Research note: Emergence of bla_NDM-13 producing Escherichia coli from a broiler chicken and farm environment in Jiangsu Province, China

Abstract

Carbapenem-resistant bacteria, in particular NDM-producing Enterobacteriaceae, have become a great threat to the global public. The bla_NDM-13 is a novel bla_NDM variant with increasing dydrolytic activity against cefotaxime. This study aimed to investigate the molecular characteristics of bla_NDM-13-positive E. coli in a chicken farm located in Jiangsu Province of China. Six bla_NDM-13-positive E. coli strains were isolated from 83 samples, including cloacal samples (n = 4), cages (n = 1), and water dispensers (n = 1). Antimicrobial susceptibility testing showed that these isolates exhibited pan-resistance to β-lactams (meropenem MIC≥32 μg/mL), colistin, and six other antibiotic classes, retaining susceptibility only to tigecycline. Illumina sequencing showed that isolates harbored multiple resistance genes, with fosA3, aph(3′)-IIa, oqxA, oqxB, and aph(6)-Id being present in all strains. Notably, blaCTX-M-65 (extended-spectrum β-lactamase), mcr-1.1 (colistin resistance), and qacE (disinfectant genes) were detected in 50 %, 66.67 %, and 66.67 % of bla_NDM-13-positive E. coli strains, respectively. MLST classified the isolates into ST155 (n = 3), ST93 (n = 2), and ST1158 (n = 1), with bla_NDM-13-positive ST155 E. coli isolates found to be horizontal transmission between chicken and environmental surface samples. Three representative isolates were subjected to Nanopore long-read sequencing; the bla_NDM-13 was located on the IncFII plasmid, which showed high homology with clinical (pB5-1) and poultry-derived plasmids (YZLc23-1-NDM-13-96k), harboring conserved resistance gene clusters (IS91-blaNDM-13-bleMBL-IS50R-aph(3′)-IIa-IS26-IS6-fosA3), and confirmed horizontal transfer via conjugation. The conjugants were stably inherited after 15 days of passage and exhibited no significant differences in growth rates when compared to E. coli J53. This study identifies bla_NDM-13-carrying multidrug-resistant E. coli clones in poultry production systems, transmitted via poultry-environment interactions and plasmid-mediated gene transfer. Farm-level surveillance targeting high-risk transmission routes is essential to control antimicrobial resistance and protect poultry health.

Introduction

The emergence and dissemination of carbapenemase-producing Enterobacterales (CPE) pose a global health threat because of the misuse and overuse of carbapenems (Roberts et al., 2020). The prevalence of carbapenemase-resistant Enterobacaterales (CRE) can be related to the encoding of carbapenem hydrolase genes such as Klebsiella pneumoniae carbapenemase (KPC), Verona integron-encoded metallo-ß-lactamase (VIM), oxacillinase-48 (OXA-48), and New Delhi metallo- ß lactamase (NDM) through horizontal gene transfer (HGT) (Caliskan-Aydogan et al., 2023). NDM, an enzyme capable of hydrolyzing carbapenem antibiotics, has rapidly spread worldwide since its first identification in India in 2009. To date, 75 NDM variants have been documented (https://www.ncbi.nlm.nih.gov/pathogens/refgene/#NDM) and have been widely prevalent in 50 countries around the world, including China, India, and the UK (Wu et al., 2019). The continuous discovery of NDM variants has further complicated the issue of bacterial resistance.

The NDM-13 gene was first identified in 2015 from Escherichia coli (E. coli) clinical isolates from Nepal and has two amino acid substitutions compared to NDM-1 (Shrestha B et al., 2015). To date, NDM-13 has been detected in Enterobacteriaceae, such as E. coli and Salmonella, which are not only significant pathogens in hospital-acquired infections but can also spread to humans through environmental, animal, and food chains, posing a potential threat to public health (Zhang et al., 2023; Kim et al., 2019). Based on previous studies, the bla_NDM-13 gene is mostly carried by different types of plasmids, including IncX3, IncFIB, and IncI1. These plasmid-encoded resistance genes contribute to a substantial rise in carbapenem antibiotics. Therefore, identifying specific clones or plasmids carrying bla_NDM genes is critical for understanding the epidemiology of resistance and formulating effective strategies to mitigate the spread of NDM in both community and healthcare settings.

Despite the prohibition of carbapenems in food-producing animals, recent years have witnessed an increase in global reports of NDM-positive Enterobacteriaceae identified during poultry production processes (Zhai et al., 2020). There is limited information regarding the prevalence and transmission of NDM-13-producing E. coli in broiler farm ecosystems. Hence, we selected an industrial broiler farm to investigate the prevalence and molecular epidemiological characteristics of bla_NDM-13-carrying E. coli isolated from broilers and their surrounding farm environments.

Materials and methods

Sample collection

In 2024, we selected an industrial chicken farm in Xuzhou City, Jiangsu Province, China, for sampling. A total of 83 samples were collected, consisting of 30 cloacal swabs and 53 environmental samples: soil (n = 6), drinking water (n = 7), feed (n = 8), cage swabs (n = 10), feeding trough swabs (n = 6), ventilator swabs (n = 6), water dispenser swabs (n = 6), and workers' hand swabs (n = 4). All specimens were transported to the laboratory on ice. A questionnaire survey was carried out to assess the use of antibiotics on the farm. The antibiotics administered to the farm do not necessarily reflect those used for this batch of chickens. The antibiotics utilized on the farm included amoxicillin, tetracycline, tylosin, enrofloxacin, lincomycin, florfenicol, and doxycycline, which were regularly incorporated into the feed and drinking water for disease treatment purposes.

Sample processing and bacterial isolation

Water samples (600 mL) were filtered through 0.45 μm membranes. Feed (5 g) and soil (5 g) were homogenized in 45 mL LB broth. Swabs and pre-processed samples were enriched in LB broth supplemented with 2 μg/mL meropenem and incubated overnight at 37°C with shaking (180 rpm). After enrichment, 100 μL of each sample was plated on MacConkey agar containing 2 μg/mL meropenem and incubated at 36±1°C for 18–24 h. Distinct colonies were purified and identified by PCR amplification. The 16S rRNA and blaNDM genes of the strains were amplified and sequenced. blaNDM: F: 5′-GGTTTGGCGATCTGGTTTTC-3′, R: 5′-CGGAATGGCTCATCACGATC-3′, 621 bp; 16s rRNA: F: 5′-ATGAAACGAGTTATTACCCTGTT-3′, R: 5′-CGCCAATAATCGATTGCACATT-3′, the fragment size was 857 bp.

Antibiotic susceptibility testing

The minimal inhibitory concentrations (MICs) of the bla_NDM-13-positive E. coli strains were determined by the broth microdilution method against the following antimicrobial agents: ampicillin (AMP), amoxicillin/clavulanic (2:1, AMC), cefoxitin (FOX), ceftiofur (CEF), meropenem (MEM), florfenicol (FFC), kanamycin (K), gentamicin (GEN), tigecycline (TGC), tetracycline (TE), doxycycline (DOX), polymyxin E, ciprofloxacin (CIP), sulfisoxazole (SF), and trimethoprim/sulfamethoxazole 1:19 (SXT).The breakpoints were interpreted according to the standards of the Clinical and Laboratory Standards Institute of America (CLSI) and the European Committee on Antimicrobial Susceptibility Testing, with E. coli ATCC25922 used as the positive control.

Whole genome sequencing and assembly

Bacterial genomic DNA was extracted according to the instruction manual of the NucleoBond® HMW DNA kit (MN NucleoBond, Germany, 740160.20), and the purity and concentration of genomic DNA were determined via Qubit4.0 (Thermo, Q33226) and Nanodrop (SMA4000, Taiwan, China). DNA integrity was assessed by 0.75 % agarose gel electrophoresis. Library preparation and next-generation sequencing were performed by Sangon Biotech (Shanghai) Co., Ltd. Subsequently, the libraries were pooled and loaded onto a DNBseq-T7 sequencer (BGI, Shenzhen, China) using a 2 × 150 bp paired-end sequencing kit, following the manufacturer's instructions. Three representative isolates were further sent out for single-molecule nanopore DNA sequencing on MinION Flow Cell (ONT, R9.4.1).

Bioinformatics analysis

The acquired resistance genes, SNP, and multilocus sequence typing (MLST) were analyzed using ResFinder 4.7.2, CSIPhylogeny, and MLST 2.0 (https://www.genomic.epidemiology.org/services/). The phylogenetic trees were visualized and embellished using ChiPlot. Circular comparisons between plasmids were performed using the BRIG v0.95 tool. Circular plots of plasmids were generated using Proksee. To visualize the genetic comparison features, we used Easyfig v2.2.5 to generate linear comparison figure.

Conjugation experiment

Isolates containing the bla_NDM-13 gene were used as donor bacteria and azide-resistant E.coli J53 as recipient bacteria. The donor and recipient bacteria were inoculated into LB broth medium and incubated at 35 °C at 200 r/min to 0.5 McFarland culture density, respectively. Then, 100 μL of each was taken into fresh LB broth, mixed, and incubated at 35°C for 16-18 h. 100 μL of the mixed cultures was spread on agar plates containing 4 mg/L meropenem and 150 mg/L sodium azide. The transformants were selected and confirmed by PCR.

Stability of plasmid and growth rate determination

The plasmid stability of transconjugants was determined by a passaging experiment as a previous study. Three single clones of transconjugants were inoculated into 2 mL antibiotic-free LB broth. Following overnight incubation, the culture was diluted at a ratio of 1:1000 into 2 mL of antibiotic-free LB broth. Dilution and serial passage were performed every 24 h for 15 consecutive days. Bacterial enumeration was performed by inoculating samples onto MacConkey agar plates as well as MacConkey agar plates containing 2 μg/mL of meropenem.The growth rate of the transconjugant was determined as previously described. Growth curves were determined by measuring the optical density at 600 nm using a spectrophotometer.

Results and discussion

Prevalence of bla_NDM-13 carrying E. coli in a chicken farm

In this study, six bla_NDM-13-positive E. coli strains were isolated from 83 samples collected on a chicken farm, comprising four cloacal swabs (CSXZ1, CSXZ2, CSXZ3, and CSXZ4), one cage swab (CSXZ5), one water dispenser swab (CSXZ6).The isolation rates were found to be 13.33 % (4/30) for cloacal samples and 3.77 % (2/53) for environmental samples, with all samples testing negative for bla_NDM-1 and bla_NDM-5. Notably, the carriage rate of bla_NDM-13-positive strains in chicken feces was higher than the 7.89 % rate of bla_NDM-positive strains reported by Wen et al. (2023), highlighting a significant of zoonotic transmission risk from chickens and farm environments to humans.

Antimicrobial susceptibility testing and antibiotic resistance genes

The bla_NDM-13-positive E. coli strains exhibited a concerning multidrug-resistant profile, demonstrating complete resistance to all tested β-lactams (including meropenem with MICs up to 64 μg/mL), aminoglycosides (kanamycin, gentamicin), fluoroquinolones (ciprofloxacin), tetracyclines, sulfonamides, and florfenicol. However, these strains exhibited universal susceptibility to tigecycline while showing intermediate resistance to polymyxin E (33.3 %) and doxycycline (16.7 %). Notably, the strains harboring bla_NDM-13 were susceptible to tigecycline, which is consistent with previous research findings indicating that bla_NDM-positive strains are generally sensitive to this antibiotic. This suggests that tigecycline continues to be an effective treatment option for infections caused by these bacterial strains.

Genomic analysis revealed an extensive resistome consisting of 36 resistance genes, including aminoglycoside-modifying enzymes (aph(3′)-IIa, aph(3′)-Ib, and aph(6)-Id), efflux pumps (oqxAB), as well as fosfomycin (fosA3) and bleomycin (bleMBL) resistance determinants. Additional genes conferring resistance to β-lactams (blaTEM-1B, blaCTX-M-65, blaOXA-10), colistin (mcr-1.1), sulfonamides (sul1-3), and trimethoprim (dfrA14 and dfrA27) were also identified. Additionally, four bla_NDM-13-positive strains exhibited resistance to polymyxin E and carried the mcr-1.1 gene. Polymyxins (e.g.,colistin) represent last-resort therapeutics for carbapenem-resistant infections. Zhang et al. isolated a strain of E. coli carrying both bla_NDM-13 and mcr-1.1 from the urine of a 45-year-old cervical cancer patient (Zhang et al., 2023). Consequently, bla_NDM-13/mcr-1.1-co-harboring strains present serious public health risks requiring ongoing surveillance. Research found that a correlation between the presence of qacE and qac∆E genes and the utilization of disinfectants. The high prevalence of the qacE disinfectant resistance gene (66.7 %) in bla_NDM-13-positive E. coli isolates suggests a reduced effectiveness of quaternary ammonium disinfectants commonly used in poultry facilities. Given the identification of qacE-positive strains in the cage, we recommend implementing chlorhexidine-based disinfection protocols. This measure may mitigate the risks of prolonged colonization at the poultry-environment interface and enhance biosecurity on farms.

MLST and phylogenetic analysis

Six isolates were analyzed using whole-genome sequencing on the Illumina HiSeq 2500 platform, revealing three sequence types (STs): ST155 (n= 3; CSXZ3, CSXZ5, CSXZ6), ST93(n=2; CSXZ1 and CSXZ4), and ST1158 (n=1; CSXZ2), which demonstrated genetic diversity among bla_NDM-13-carrying E. coli strains, with no predominant ST. Among the E. coli isolates, ST155, ST1158, and ST93 were identified in diverse sources, including humans, food, poultry, the environment, and livestock. ST1158 demonstrates relatively low prevalence and, according to the EnteroBase database, has not been previously reported in China. To investigate clonal relationships among bla_NDM-13-positive E. coli, we analyzed our six strains alongaside four bla_NDM-13-positive strains from the NCBI database and selected 12 ST155, 10 ST1158, and 12 ST93 E. coli genomes from the EnteroBase database (seen in Fig. 1). The reference strain utilized for alignment and single nucleotide polymorphism (SNP) identification was YZLc23-1 (GenBank accession number CP123266.1), which was isolated from a live poultry market in Yangzhou, China. The phylogenetic tree revealed that the SNP differences among these strains ranged from o to 46,180 (Fig. 1). The strains isolated in this study exhibited SNP differences ranging from 11,208 to 46,058 when compared to the other four bla_NDM-13-positive E. coli strains, suggesting distant genetic relatedness. In this chicken farm, strains CSXZ1, CSXZ2, CSXZ3, and CSXZ4 were isolated from cloacal samples of chickens, representing ST155, ST93, and ST1158 ST types, with SNP differences reaching to 22,855, suggesting multiple independent introductions of distinct clones into the flock. The three ST155 strains (CSXZ3, CSXZ5, and CSXZ6) exhibited identical SNP profiles (0 SNP differences), indicating clonal transmission between chicken cloacal samples and farm environments, including cages and feed troughs. This finding provides direct evidence of bacterial circulation within the poultry production system. In this study, although no bla_NDM-13-positive strains were isolated from human samples, these findings suggest the potential for clonal transmission among humans, animals, and the environment within this farm.

Fig. 1.

Phylogenetic comparison of bla_NDM-13-positive isolates with EnteroBase ST-matched strains based on SNP analysis.

Characterization of bla_NDM-13-carrying plasmids

Bioinformatic analysis identified three bla_NDM-13-harboring plasmids: pCSXZ1-NDM-13 (109,022 bp, 53.86 % GC content), pCSXZ2-NDM-13 (108,509 bp, 53.24 % GC), and pCSXZ5-NDM-13 (126,837 bp, 47.79 % GC), all of which belong to the IncFII replicon type. Notably, pCSXZ1-NDM-13 exhibited 99.99 % identity (87 % coverage) to the clinical E. coli plasmid pB5-1 (CP176052.1) from Anhui, China, while pCSXZ2-NDM-13 showed 99.19 % identity (86 % coverage) with the poultry-derived plasmid YZLc23-1-NDM-13-96k (CP123269.1), and pCSXZ5-NDM-13 demonstrated near-complete identity (99.98 %) with pB5-1 (seen in Fig. 2) . The presence of pCSXZ1-NDM-13, pCSXZ2-NDM-13, and pYZLc23-1-NDM-13-96 from chicken cloacal samples, alongside plasmids pCSXZ5-NDM-13 and pB5-1 from environmental sources, suggest that IncFII-type plasmids harboring bla_NDM-13 may have the potential for transmission between animals and their environments.

Fig. 2.

Comparative analysis of seven bla_NDM-13-contaning plasmids.

In this study, all three representative plasmids exhibited a conserved resistance gene cluster (IS91-blaNDM-13-ble-cds-cds-IS50R-aph(3′)-IIa-cds-IS26-IS6-fosA3-fidL-cds-cds-IS26-IS26) that was identical to those found in plasmids pB5-1 and pYZLc23-1-NDM-13-96k (seen in Fig. 3). Our findings differ from previous reports positioning ISAbla125 upstream of bla_NDM-13, suggesting potential genomic variations in bla_NDM-13-harboring plasmids. NCBI database analysis revealed conserved genetic architectures in both pYZLc23-1-NDM-13-96 (Jiangsu isolate) and pB5-1(Anhui isolate), indicating regional dissemination of this plasmid structure. The bla_NDM-13-carrying plasmids identified in this study can be transferred between bacteria via conjugation, revealing that IS91, IS50R, IS6 and IS26 are important roles in the transfer of bla_NDM-13. Furthermore, the stability of the plasmid was confirmed after 15 generations of cultivation in LB broth absent of antibiotics, suggesting that the IncFII plasmid carrying bla_NDM-13 gene can be stably inherited in the recipient strain. The growth rate of the transconjugant was no decreased, compared to the recipient strain E. coli J53, indicating that the bla_NDM-13-harbouring IncFII plasmid had little effect on bacterial growth and fitness.

Fig. 3.

Comparative analysis of genetic context of bla_NDM-13-caryying plasmids in this study with other bla_NDM-13-carrying plasmids. The shading intensity indicates the degree of nucleic acid homology. The direction of the arrows denotes the direction of gene coding. Arrows of different colors represent distinct functional genes: red for antibiotic resistance-encoding genes, blue for transposon and integron-associated genes, green for insertion sequences, purple for the bla_NDM-13 gene, and black for all other genes.

These findings indicate that IncFII plasmids play a significant role in dissemination bla_NDM-13 in China, highlighting the need for intensified surveillance to establish the epidemiological prevalence of bla_NDM-13-carrying IncFII plasmids in clinical and poultry production settings.

CRediT authorship contribution statement

Mengjun Tang: Writing – original draft. Qian Zhou: Methodology. Xiaoyan Zhang: Formal analysis. Xiujun Tang: Investigation. Junxian Lu: Investigation. Yushi Gao: Supervision.

Disclosures

All of the authors declared that they have no conflict of interest. The contents of this manuscript have not been copyrighted or published previously. The contents of this manuscript are not now under consideration for publication elsewhere. The contents of this manuscript will not be copyrighted, submitted, or published elsewhere while acceptance by Poultry Science.