First Detection of bla NDM-1 -Haboring IncHI2 Plasmid in Escherichia coli Strain Isolated from Goose in China

Introduction

E scherichia coli (E. coli) is a zoonotic pathogen that causes both enteric and extraenteric infections. Antimicrobial therapy is the primary method used to treat bacterial infections (Riley, 2014). Carbapenem is a broad-spectrum penicillin antibiotic that has been used as the last line of defense against multidrug-resistant (MDR) gram-negative infections (Moussounda et al., 2017). Carbapenemase-producing Enterobacteriaceae have been reported worldwide and have become an urgent threat to public health (Potter et al., 2016). New Delhi metallo-β-lactamase 1 (NDM-1), a metallo-β-lactamase (MBL) capable of hydrolyzing all β-lactams except monobactams, was first identified in a carbapenem-resistant Klebsiella pneumoniae strain recovered from a Swedish patient hospitalized in India in 2008 (Li et al., 2018; Yong et al., 2009).

Since then, NDM-1 has been identified in various species, and 44 variants of NDM have been identified in GenBank (Hornsey et al., 2011; Khajuria et al., 2016; Khan and Parvez, 2014; Li et al., 2021; Sun et al., 2020). The rapid spread of NDM is mainly supported by plasmids, such as IncX, IncF, IncA/C, and IncHI (Feng et al., 2018; Pedersen et al., 2018). Currently, bla _NDM is extensively disseminated in clinical settings, environments, and animal sources (Bose et al., 2022).

The goose breeding industry is particularly developed in Jiangsu, China. The breeding mode of geese in farms is different from that of geese in other poultry farms, because many pools are built on goose farms to support their growth. Owing to the use of antibiotics and the possible dissemination of resistant bacteria through the water system on goose farms, bacteria have complex antibiotic resistance (Cen et al., 2021; Zhang et al., 2021). However, there are few reports of bla _NDM originating from geese. In this study, we identified seven bla _NDM-positive E. coli strains. Among these, we identified a novel bla _NDM-1-haboring IncHI2 plasmid in E. coli isolated from the feces of geese in Jiangsu province, China. To the best of our knowledge, this is the first report of an E. coli isolate from geese carrying this plasmid.

Materials and Methods

Results and Discussion

Antimicrobial susceptibility testing showed that the lowest resistance rate of the 1013 strains to meropenem was 0.69%, followed by amikacin (8.88%), which was low at 10%. The rate of resistance to tetracycline was the highest (72.36%), followed by ampicillin (71.67%), naphthyric acid (69.79%), compound sulfamethoxazole (65.65%), and streptomycin (52.52%).

Carbapenem-resistant Enterobacteriaceae (CRE) infections are among the most serious health threats to humans and animals worldwide, NDM variants continue to emerge, leading to clinical difficulties in the treatment of infections (Saeed et al., 2019). Seven CRE isolates were concentrated in Jiangsu, Anhui, and Henan provinces. Although the number of NDM-positive strains detected among the 1013 E. coli strains was low, the host sources were extensive. The CRE strains included two strains from goose samples (C1147 and C1283), two strains from pig samples (C2621 and C2778), one strain from chicken samples (C1287), one strain from sheep samples (C1289), and one strain from duck samples (C1588).

The prevalence of bla _NDM-positive CRE in humans is high (Zhang et al., 2017). Recently, animal-borne NDM bacteria have been reported (Ghatak et al., 2013; Shaheen et al., 2013). Wang et al. (2017) identified 161 bla _NDM-positive strains among 245 CRE isolates from Shandong province in China. All NDM-positive strains were resistant to multiple antibiotics, including ampicillin, cefazolin, cefotaxime, meropenem, tetracycline, and trimethoprim-sulfamethoxazole, but all were susceptible to amikacin, which is consistent with the findings of Kashaf and David's report (Junaid, 2021; Mantilla-Calderon et al., 2016).

Multilocus sequence typing (MLST) of bacteria is often used to study molecular epidemiology. It has high resolution and can be used for the analysis and comparison of pathogens in different countries and regions (Giufrè et al., 2021; Wang, et al., 2021; Waters et al., 2012). According to the sequencing data, the seven NDM-positive strains belonged to five different STs, namely ST156, ST6388, ST7386, ST10, and ST5229, indicating that clonal spread was not the major reason for the spread of bla _NDM in E. coli (Table 1).

Two NDM variants were identified bla _NDM-1 (n = 1) and bla _NDM-5 (n = 6). E. coli ST156 has not been the predominant MDR clone observed worldwide in the past, but it is associated with the distribution of bla _NDM-1 and bla _CTX-M-15 in humans and poultry (Giufrè et al., 2012; Mushtaq et al., 2011). All of the bla _NDM-5-positive strains contained the IncX3 plasmid. The bla _NDM-5-bearing E. coli isolates were first identified in goose farms and their surroundings in 2019 (Liu et al., 2019). We identified a novel bla _NDM-1-haboring IncHI2 plasmid in E. coli ST156 isolated from the feces of geese in Jiangsu province, China.

A conjugation assay was performed to investigate the transferability of bla _NDM-1 gene (Cabezón et al., 2015). The transconjugant was successfully selected on MacConkey agar plates supplemented with streptomycin (3000 mg/L) and meropenem (2 mg/L), indicating that the bla _NDM-1 gene in strain C1147 was transferable. The E. coli C600-pC1147 transconjugant harbored only bla _NDM-1 with frequencies of 10⁻⁵ per donor (Fig. 1), and 256 times higher MICs than that of the parental strain E. coli C600 (Table 2).

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FIG. 1.

Identification of C1147 strains and transconjugants by PCR. PCR, polymerase chain reaction.

The complete pC1147 was obtained using genome sequencing. pC1147 was 238291 bp long with bla _NDM-1, aac(6′)-Ib-cr, aph(3′)-Ia, aph(4)-Ia, bla _OXA-1, fosA3, aac(6′)-Ib-cr, sul1, dfrA12, and ble _MBL resistance genes (Fig. 2). pC1147 has a typical IncHI2 plasmid backbone that contains replication, conjugative transfer, maintenance, and stability regions, making this plasmid to be self-transmissible. Comparison of pC1147 with eight strains harboring IncHI2 plasmids using BRIG30 showed a sequence coverage of 80–88% (Fig. 3), which was the highest in the backbone structure region, and showed significant differences in the regions harboring drug-resistance genes.

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FIG. 2.

Plasmid map of plasmid pC1147 (GenBank Accession No. CP102295). The innermost circle is GC content (46% GC), the second circle is GC skew, the third circle of arrows represents the position and transcriptional direction of the open reading frames. Resistance genes are indicated by fuchsia arrows. The remaining sequence segments are indicated by red arrows. GC, guanine and cytosine.

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FIG. 3.

Sequence alignment analysis of pC1147 (GenBank Accession No. CP102295) plasmids. Alignment was performed using BRIG30, pC1147 was used as the reference. GenBank accession numbers of other plasmids are CP022735, KU743384, KX129782, LT599829, KY421937, KU341381, KX084394, and KX856066, respectively. Outer circle arrow represents the position and transcriptional direction of the open reading frames in pC1147. Resistance genes are indicated by fuchsia arrows. The remaining sequence segments are indicated by red arrows.

Linear comparisons of the resistance gene background on the pC1147 plasmid with other plasmids (Fig. 4) showed that the genetic environment of bla _NDM-1 was similar to that of bla _NDM-1 in pCf75 (Citrobacter freundii, NZ_CP047308) (Li et al., 2020) and p6061604-KPC (E. coli, MN823987), which has the structure sul1-emrE-catB-bla _NDM-1-ble _MBL-trpF-folP. However, pC1147 had an additional insertion sequence (IS26 and Tn2) compared with them, thus constituting a new genetic environment IS26-sul1-emrE-catB-bla _NDM-1-ble _MBL-trpF-folP-Tn2.

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FIG. 4.

Linear comparisons of pC1147 (GenBank Accession No. CP102295) with other plasmids. Regions of >99% homology are shaded in gray. Solid lines indicate the plasmid backbone. The extents and directions of antibiotic resistance genes and other genes are indicated. Insertion sequences are shown as boxes labeled with “IS.”

Conclusion

Carbapenems are currently approved only for human use and are prohibited for animals (Liu et al., 2019; Paul et al., 2022). In our study, an IncHI2 plasmid-harboring bla _NDM-1 was observed in an E. coli strain isolated from a goose sample in China for the first time. The presence of this strain isolated from geese indicates that E. coli-producing NDM-1 can be transmitted through the waterfowl system. The occurrence of bla _NDM-1-positive E. coli in animals and the surrounding environment indicates that the use of these antibiotics is unreasonable and may pose a serious threat to public health in the long run. Therefore, further studies are required to elucidate the extent to which routine of E. coli produces NDM-1 in the waterfowl system.