Occurrence and characterization of NDM-5-producing Escherichia coli from retail eggs

The New Delhi Metallo-β-lactamase (NDM) producing Enterobacterales has been detected from diverse sources but has rarely been reported in retail eggs. In this study, 144 eggshell and 96 egg content samples were collected in 2022 from Guangdong province and were screened for NDM-producing strains. Four Escherichia coli strains (ST3014, ST10, ST1485, and ST14747) recovered from two (1.39%, 2 of 144) eggshells and two (2.08%, 2 of 96) egg content samples were identified as bla_NDM−5-positive strains. Oxford Nanopore MinION sequencing and conjugation assays revealed that the bla_NDM−5 gene was carried by IncX3 (n = 1), IncI1 (n = 1), and IncHI2 (n = 2). The IncI1-plasmid-carrying bla_NDM−5 displayed high homology with one plasmid pEC6563-NDM5 from the human clinic, while the IncHI2 plasmid harboring bla_NDM−5 shared highly similar structures with plasmids of animal origin. To the best of our knowledge, this is the first report on the identification of bla_NDM−5-positive bacteria in retail eggs. NDM-producing E. coli could be transmitted to humans by the consumption of eggs or direct contact, which could pose a potential threat to human health.

1 Introduction

Carbapenemase-resistant Enterobacterales (CRE) have increased rapidly over the last decades and have become an urgent public health threat (El-Gamal et al., 2017). Carbapenem resistance in Enterobacteriaceae is attributed to three dominant carbapenemase enzymes, including New Delhi metallo-beta-lactamases (NDM), Klebsiella pneumoniae carbapenemases (KPC), and carbapenem-hydrolyzing oxacillinase-48-type β-lactamases (OXA-48) (Iovleva and Doi, 2017). Among these CRE, NDM-producing strains are highly prevalent around the world, especially in China and South Asia (Wu et al., 2019). At present, 47 variants of NDM have been identified (https://www.ncbi.nlm.nih.gov/pathogens/refgene/#NDM); of these, NDM-1 and NDM-5 remain the most prevalent carbapenemases (Shen et al., 2022; Saravanan et al., 2023). The NDM-5 possesses higher carbapenemase activity than NDM-1 (Hornsey et al., 2011). Currently, the bla_NDM−5 gene has been disseminated to various bacterial species (e.g., Escherichia coli, K. pneumoniae, and Klebsiella aerogenes), with E. coli as the main bacterial host (Nordmann and Poirel, 2019; Jean et al., 2022; Ma et al., 2023).

Plasmid-mediated transmission has facilitated the widespread distribution of the bla_NDM−5 gene among bacteria from various environmental sources and geographical regions. The bla_NDM−5 gene has been found in an array of plasmid replicon types, such as IncX3, IncFII, IncF, IncN, and IncHI2 (Nordmann and Poirel, 2019; Jean et al., 2022; Lv et al., 2022). The IncX3 has long been recognized as the primary carrier for transmission of the bla_NDM−5 gene (Shen et al., 2022); however, recently, there is an increase in IncHI2 plasmid as a carrier of bla_NDM−5 in China (Ma et al., 2021; Zhao Q. et al., 2021; Lv et al., 2022; Wang et al., 2022; He et al., 2023). Alarmingly, IncHI2 has also been found to carry multiple antibiotic resistance genes, including colistin resistance gene (mcr), extended-spectrum beta-lactamase, and quinolone resistance genes (Webb et al., 2016; Mmatli et al., 2022).

Although carbapenems have not been approved for food animals, CRE has been continuously detected in pigs, poultry, and animal-derived foods, especially from chickens and poultry products. Eggs are important poultry products that play an essential role in the daily healthy diet of human beings and are the most consumed food all over the world [https://www.who.int/zh/news-room/fact-sheets/detail/salmonella-(non-typhoidal)]. Poultry eggs are also considered as reservoirs and transmission vectors of resistance genes, such as mcr-1, fosA, qnrS1, bla_CTX−M−1, bla_IMP, and bla_{OXA−48−like} (Benameur et al., 2018; Kapena et al., 2020; Zhang et al., 2021, 2022b; Kanaan et al., 2022; Li et al., 2022). Resistant bacteria and genes in eggs have the risk of spreading to humans through various ways, such as hand-to-egg contact and storing unwashed eggs in fridge. However, the occurrence of clinically important resistant bacteria, NDM-producing Enterobacterales, in eggs has rarely been studied. Hence, we investigated the prevalence of NDM-producing Enterobacterales among egg samples recovered from markets in Guangzhou and characterized the molecular traits of bla_NDM-positive isolates.

2 Methods

2.1 Sampling

From June to September 2022, 144 non-repetitive egg samples were randomly collected from 29 farmer markets located in four districts (Tianhe, Baiyun, Yuexiu, and Haizhu) of Guangzhou. To ensure diversity in the sampling and prevent repeated sampling from a singular supplier, we selected different stalls within each market, with a maximum of three eggs procured from any single stall. Each sample was placed in a separate sterile sample bag, and all samples were transported to the laboratory in a cool box within 8 h.

2.2 Bacterial isolation and detection of carbapenemase-encoding genes

For the isolation of bacteria from eggshells, the surface of eggs was wiped with a sterile swab, and then, the swab was placed into 4 ml sterilized Luria–Bertani (LB) broth medium for enrichment cultivation at 37°C overnight. For the isolation of bacteria from egg content, the eggshell was wiped with gauze with 70% ethanol, followed by being homogenized. During the processing of the first batch, 48 cracked eggs were collided and discarded to avoid cross-contamination (detailed information about all the samples is shown in Supplementary Table S1), and then, the remaining eggs were opened to extract the whole egg content. In total, 1 ml of egg content was dispensed into 4 ml sterilized Luria–Bertani (LB) broth medium and enriched at 37°C overnight with shaking. The overnight cultures of each sample were incubated on MacConkey agar plates supplemented with 0.5 mg/L meropenem, and the plates were incubated at 37°C for 16 h. One to three colonies with different morphologies in each plate were selected for the detection of carbapenemase-encoding genes, bla_NDM, using PCR and DNA sequencing (primers are shown in Supplementary Table S2). All bla_NDM-positive isolates were collected for species identification by direct smear and matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry (MALDI-TOF MS; Bruker Daltonik GmbH, Bremen, Germany).

2.3 Antimicrobial susceptibility testing

According to the recommendations of the Clinical and Laboratory Standards Institute, the minimal inhibitory concentrations (MICs) of 19 antimicrobials against NDM-positive isolates were determined using the agar dilution method or broth microdilution (colistin and tigecycline) method. E. coli ATCC 25922 was used as a quality control strain. The results of MICs were interpreted according to CLSI (M100-S30) criteria and EUCAST (http://www.eucast.org/clinical_breakpoints/).

2.4 Plasmid transferability and stability

Conjugation experiments were performed by broth mating using bla_NDM-positive strains as the donor and a sodium azide-resistant (MIC > 2,000 μg/ml) E. coli J53 strain as the recipient. In detail, the donor and recipient strains were incubated separately in LB broth for 4 h, followed by mating the bacterial cultures with a ratio of 1:1 and incubating at 37°C without shaking overnight. A 50-μL overnight mixture was plated onto MacConkey agar plates containing 0.5 mg/L meropenem and 150 mg/L sodium azide, and incubated for 18 h to count and select transconjugants. Conjugation frequency was calculated as the number of transconjugants per recipient. Chemical transformation experiments were performed in those cases that bla_NDM-positive strains failed to conjugate. All transconjugants and transformants were confirmed by PCR (primers are shown in Supplementary Table S2) and antimicrobial susceptibility testing.

The stability of bla_NDM−5-carrying plasmids in host bacteria was performed by a passage in the absence of antibiotic Luria broth (LB). Three single clones of each bla_NDM−5-positive strain were grown in 3 ml LB without antibiotic treatment overnight at 37°C. The overnight culture was daily diluted 1:100 in fresh LB broth for 15 days. Cultures were collected at the end of each of 3 days for streaking on antibiotic-free MacConkey agar plates. Then, 100 colonies were selected, and the presence of bla_NDM−5 and the corresponding plasmids was verified by PCR amplification of bla_NDM−5 and repA (primers are shown in Supplementary Table S2). Plasmid retention was calculated as the ratio of strains with bla_NDM−5 and repA and over 100 colonies.

2.5 Whole-genome sequencing and bioinformatics analysis

Genomic DNAs of four NDM-positive isolates were extracted by using HiPure Bacterial DNA Kit (Magen, Beijing, China), according to the manufacturer's instructions. Whole genomic DNA was sequenced using the Illumina NovaSeq 6000 and MinION platform (Nanopore, UK). Hybrid assembly of complete genomes was carried out using the Unicycler version 0.4.8 (Wick et al., 2017). MLST v2.19 (https://github.com/tseemann/mlst) was applied to the verified sequence type (ST). Center for Genomic Epidemiology (CGE) (http://genomicepidemiology.org/services/) and PubMLST were used to identify antimicrobial resistance genes (ARGs) and plasmid replication types. Prokka software was used to annotate the draft genome (Seemann, 2014). EasyFig tool (http://mjsull.github.io/Easyfig/) was used to draw the genetic context of bla_NDM and plasmid alignments and comparisons (Sullivan et al., 2011).

2.6 Accession numbers

The complete sequences of NDM-positive isolates have been deposited in the GenBank database under accession numbers PRJNA983957.

3 Results

3.1 Prevalence of NDM-producing enterobacterales in egg samples

From the 144 eggs, 144 eggshell and 96 egg content samples were collected, and 4 bla_NDM-positive isolates were recovered from 2 (1.39%, 2/144) eggshell samples (GD22SC3180PM and GD22SC3312PM) and 2 (2.08%, 2/96) egg content samples (GD22SC3148PM and GD22SC4181PM). These strains were further identified as E. coli by MALDI-TOF MS. All the bla_NDM genes were identified as bla_NDM−5.

3.2 Antimicrobial resistance patterns of bla_NDM-5-positive E. coli strains

Antimicrobial susceptibility testing showed that all four isolates were resistant to most of the antimicrobials, including β-lactams (ampicillin, cefoxitin, cefotaxime, ceftazidime, cefquinome, and imipenem), aminoglycosides (apramycin, neomycin, and streptomycin), fosfomycin, florfenicol, tetracycline, and sulfamethoxazole. However, all isolates remained susceptible to amikacin, colistin, and tigecycline (Supplementary Table S3).

3.3 Genotyping and genetic background of bla_NDM-5-positive E. coli strains

The four bla_NDM−5-positive E. coli strains were subjected to short- and long-read sequencing to acquire complete genomes. Sequence analysis revealed that GD22SC3180PM, GD22SC3312PM, GD22SC3148PM, and GD22SC4181PM belonged to ST14747, ST10, ST3014, and ST1485, respectively (Table 1). All bla_NDM-positive isolates harbored multiple antimicrobial resistance genes (ARGs) and plasmid replicon types (Table 1). Aminoglycoside resistance gene, β-lactam resistance gene, florfenicol resistance gene floR, macrolide resistance gene mdf(A), and sulfonamide resistance gene dfrA were detected in all the bla_NDM−5-positive E. coli strains. In addition, the fluoroquinolone resistance gene qnrS and rifampicin resistance gene arr-3 were identified in two isolates (GD22SC3312PM and GD22SC4181PM). GD22SC3180PM additionally harbored tetracycline resistance gene tet(A), fosfomycin resistance gene fosA3, and lincomycin resistance gene lnu(F).

TABLE 1

Table 1. Characteristics of bla_NDM−5-positive isolates.

3.4 Characterization of bla_NDM-5-carrying plasmids

Bioinformatics analysis revealed that there were three types of replicons carrying the bla_NDM−5 gene, namely, IncX3 (pHN22SC3148), IncI1 (pHN22SC3180), and IncHI2 (pHN22SC3312 and pHN22SC4181) (Table 1). The complete sequence of IncI1 plasmid pHN22SC3180 is a 107,617 circular molecule with a GC content of 47% (Figure 1A). The backbone regions of the pHN22SC3180 displayed the highest similarity to plasmid pEC6563-NDM5 (CP095858.1, urine, Homo sapiens, E. coli, Zhejiang, China), with 100% identity and 99.0% coverage (Figure 1A) (Zhang et al., 2022a), but had low similarity to the other four bla_NDM−5-bearing IncI1 plasmids deposited in the NCBI database (Supplementary Table S4). The plasmid pHN22SC3312 (IncHI2) was 263,640 bp in size with a GC content of 54% (Figure 1B) and had a high degree of homology with pHNGD64-NDM (MW296099.1, pig, E. coli, Guangdong, China) with 100% identity and 98.0% coverage, pNDM33-1 (CP076648.1, duck, E. coli, Guangdong, China) with 99.99% identity and 99.0% coverage, and pHNBYF33-1 (CP101733.1, fish, E. coli, Guangdong, China) with 99.99% identity and 98.0% coverage (Figure 1B). Furthermore, in addition to bla_NDM−5, the variable region of pHN22SC3312 contained several antibiotic resistance genes [e.g., bla_OXA−10, tet(A), qnrS1, floR, sul3, aadA2, and aph(4)-Ia].

FIGURE 1

Figure 1. Comparison and stability of bla_NDM−5-carrying plasmids. (A) bla_NDM−5-harboring IncI1 plasmids in this study with other similar plasmids. p1108-emrB (NZ_MG825377.1), p2_025970 (CP036181.1), pPNUSAS039582_1 (CP093089.1), and pEC6563-NDM5 (CP095858.1). (B) bla_NDM−5-harboring IncHI2 plasmids in this study with other similar plasmids. p8C59-NDM (MT407547.1), pHNBYF33-1 (CP101733.1), pHNGD64-NDM (MW296099.1), and pNDM33-1 (CP076648.1). (C) Stability of bla_NDM−5-carrying plasmids in their corresponding host bacteria. Error bars represent standard deviations (n = 3).

3.5 The biological features of bla_NDM-5-carrying plasmids

To evaluate the transferability of the bla_NDM−5 gene, all four bla_NDM−5 positive strains were conducted on a conjugation assay. The bla_NDM−5-carrying plasmids were successfully transferred to recipients E. coli J53 at a frequency of 10⁻⁵-10⁻⁶. The imipenem MICs of the transconjugants were 2–4 μg/ml, which were 32–64-fold the MICs of the recipient (Supplementary Table S3). To evaluate the stability of bla_NDM−5-carrying plasmids, we performed passage with the four bla_NDM−5-positive strains in antibiotic-free Luria broth. The stability of the IncX3 plasmid pHN22SC3148 was 100% in the absence of antibiotic after 15 days (i.e., ~150 generations) in the natural host GD22SC3148PM, while IncI1 plasmid pHN22SC3180 and IncHI2 plasmids, pHN22SC3312 and pHN22SC4181, were gradually lost from their corresponding host strains after 7 days of passage, with 93.40, 31.60, and 30.90% retention after 15 days, respectively (Figure 1C). Thus, in the absence of antibiotic selection, IncX3 plasmid pHN22SC3148 is stable in the original isolate, and the other three plasmids (IncHI2 and IncI1) are less stable in the host strains.

3.6 Genetic environments of bla_NDM-5 genes in IncI1 and IncHI2

The genetic context of the bla_NDM−5 gene in IncI1 plasmid pHN22SC3180 was IS26-IS3-ISAba125-IS5-bla_NDM−5-ble_MBL-trpF-dsbD-IS26, which was similar to the genetic environment recently discovered in IncI1 plasmid pEC6563-NDM5-carrying bla_NDM−5 (GenBank accession no. CP095858.1) (Figure 2). Although the bla_NDM−5 region (IS26-IS3-ISAba125-IS5-bla_NDM−5-ble_MBL-trpF-dsbD-IS26) was surrounded by two copies of IS26 with the same direction, no circular intermediate was obtained in this study, similar to previous report (Zhang et al., 2022a). Moreover, the genetic contexts of bla_NDM−5 in IncHI2 plasmid pHN22SC3312, IS3000-ISAba125-IS5-bla_NDM−5-ble_MBL-trpF-ble_MBL-IS26-dsbD-IS3000, were highly similar to other bla_NDM−5-harboring IncHI2 plasmids, pNDM-M121 (GenBank accession no. CP083586.1), pHNBYF33-1 (GenBank accession no. CP101733.1), and pNDM33-1 (GenBank accession no. CP076648.1), except for the presence of two copies of ble_MBL downstream of bla_NDM−5 in pHN22SC3312 in this study (the information of all egg samples is shown in Supplementary Table S1).

FIGURE 2

Figure 2. The genetic environment of bla_NDM−5 genes.

4 Discussion

To date, bla_NDM-positive Enterobacterales have been identified in various sources, including food animals, pets, human beings, animal foods, vegetables, and the environment (Zhai et al., 2020; Huang et al., 2023; Ma et al., 2023). However, there are few reports of NDM-producing bacteria in egg sources, except for one study, which reported the presence of bla_NDM-positive Salmonella enterica in eggs from Iraq (Kanaan et al., 2022). To the best of our knowledge, this is the first report of NDM-5-producing Enterobacterales in retail egg samples from China. As eggs are an important food in the human diet and its consumption continues to increase, the NDM-positive Enterobacterales in eggs have the risk of spreading to humans via the food chain and even hand–egg contact.

Previous studies revealed that IncX3 is the most epidemiologically successful vehicle for spreading bla_NDM-5 (Zhang et al., 2019; Zhao Q. Y. et al., 2021; Ma et al., 2023). bla_NDM−5-bearing IncX3 plasmids are widely distributed in animals, human beings, and environments worldwide (Lv et al., 2022; Ma et al., 2023). The IncX3 plasmids carrying bla_NDM−5 in this study further confirmed the importance of the IncX3 plasmid by acting as a vehicle for bla_NDM−5 transfer. The bla_NDM−5-positive IncX3 plasmids can be stably inherited in the original isolate (Figure 1C), which may partly explain the rapid global dissemination of bla_NDM−5-bearing IncX3 plasmids (Ma et al., 2020).

In this study, we also detected the IncI1 plasmid (pHN22SC3180) and IncHI2 plasmid (pHN22SC3312) carrying the bla_NDM−5 gene. IncI1 is an epidemic plasmid and can carry many resistance genes, especially the extended-spectrum beta-lactamase gene bla_CTX, which has widely spread in patients and animals (Yang et al., 2014; Chong et al., 2018; Carattoli et al., 2021; Liu et al., 2021). However, the reports of the bla_NDM−5-bearing IncI1 plasmid are few, and bla_NDM−5-bearing IncI1 plasmid has just been detected in isolates from clinical and duck samples in China (Zhao Q. Y. et al., 2021; Dong et al., 2022; Zhang et al., 2022a). Of note, by searching through the NCBI database, we found only five bla_NDM−5-bearing IncI1 plasmids, four of which were clinical samples isolated from China in recent years, implying that the prevalence and risk of bla_NDM−5-bearing IncI1 in the clinic might be underestimated and need further investigation.

IncHI2 is a wide host plasmid and acts as an important vector for the dissemination of multiple ARGs, especially mcr-1 (Webb et al., 2016; Liu and Liu, 2018; Wu et al., 2018; Cao et al., 2020). To date, IncHI2-type plasmids carrying bla_NDM−5 have only been detected in strains recovered from chicken, duck, pig feces, and freshwater fish (Ma et al., 2021; Zhao Q. Y. et al., 2021; Lv et al., 2022). These IncHI2-bla_NDM−5 plasmids mainly spread regionally in Guangdong province in China but have also spread to other regions (Wang et al., 2022; He et al., 2023). The high similarity of the bla_NDM−5-bearing IncHI2 plasmids in eggs and other origins suggested that these plasmids are spreading. However, IncHI2-type plasmids carrying bla_NDM−5 are not stable in bacterial hosts (Figure 1C). The increasing occurrence of IncHI2-type plasmids carrying bla_NDM−5 in China might be associated with the co-selection by other antimicrobials as IncHI2 plasmids usually carry various antimicrobial resistance genes.

While our findings indicate a slightly higher prevalence of bla_NDM-positive isolates in egg contents (2.08%, 2/96) compared with eggshells (1.39%, 2/144), this study is not without limitations. The exclusion of 48 egg content samples may influence the overall contamination rates. Furthermore, the sample size, hovering around a hundred, does not offer a comprehensive representation of the prevalence of bla_NDM in egg samples, highlighting the need for continuous surveillance.

5 Conclusion

In summary, to the best of our knowledge, we report the first case of Enterobacterales carrying bla_NDM−5 of retail eggs in China. The bla_NDM−5-bearing plasmids displayed high homology with those of plasmids from other sources. Of note, the IncHI2 plasmids carrying both carbapenem and multiple resistance genes showed an increasing trend that pose another threat to human health. Considering the clinical importance of carbapenem together with the fact that the consumption of eggs is substantial in our diet, the carbapenem resistance in eggs has the risk to spread to humans through the food chain or contact with the contaminant. Continued monitoring of carbapenem resistance in eggs is urgently needed.

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 in the article/Supplementary material.

Author contributions

Y-YL: Investigation, Writing—original draft, Writing—review & editing. TL: Formal analysis, Investigation, Writing—original draft. HY: Investigation, Writing—original draft. CY: Investigation, Writing—original draft. LL: Formal analysis, Writing—original draft. JC: Formal analysis, Writing—original draft. HD: Formal analysis, Writing—original draft. XG: Formal analysis, Writing—original draft. J-HL: Conceptualization, Funding acquisition, Writing—review & editing.

Funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This study was supported by the National Key Research and Development Program of China (No. 2022YFC2303900), the National Natural Science Foundation of China (grants no. 32141002), and the Local Innovative and Research Teams Project of Guangdong Pearl River Talents Program (2019BT02N054).

Conflict of interest

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.

Publisher's note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb.2023.1281838/full#supplementary-material

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