rmtB-positive E. coli strains in duck farms. However, the detection percentage in 2021 decreased, which may be due to the policy of limiting and reducing the use of antibiotics in China and duck farms reducing the use of antimicrobial drugs. At the same time, the size of the duck farm and the breeding environment can lead to differences in the detection rate.

AMR is an increasingly serious problem worldwide. From the results of antimicrobial susceptibility tests, most of the rmtB-positive E. coli isolates were MDR strains, and 99.4% of the strains were resistant to more than 10 drugs. Those isolates displayed high levels of resistance to gentamicin, neomycin, amikacin, ampicillin, amoxicillin, cefotaxime; sulfamethoxazole-trimethoprim, doxycycline, florfenicol, enrofloxacin, and ciprofloxacin. Compared with other studies, the levels of AMR were even higher (Yang et al., 2015a; Xia et al., 2016; Long et al., 2019; Fournier et al., 2022). This study indicated that MDR in ducks and the surrounding environment was extremely serious. Some duck farms at one time have used florfenicol, amoxicillin, and gentamicin to treat bacterial infections and prevent diseases. This phenomenon may be why the rmtB-harboring E. coli isolates showed high resistance to those antimicrobial drugs.

PFGE analysis showed a vertical clonal relationship among rmtB-positive E. coli strains from the same duck farm, but most strains isolated from different duck farms had nothing to do with epidemiology. The rmtB gene is mainly transferred horizontally by moving plasmids, which was also reported in other studies (Ho et al., 2013). Conjugative plasmids included IncFII plasmids, especially F33:A-:B- plasmid, which played a considerable role in the horizontal transmission of rmtB in this study. IncFII plasmids with the rmtB gene had high conjugative frequency in the conjugation experiment, stabilization in continuous cultivation, and relative fitness advantage in competitive experiments (Chen et al., 2007). These features helped promote rmtB widely and horizontally disseminate among Enterobacteriaceae, especially among E. coli. Therefore, we need to attach great importance to the horizontal transmission of the IncFII plasmid, which will greatly reduce the antibacterial effect of clinical infection.

Most rmtB-positive transconjugants simultaneously detected the ESBL genes blaTEM and blaCTX-M. Those transconjugants concurrently showed high resistance to AAs and ESBL antibiotics. This result suggested that rmtB horizontally codisseminated with ESBL-encoding genes to diverse bacteria through bacterial conjugation with mobile plasmids, similar to the reports of another study (Xia et al., 2016). The coexistence of some resistance genes on the identical plasmid may contribute to the spread and efficient persistence under diverse antimicrobial selection pressure.

In this study, rmtB-carrying E. coli isolates commonly cocarried AAs-, ESBL-, sulfonamide-, florfenicol-, tetracycline-, quinolone-, and fosfomycin-associated resistance genes and various virulence genes. These E. coli strains are highly adaptable to the environment, which makes them easily spread horizontally between food animals and the environment. This study emphasized that ducks play an important role as a reservoir of MDR genes. The rmtB gene is likely to be transmitted to human intestinal flora through the food chain and duck farm environment. Although this hypothesis needs further evaluation, the transmission of MDR genes from food animals to humans through contact with cultured animals or eating contaminated food is often reported (Camelena et al., 2020). Therefore, we must pay close attention to the possible risk of the transmission of MDR genes from the environment and food animals to humans, as well as further evaluate the influence of plasmid-mediated rmtB on human, animal, and environmental health (“One Health”).

A high degree of genetic similarity was noted between the genetic background (types I, III, and V) of the flanking region of rmtB and other Enterobacteriaceae carrying rmtB gene plasmids. The query coverage rate exceeded 99%, and nucleotide homology exceeded 99%. These homologous rmtB gene regions appeared in Salmonella enterogenes from ducks, K. pneumoniae, and Enterobacter cloacae from humans, indicating that the rmtB gene cluster spread to other Enterobacteriaceae from different origins. RmtB-qepA, rmtB-blaTEM, rmtB-blaTEM-sul1, and rmtB-blaTEM-blaCTX-M gene combinations were closely affiliated with plasmids in Enterobacteriaceae. The exchange of the rmtB gene cluster was likely to occur in human and animal Enterobacteriaceae (Amin et al., 2019). The coexistence of rmtB, blaTEM, blaCTX-M, and qepA on the same plasmid was likely to promote the spread of AA, ESBL, and quinolone resistance in different animal and human sources.

The spread of AMR genes is usually frequently associated with mobile genetic elements such as plasmids, transposons, and integrons (Uchida et al., 2019). Several mobile elements are affiliated with the transfer of rmtB genes, including Tn2, IS26, ISCR1, ISCR3, IS903, and ISKpn43, which are often located in IncFII plasmids (Fang et al., 2019; Camelena et al., 2020; Wang et al., 2020). In this study, rmtB-flanking region genetic contexts were complicated with diverse AMR genes and horizontal transfer elements. We found that the insertion sequences (including IS3, IS6, IS26, IS1380, ISCfr1, ISCR1, and ISCR3) and integron Tn3 played an important role in the horizontal dissemination of rmtB gene. Illumina technology for WGS was deficient in analyzing the complete rmtB-flanking region genetic context, maybe owing to a great deal of complicated insertion sequences (ISs) and mobile genetic elements (Taylor et al., 2021). Those mobile genetic elements contribute to the spread of AMR genes and generate complex genetics by homologous recombination. Therefore, this study can provide strong evidence for the horizontal transmission of rmtB-positive E. coli strains in ducks and the environment by ISs (especially IS26, ISCR3, and ISCR1).

In this study, STs were multiplex including 23 classes; ST48 was the most prevalent ST in the sequenced 46 rmtB-positive E. coli strains. It differed from 2 studies that found that ST405 was the main ST in rmtB-carrying E. coli isolates recovered from Parisian and Algerian hospitals (Amin et al., 2019; Taylor et al., 2021). This study found rmtB-positive E. coli strains with the same ST48 in fecal sample, duck liver sample, and soil sample from an identical duck farm. Thus, the ST48 rmtB-positive strains, known to be involved in MDR, could potentially and clonally spread among ducks and the surrounding environment. Duck-associated isolates were closely related to environment-associated strains isolated from an identical duck farm from the analysis of SNP difference. RmtB-positive E. coli strains could easily disseminate through pond water, soil, feed, and feces in duck farms and clonally spread between ducks and environment isolates in the same duck farm, which was in agreement with the previous report (Wang et al., 2021).

CONCLUSIONS

This study investigated the molecular epidemiological characteristics of the rmtB gene in E. coli strains isolated from duck farms in Guangdong Province, China. The isolation rate of rmtB-carrying E. coli isolates in duck farms increased yearly but decreased in 2021. Surprisingly, duck- and environment-associated isolates similarly had severe MDR. RmtB was strongly correlated with other ESBL-encoding genes, sulfonamide resistance genes, and PMQR genes. IncFII plasmids; insertion sequences containing IS26, ISCR1, and ISCR3; and integron Tn3 promoted rmtB gene horizontal dissemination among E. coli. Clonal and horizontal spread occurred among ducks and the surrounding environment, with a high degree of efficiency. These results indicated the need to enhance the surveillance of monitoring the transmission of this MDR E. coli in duck farms.

ACKNOWLEDGMENTS

This work was supported by the Natural Science Foundation of Guangdong Province of China [2021A1515011159].

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 at: https://www.ncbi.nlm.nih.gov/genbank/, PRJNA885617.

Ethics Statements: Studies involving animal subjects. The animal study was reviewed and approved by the South China Agriculture University (SCAU) Animal Ethics Committee. All animals were sampled under authorization from the Institutional Animal Care and Use Committees (IACUCs) of SCAU.

Author Contributions: Guihua Li and Xiaoshen Li wrote the first draft of the manuscript. Wenguang Xiong, Dongping Zeng, and Zhenling Zeng contributed to conception and design of the study. Guihua Li, Xiaoshen Li, Jianxin Hu, Yu Pan, Zhenbao Ma, Lingxuan Zhang performed the sampling and statistical analysis. All authors contributed to manuscript revision, read, and approved the submitted version.

DISCLOSURES

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Footnotes

Supplementary material associated with this article can be found in the online version at doi:10.1016/j.psj.2023.102579.

Appendix. Supplementary materials

mmc1.xlsx (44KB, xlsx)

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