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 blaNDM−5-positive strains. Oxford Nanopore MinION sequencing and conjugation assays revealed that the blaNDM−5 gene was carried by IncX3 (n = 1), IncI1 (n = 1), and IncHI2 (n = 2). The IncI1-plasmid-carrying blaNDM−5 displayed high homology with one plasmid pEC6563-NDM5 from the human clinic, while the IncHI2 plasmid harboring blaNDM−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 blaNDM−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.

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 betalactamase, 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/newsroom/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., 2021Zhang et al., , 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-toegg contact and storing unwashed eggs in fridge.However, the occurrence of clinically important resistant bacteria, NDMproducing 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 NDMpositive isolates.

Methods . 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.

. 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-timeof-flight mass spectrometry (MALDI-TOF MS; Bruker Daltonik GmbH, Bremen, Germany).

. Antimicrobial susceptibility testing
According to the recommendations of the Clinical and Laboratory Standards Institute, the minimal inhibitory concentrations (MICs) of 19 antimicrobials against NDMpositive 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/).

. Plasmid transferability and stability
Conjugation experiments were performed by broth mating using bla NDM -positive strains as the donor and a sodium azideresistant (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.

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

. The biological features of bla NDM--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  N.D. means that conjugation frequency cannot be measured.
Frontiers in Microbiology frontiersin.org

FIGURE
The genetic environment of bla NDM− genes.
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.

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 handegg 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−5positive 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−5bearing 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−5bearing 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−5bearing 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.

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.