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Transmission patterns of multiple strains producing New Delhi metallo-β-lactamase variants among animals and the environment in live poultry markets

One Health Advances volume 2, Article number: 12 (2024) Cite this article

Abstract

The widespread transmission of blaNDM among livestock and the live poultry industry attracts considerable public attention. However, studies have not yet addressed its impact on public health in live poultry markets (LPMs). Herein, we investigated the prevalence and genomic epidemiology of blaNDM-positive bacteria in various niches, and explored the transmission patterns of blaNDM within LPMs. Samples were collected between 2019 and 2022 from two LPMs in China. blaNDM was most prevalent in wastewater (35/66, 53.03%). All vegetable samples were negative for blaNDM. blaNDM was mainly distributed among Escherichia coli (266/336, 79.17%), Klebsiella pneumoniae (62/336, 18.45%), and Acinetobacter baumannii (3/336, 0.89%). Some novel hosts, including Pseudomonas monteilii and Pseudomonas otitis, were also identified. Diverse variants blaNDM-1, blaNDM-5, blaNDM-9, blaNDM-13, and blaNDM-27 were identified. The blaNDM-positive E. coli ST2659 was dominant. blaNDM was found to coexist with mcr-1 (4/51, 7.84%). Horizontal gene transfer plays a vital role in blaNDM transmission within the LPMs. Some blaNDM-harboring clones transfer among animals and the environment through the food chain and close contact. More efforts are needed to curb the transmission trend of blaNDM among humans, animals, and the environment within LPMs.

Introduction

According to a recent review published in The Lancet, over 1.2 million people died directly from Antimicrobial Resistance (AMR) infections, and an additional 4.95 million deaths were associated with AMR [1]. The emergence of infectious diseases and the spread of AMR severely threaten public health, and hundreds of thousands of people worldwide die from infections caused by multidrug-resistant organisms [2, 3]. Horizontal gene transfer via mobile genetic elements, such as plasmids [4], insertion elements [5], and transposons [6], contributes strongly to the emergence and global spread of multidrug-resistant bacteria [7]. Furthermore, the exchange of antibiotic resistance genes (ARGs) between pathogens and commensal microorganisms from diverse ecological niches increases antibiotic resistance [8]. Over the past decade, numerous studies have investigated the occurrence and spread of ARGs and antibiotic-resistant bacteria in humans, animals, and the environment [9].

Live poultry markets (LPMs), where various live animals are gathered for trading, are highly important for the wholesale and retail of live poultry in China and other Asian countries, and represent a substantial interface between humans, animals, and the environment [10]. LPMs account for about 50% of the live poultry supply to consumers through a complex and non-uniform transportation system, which plays a vital role in live poultry trading [11]. The live animals are imported from different regions and housed at a high density in a relatively compact space alongside other poultry gathered from other places, providing an optimal setting for the transmission and persistence of infectious agents [12]. Hence, LPMs are potential areas where live poultry-associated pathogens, such as some avian influenza viruses, can spread and evolve, causing infection and death in humans [13, 14]. Workers who have been exposed to live poultry for a long term are at a higher risk of contracting avian influenza virus than the consumers [15]. Wang et al. recently showed that LPMs are a huge reservoir of ARGs. Moreover, their diversity here was higher than that in farms, and more abundant ARGs were found in LPM workers than in those who had no contact with LPMs, confirming that live poultry trade promotes the spread of ARGs [16, 17].

Carbapenems, with their broad-spectrum antibacterial activity, are crucial to treat clinical infections in humans caused by multidrug-resistant bacteria. However, overuse and misuse has led to the rapid worldwide proliferation of carbapenem-resistant Gram-negative bacteria [18, 19]. blaNDM represents a typical mobile carbapenem-resistant gene, encoding New Delhi metallo-β-lactamase (NDM), that is often located on plasmids [19]. It encodes a metallo-β-lactamase capable of hydrolyzing most β-lactam antibiotics, particularly meropenem, imipenem, and ertapenem, all three of which are clinically important drugs in the treatment of multidrug-resistant infections. The global prevalence of bacteria carrying blaNDM represents a major public health concern.

blaNDM-1 was first detected within Klebsiella pneumoniae isolated from a Swedish patient in India in 2008 [20]. Ever since, the number of blaNDM-positive strains isolated from humans, animals, and environment around the world has increased dramatically, posing a major global public health problem [21]. Shen and his team [22] investigated the presence and the genetic environment of blaNDM in slaughterhouses and large-scale livestock and poultry farms in China. Li et al. analyzed the transmission pattern of blaNDM-harboring carbapenem-resistant E. coli between humans and backyard animals [23]. Li and his colleagues studied the transmission characteristics of blaNDM in the pork production chain [24], while Wang et al. conducted epidemiological research on blaNDM in the live poultry industry chain [25]. However, reports on the distribution and transmission characteristics of blaNDM in LPMs are scarce. Here, we applied a One Health approach to identify the prevalence and transmission mechanism as well as the potential transmission route of blaNDM within various niches in LPMs using a combination of whole-genome sequencing (WGS) and bioinformatic tools.

Results

Prevalence of bla NDM-positive samples in diverse niches

In this study, a total of 593 samples were collected from LPMs A and B in Yangzhou between 2019 and 2022. Of them, 203 samples (336 strains) tested positive for blaNDM, resulting in a positive rate of 34.23% (203/593). The isolation rate of blaNDM-positive samples was higher in LPM B (73/159, 45.91%) compared with LPM A (130/434, 29.95%) (Table S1). Since animals in the LPMs hailed from multiple regions in Jiangsu, a map depicting the sample sources was created (Fig. 1). The isolation rates of blaNDM-positive samples varied across locales. The rates were relatively higher in Huai’an (27/56, 48.21%) and Yancheng (38/89, 42.70%) (Table S3), while Nanjing demonstrated the lowest prevalence rate (2/31, 6.45%). Among the seven areas, Yangzhou contributed the largest number of blaNDM-bearing strains (Fig. 1). In terms of the sources of blaNDM-positive samples, 112 were isolated from chickens (112/312, 35.90%), 18 from ducks (18/30, 46.15%), 7 from pigeons (7/27, 25.93%), 2 from geese (2/17, 11.76%), 1 from soil (1/32, 3.13%), 35 from wastewater (35/66, 53.03%), and 28 from dust (28/87, 34.06%) (Table S4). However, all 13 samples collected from vegetables were blaNDM-negative, and no carbapenem-resistant E. coli were recovered. The following species detection rates were found among blaNDM-positive strains: E. coli (266/336, 79.17%), K. pneumoniae (62/336, 18.45%), and A. baumannii (3/336, 0.89%) (Table S5). The five remaining strains included one each of Acinetobacter bereziniae, Enterobacter cloacae, Providencia rettgeri, Pseudomonas monteilii, and Pseudomonas otitidis.

Fig. 1
figure 1

Prevalence and distribution of blaNDM-positive isolates in live poultry markets (LPMs). A Distribution of different species of blaNDM-positive isolates. B Prevalence of blaNDM-positive isolates from different regions in Jiangsu province. The prevalence of blaNDM-positive samples is marked in red on the map; the darker the color is, the higher the percentage of blaNDM-positive samples. The map is based on the standard map GS(2019)3266 without further modifications

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Antimicrobial susceptibility profiles and resistance and virulence genes

All 336 blaNDM-bearing strains were evaluated for their susceptibility toward a series of β-lactam and non-β-lactam antibiotics (Table S6). Among β-lactam antibiotics, the highest levels of resistance were observed against meropenem (100%), imipenem (100%), ceftiofur (100%), and carbenicillin (100%), while > 95% of these isolates showed resistance to ampicillin and ceftazidime. Diverse resistance patterns against routinely used non-β-lactam antibiotics were also observed. More than 80% of these isolates were non-susceptible to kanamycin, ciprofloxacin, and gentamicin. Interestingly, blaNDM-harboring isolates from animals displayed higher rates of resistance to tigecycline, colistin, kanamycin, and amikacin compared to those from the environment, while the opposite was true regarding resistance to ciprofloxacin (Table S6). The rate of resistance was relatively low to tigecycline (20.53% in animal isolates, 8.33% in environmental isolates) and colistin (7.44% in animal isolates, 2.38% in environmental isolates).

In most instances, the phenotype could be explained by the presence of the appropriate resistance genes. Hence, 51 representative blaNDM-harboring isolates were selected and subjected to WGS. These strains were isolated from diverse sources, including different animal feces, wastewater, soil and car surface samples. A total of 77 ARGs were identified, conferring resistance to aminoglycosides, rifampicin, β-lactams, carbapenems, phenicols, macrolides, lincosamides, streptomycin, fosfomycin, colistin, fluoroquinolones, sulfonamides, and tetracyclines, proving that these LPMs were huge reservoirs of ARGs. Among these, dfrA, which confers resistance to the veterinary drug trimethoprim/sulfamethoxazole, and floR, which confers resistance to florfenicol, were commonly associated with blaNDM, with carriage rates of 90.20% (46/51) and 92.16% (47/51), respectively. Compared with other ARGs, the association with blaNDM was higher for the spectinomycin resistance gene aadA (43/51), the sulfonamide resistance gene sul2 (37/51), the plasmid-mediated quinolone resistance gene qnrS1 (29/51), and the tetracycline resistance gene tet(A), which undoubtedly increases the risk of their co-transmission. Notably, the colistin resistance gene mcr-1 was detected only in four strains, whereas the tigecycline resistance gene tet(X) was not identified in any strain, indicating that these isolates might either be inherently resistant or possess other resistance mechanisms. Furthermore, almost half of the E. coli isolated from animals and the environment carried multiple virulence genes (iucABCD, iutA, iroBCDN etc.), which serve as good indicators of bacterial pathogenic potentials [1] (Fig. S1).

Comparative susceptibility of bla NDM-positive isolates to β-lactam antibiotics

Besides blaNDM, other β-lactamase genes, such as blaOXA-1 and blaOXA-10, were detected. Sequence analysis revealed that blaNDM from 50 of the 51 isolates differed from blaNDM-1 by point mutations (Table 1). Among the variants, blaNDM-5, was the most prevalent, being detected in 42 isolates. blaNDM-27, a new variant of NDM that was detected in two isolates of E. coli and K. pneumoniae from chickens, differed from blaNDM-1 by amino acid substitutions at positions 95 (Asp➔Asn) and 233 (Ala➔Val).

Table 1 Amino acid substitutions at various positions among NDM variants
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To further determine whether the susceptibilities to β-lactam antibiotics were similar for blaNDM-27 and other variants, we performed conjugation experiments and gene cloning assays. E. coli isolates YN1-3, YM3-1, YN23-1, and YN4-1, and P. rettgeri YN5-3, which were positive for different blaNDM variants (NDM-5, NDM-9, NDM-13, NDM-27, and NDM-1, respectively), were cloned full-length in E. coli BL21(DE3), giving rise to BL21(DE3) colonies carrying the recombinant plasmid blaNDM. The four blaNDM variants from E. coli isolates conjugated successfully with E. coli C600 as the recipient strain, but blaNDM-1 from P. rettgeri failed to do so after three attempts, probably due to species differences. We discovered that blaNDM-27-positive transconjugants and transformants showed 2–4-fold higher MIC values toward meropenem compared with other blaNDM variants (Table 2). Besides, the MIC values of blaNDM-13- and blaNDM-5-carrying transformants to meropenem were slightly higher than those of blaNDM-1- and blaNDM-9-carrying transformants. No other variant-specific differences in MIC values were observed toward other β-lactam antibiotics.

Table 2 Antimicrobial susceptibility testing of blaNDM-positive clinical strains and E. coli strains harboring natural and recombinant blaNDM-carrying plasmids
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Phylogenetic analysis

Given the high isolation rate of blaNDM-positive E. coli and K. pneumoniae in the LPMs as well as their widespread distribution in different animals and environment, 51 representative blaNDM-positive strains were chosen for WGS (Table S7). Furthermore, phylogenetic trees were generated using the blaNDM-positive strains from this research and those retrieved from NCBI. We created different phylogenetic trees for the E. coli and K. pneumoniae variants to analyze the transmission characteristics of blaNDM genes in these two species.

Based on core genome SNPs, we built a phylogenetic tree comprising 224 blaNDM -positive E. coli strains, including 39 from this research and 185 from NCBI (Fig. 2). The isolates retrieved from NCBI came from clinical, animal, and environmental samples from different countries and regions. The 224 E. coli strains were divided into eight clades using rhierbaps (https://rdrr.io/cran/rhierbaps/man/hierBAPS.html), and the distinctions between the clonal groups were obvious. The 39 E. coli strains from this study were assigned to A (11/39, 28.20%), B1 (21/39, 53.85%), D (4/39, 10.26%), E (2/39, 5.13%), and F (1/39, 2.56%), according to Clermont’s method (http://clermontyping.iame-research.center/), and these strains were scattered among the eight clonal groups. Overall, the phylogroup determination performed by Clermont’s method was generally consistent with the division of clades. Within these strains, blaNDM-5 was the most prevalent variant (144/224, 64.29%), followed by blaNDM-1 (27/224, 12.05%) and blaNDM-9 (23/224, 10.27%). MLST analysis revealed that the 39 E. coli strains from this study presented 22 distinct sequence types, with ST2659 (4/39, 10.32%), ST155 (3/59, 5.26%), ST2473 (3/59, 5.26%), and ST162 (3/59, 5.26%) being the most prominent. This is inconsistent with a previous study that reported ST167 and ST101 as the most common E. coli sequence types [26]. However, these two sequence types were indeed more prevalent among the E. coli strains retrieved from NCBI, with detection rates of 6.48% (12/185) and 3.78% (7/185), respectively. Notably, some same sequence type strains with a low number of SNPs (< 150) were found widely distributed in humans, animals, and the environment, suggesting that blaNDM genes were probably mediated by the clonal spread of dominant sequence type strains. Furthermore, chicken-derived E. coli YCLc26-1 from LPM A displayed 38 SNPs relative to duck-derived E. coli YZMd12-2 from the same LPM. The pigeon-derived YZMp22-1 isolate demonstrated no SNPs relative to chicken-derived YZMc16-1, implying that clonal transmission of blaNDM-positive E. coli was quite common among various animals in the LPMs. In addition, two blaNDM-5-positive E. coli isolates from wastewater and chicken feces were closely related to two blaNDM-9-carrying E. coli strains from clinical samples in France. These data further evidence the widespread dissemination of blaNDM across humans and animals worldwide.

Fig. 2
figure 2

Phylogenetic analysis of 224 blaNDM-positive E. coli strains. A phylogenetic maximum-likelihood tree was generated using iTOL software based on single nucleotide polymorphism (SNP) analysis performed using the snp-dists tool (see Table S8 and S9 in the supplemental material). A total of eight clades (pink, green, gray, dark gray, blue, dark blue, orange, and purple) were identified. Strains depicted in dark blue are from this study. Strains positive for blaNDM-1, blaNDM-4, blaNDM-5, blaNDM-7, blaNDM-9, blaNDM-13, blaNDM-27, and blaNDM-29 are indicated by green circles, blue triangles, red squares, pink triangles, purple asterisks, yellow hooks, blue circles, and pink squares, respectively. The origins of blaNDM-positive samples in this study are marked outside the outermost circle

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For K. pneumoniae, we constructed a phylogenetic tree using the 10 isolates from this investigation and 44 blaNDM-positive K. pneumoniae strains from NCBI (Fig. S2). The 54 strains were divided into five clades. The clinical samples were mainly concentrated in clades 1 and 2, whereas the animal samples were primarily clustered in clades 4 and 5. The clades could not be discernibly distinguished. Core SNP-based phylogenetic analysis revealed that all isolates from this research shared 157 SNPs and clustered in the same clade, regardless of whether they were isolated from wastewater, chicken feces, soil, or car dust, implying that blaNDM-positive K. pneumoniae not only transmitted clonally between animals and the environment in the LPMs, but could also disseminate through vehicles. Moreover, all these strains belonged to the same sequence type, ST5241, and harbored highly identical plasmid replicons, ARGs, and virulence genes, suggesting that K. pneumoniae ST5241 could be present in various niches in the LPMs. Hence, effective strategies must be devised to reduce the prevalence and dissemination of this K. pneumoniae strain.

Transmissibility and characteristics of bla NDM-harboring plasmids

Plasmids carrying blaNDM from 225 strains were successfully transferred into E. coli C600 or E. coli J53. The conjugation frequency of the transconjugants ranged from 109–102 (Table S6). Among all the plasmid replicon types tested in this study, the highest conjugation frequency of 106–104 was exhibited by IncX3-type plasmids (Table S12).

To further investigate the genetic background of blaNDM-bearing bacteria and its role in dissemination, seven strains carrying different blaNDM variants from various clades in the phylogenetic tree were completely sequenced to reveal plasmid characteristics (Table S7). The expression of all seven blaNDM genes was mediated by plasmids, but these genes did not coexist with mcr-1 (Fig. 3). Three almost distinct transmissible blaNDM-5-bearing plasmids from chicken-derived E. coli strains YZMc10-2, YZMc17-1, and YZLc1-3 were detected, with sizes ranging from 46–245 kb. Replicon analysis revealed that these plasmids belonged to different Inc types, including IncHI2-HI2A, IncFII, and IncX3, respectively. pYZMc10-2_NDM-5_245k, with the most abundant resistance genes and the highest conjugation frequency, shared over 99% identity and 100% coverage with a chicken-derived blaNDM-5-positive plasmid isolated in 2020 (MT407547), indicating the potential long-term occurrence of IncHI2-HI2A blaNDM-5-positive plasmids (Fig. 3A). In addition, pYZMc3-1_NDM-9_226k shared relatively low coverage rates of 67% and 13% with blaNDM-9-positive plasmids within Salmonella enteritidis from chickens and E. coli from retail meat, respectively. However, all three blaNDM-9 positive plasmids harbored a multidrug-resistant region of approximately 9 kb, containing multiple mobile genetic elements, which could be transferred and integrated into other plasmids via circular intermediates (Fig. 3B). pYZLc4-1_NDM-27_46k, which is 46,283 bp long and carries the blaNDM-27 variant, has never been reported previously. This blaNDM-27-carrying plasmid was successfully transferred into E. coli C600, and the conjugation frequency was 4.47 × 10−6. It belongs to the IncX3 plasmid type, which is the most common plasmid type worldwide [5]. The blaNDM-positive plasmids that were isolated from Vietnamese chickens (LC570846), Chinese chickens (MH286946), and Chinese ducks (MK628734), and retrieved from NCBI shared up to 100% homology with pYZLc4-1_NDM-27_46k, indicating that IncX3-type blaNDM-positive plasmids are relatively conserved in different ecological niches (Fig. 3C). The backbone of the blaNDM-13-carrying plasmid was relatively simple, and showed 85% coverage and 99.88% homology with p1_020022 isolated from a clinical sample in Sichuan (CP032880). The two plasmids differed only in their multi-drug resistance region, indicating that this region in the blaNDM-13-carrying plasmid was mobile and could be mediated by the insertion sequence to spread among different Enterobacteriaceae species (Fig. 3D). Notably, the non-transferable pYZLc5-3_NDM-1_129k plasmid possessed a plasmid-free replicon similar to the plasmid backbone of blaNDM-negative strains that were isolated from Nantong pig farms (CP047346) and Shandong chicken farms (CP073357) in 2018. Further analysis revealed that the two blaNDM-1-negative bacteria lacked the conserved structure of blaNDM-1-bleMBL-trpF-tat mediated by ISAba125, highlighting the significant role of the insertion sequence in mediating the transfer of ARGs (Fig. 3E).

Fig. 3
figure 3

The genetic environment of blaNDM-positive plasmids. The GC skew and GC content are indicated from the inside out. The arrows represent the positions and transcriptional directions of the open reading frames (ORFs). Genes are differentiated by color. A, B, C, D, E, and F represent different blaNDM-bearing plasmids with different blaNDM variants (blaNDM-5, blaNDM-9, blaNDM-27, blaNDM-13, and blaNDM-1)

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The diversity of bla NDM variant-bearing genetic contexts

To further analyze the transmission mechanism of blaNDM-harboring plasmids, we conducted a comprehensive analysis of the core genetic environment of 51 blaNDM-positive strains that were subjected to sequencing. The region downstream of blaNDM, blaNDM-bleMBL-trpF-tat, is fairly conserved (Fig. 4). Based on sequence homology, we categorized the core genetic environment into six types: G1 (1/51), G2 (1/51), G3 (5/51), G4 (2/51), G5 (28/51), and G6 (14/51).

Fig. 4
figure 4

Different types of genetic environments of various blaNDM genes. Major types of blaNDM-carrying genetic contexts among the 51 blaNDM-bearing plasmids

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The G1 type was flanked by two inverted IS26 elements, and the upstream 10,473-bp region shared high homology with the region found in S. enteritidis C629 (CP015725) (Fig. 3B), encompassing 13 genes from the truncated ISAba125 to intI. Moreover, the G1 type harbored a multidrug resistance region, sul1-ΔqacE-hp-aadA2-dfrA12, between ISCR1 and intI, conferring resistance to sulfonamides, aminoglycosides, and trimethoprim. On the other hand, the G2 type, with an 18,625-bp-long backbone, carried the cassette ISAba125-blaNDM-bleMBL-trpF-Δtat-ISCR1-sul1-ΔqacE-hp-arr-3, which was also found in the blaNDM-1-positive plasmid pHFK418-NDM from Proteus mirabilis (MH491967) (Fig. 3D), along with the truncated transposons TnAs3 and ISShes11 downstream. The G3 type, which was associated with five blaNDM-13-positive strains isolated from chicken feces and car dust, was characterized by the presence of IS1294 upstream of blaNDM-13, the absence of ISCR1 downstream, and the presence of truncated IS50R and IS26. Both the G4 and G5 genetic contexts contained IS3000 and IS26, and blaNDM-5 could be integrated into the E. coli plasmid through an IS3000-mediated replicative transposon [27]. IS26 has also been implicated in capturing and mobilizing the transfer of blaNDM in Enterobacter species [28], indicating that insert sequences may play a vital role in the transmission of ARGs. However, while the G4 type was characterized by the presence of ISKox3 upstream of blaNDM, the G5 type included IS5 upstream of blaNDM. The G5 type served as the predominant genetic environment of the widely prevalent blaNDM-5-positive plasmids in the LPMs. These plasmids have been detected in diverse sample sources, including various animals and environments. Notably, the core genetic environment of the blaNDM-5-positive plasmid isolated from the LPMs shares 100% homology with that of the blaNDM-5-positive plasmid found in carbapenem-resistant Enterobacteriaceae isolated from humans in China [29], suggesting that the G5-type genetic environment, with the help of insertion sequences such as IS26 and IS3000, has been widely disseminated across different ecological niches in the LPMs. Furthermore, subtypes G5-1 and G5-2 were identified based on the integrity of IS3000 upstream of blaNDM-5 and the presence of the truncated transposon Tn2. In contrast, IS3000 is absent upstream of blaNDM-5 in the G6 type, while the G6-1 subtype also lacks ISAba125.

We used a Sankey diagram to elucidate the relationships between the blaNDM core genetic environment and various factors, such as species, strain source, plasmid replicon type, and blaNDM variant (Fig. 5). G5 exhibited the highest prevalence (28/51, 54.9%), followed by G6 (14/51, 27.45%). It is worth noting that E. coli exhibits a greater diversity of blaNDM bearing genetic contexts than the more homogeneous genetic context G6-1 observed in K. pneumoniae. Interestingly, IncHI2-HI2A plasmids were more prevalent (27/51) than IncX3 plasmids (13/51) in this study, whereas previous studies have reported the opposite trend in China [5]. This discrepancy might be attributed to the concentration and limitations of the sampling area. The presence of six other blaNDM-positive plasmid replicons in the environment indicated the ubiquitous nature of blaNDM in Enterobacteriaceae.

Fig. 5
figure 5

Sankey diagram combining the species, genetic environment, source of strains, plasmid replication types, and blaNDM variants. The diameter of the line is proportional to the number of isolates, which are also labeled at the consolidation points

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Discussion

Over the past decade, blaNDM-positive strains have been increasingly isolated from humans [30, 31], animals [21], and the environment [32] worldwide, raising global concern in the field of public health. LPMs represent a key human–animal interface because live animals from various areas are congregated here for trade in urban areas. However, the prevalence and transmission mechanisms of blaNDM in different ecological niches within LPMs have rarely been studied [17, 33, 34]. Therefore, the rapid spread of blaNDM-harboring strains in LPMs has not yet been considered a major threat to public health yet.

For the first time here, using a One Health approach, we have provided evidence of the potential transmission of blaNDM-positive bacteria through the food chain or through close contact between different animals and their environment in LPMs. The key findings of this study are as follows: (a) Over 65% of blaNDM genes were able to be transferred, including cross-species transmission, and the conjugative transfer frequency was concentrated at 10−5, suggesting that blaNDM has been extensively disseminated throughout the LPMs; (b) Some blaNDM-harboring strains isolated from chickens, ducks, pigeons, and car dust showed similar genotypes based on SNP analysis, suggesting that different niches within LPMs could serve as an important transmission medium for blaNDM-positive strains, facilitating the spread of ARGs across different ecological niches; (c) The blaNDM core genetic environment G5 was detected with 100% homology in 43% of the strains, isolated from various animal and environmental samples; and d) Over 20% of the strains isolated from animal samples shared the same sequence type as that of strains isolated from environmental samples, further corroborating the possibility that the different niches can act as a transmission medium. The above results indicate the possibility of clonal transmission among animals and the environment in LPMs through the food chain and close contact. Based on these findings, we concluded that LPMs are an underestimated hotspot for the spread of multidrug-resistant bacteria among animals and the environment. This conclusion underscores the critical role played by LPMs in the dissemination and evolution of drug-resistant bacteria, and highlights the need for enhancing regulatory and control measures in these niches to curb the emergence and spread of ARGs. Furthermore, these findings call for stricter monitoring of animals and the environment in LPMs to promptly detect and control potential pathogen transmission risks, ensuring the health and safety of humans and animals.

Five blaNDM variants, including blaNDM-27, a newly reported variant, were isolated from the LPMs. We found that the MIC values of blaNDM-27-positive transconjugants to meropenem were 2–4-fold higher than those of isolates carrying other variants. Amino acid substitutions were found at positions 95 and 233, with Asp being replaced by Asn and Ala by Val, respectively. Previous studies have shown that substitutions at these positions can affect enzyme kinetics and drug resistance; for example, NDM-3, which has an amino acid substitution at position 95, showed a slightly lower kcat/Km ratio compared with NDM-1 [35]. NDM-6, which has an amino acid substitution at position 233, exhibited higher drug resistance than NDM-1, particularly against meropenem and imipenem [36]. However, it had lower thermal stability than NDM-1 [37]. Therefore, substitutions at positions 95 and 233 might enhance drug resistance of blaNDM-positive strains, while compromising on their environmental stability. Further research is needed to explore whether these substitutions affect enzyme activity and thermal stability. The blaNDM variants detected in this study were distributed among various species as previously reported [38], with E. coli being the most common. These species were isolated from various animal and environmental samples. More than half of the wastewater samples contained blaNDM (35/66). This water is primarily used as drinking water for animals and domestic water for workers, increasing the risk of blaNDM transmission to humans and animals in the LPMs. The tigecycline resistance genes tet(X3) and tet(X4) have been previously detected in the workers and environment of LPMs [17], hinting at their transmission from animals to humans and the environment via plasmids, corroborating the notion of LPMs being potential reservoirs of ARGs. All niches within LPMs are at risk of supporting the dissemination of blaNDM, emphasizing the need to adopt a One Health approach to prevent the transmission of pathogenic bacteria.

Although we provide evidence of the transfer of blaNDM-positive strains between animals and the environment in LPMs, we acknowledge several limitations in this study. First, a large sample size of LPM workers was unable to be used in the present study due to sampling limitations associated with the large workload and high cost. However, phylogenetic analysis has shown that blaNDM-positive strains are widespread among humans, animals, and the environment globally. Previous research has also indicated that workers with prolonged exposure to LPM environments may harbor a higher abundance of ARGs than workers in a control group. Therefore, we hypothesized that LPM workers could carry and spread blaNDM-positive isolates through close contact or the food chain (Fig. 6). Second, considering the significant differences in the number of different animals in LPMs, it is worth noting that chickens account for up to 80% of the total number of LPMs. This disparity can largely be attributed to the eating habits prevalent in our country. To provide a more accurate reflection of the market situation, our sample collection process focused primarily on chickens. Of the 395 samples collected, 312 were derived from chicken manure, accounting for approximately 78.99% of the total samples. However, it is important to acknowledge that this sampling strategy may introduce some bias in the sample size. Furthermore, although we collected 593 samples from various ecological niches in LPMs, it is important to note that the sampling was primarily focused on two LPMs. As a result, the findings may not entirely represent the overall prevalence and transmission mechanism of blaNDM-positive bacteria in LPMs. Therefore, it is crucial to broaden our sampling scope and include more LPMs in our study. By adopting this comprehensive sampling strategy, we can gain a more comprehensive understanding of the transmission pathways of blaNDM-positive strains in this environment. By considering the actual situation, we can develop and implement appropriate measures to effectively mitigate the spread of blaNDM-positive strains in LPMs [39].

Fig. 6
figure 6

Diagram showing possible transmission routes for blaNDM-positive strains among humans, animals, and the surrounding environment in LPMs. A The production mode of LPMs. B Possible transmission routes of blaNDM-positive strains

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Conclusion

To our knowledge, this is the first and most comprehensive surveillance of blaNDM-positive bacteria in humans, animals, and environmental niches within LPMs. These bacteria were more prevalent among wastewater, ducks, chickens, and dust samples, and less prevalent among pigeons, geese, and soil samples. blaNDM genes were distributed between two main species, E. coli and K. pneumoniae. blaNDM-carrying bacteria were highly diverse and frequently associated with multidrug resistance phenotypes. Horizontal gene transfer mediated by the IncHI2-HI2A plasmid and the core genetic environment G5 formed the major transmission mechanism of blaNDM within the LPMs. Phylogenetic analysis evidenced the transmission of blaNDM-harboring bacteria between animals and the environment, and suggested that humans might play a crucial role in this transmission via the food chain and close contact.

Materials and methods

Sample collection and screening of bla NDM-positive strains

Samples were collected between April 1, 2019 and July 31, 2022, from two large-scale LPMs in Yangzhou to investigate the epidemiology of blaNDM-positive bacteria in animals and the environment (Table S2). The animals gathered in the LPMs were from different regions (Huai’an, Yancheng, Yangzhou, Taizhou, Nanjing, Nantong, and Suzhou) of the Jiangsu Province (Fig. 1, Table S3). A total of 593 non-duplicate samples were collected, comprising animal feces (chicken, n = 312; duck, n = 39; pigeon, n = 27; goose, n = 17) and environmental samples (soil, n = 32; wastewater, n = 66; vegetables, n = 13; dust, n = 87) (Table S4). Sterilized water was used as a transfer medium for the dust samples. All the samples were kept in cool boxes with ice packs (4 °C) while being transported to the laboratory for bacterial cultivation and DNA extraction.

Samples were spread onto MacConkey plates supplemented with 2 mg/L meropenem, and incubated for 18 h at 37 °C. Different colored colonies were selected from each plate for identifying carbapenem-resistant isolates. Cultures were identified using MALDI-TOF MS AximaTM [40] and 16S rRNA gene sequencing (Table S1). All confirmed carbapenem-resistant strains were investigated for the presence of blaNDM genes (Table S1).

Antimicrobial susceptibility testing

Antimicrobial susceptibility was tested using the broth dilution method. The susceptibility of carbapenem-resistant isolates was tested for antimicrobial drugs commonly used in both medical and veterinary settings, including meropenem, imipenem, ampicillin, ceftazidime, tigecycline, kanamycin, amikacin, ciprofloxacin, gentamicin, colistin, ceftiofur, and carbenicillin. Minimum inhibitory concentrations (MICs) were interpreted according to the guidelines provided by the Clinical and Laboratory Standards Institute (2021) [41], along with the breakpoint tables specified in version 12.0 of the European Committee on Antimicrobial Susceptibility Testing. As a control, we employed E. coli American Type Culture Collection 25922 as a quality control measure.

Plasmid conjugation assay

To investigate the transferability of blaNDM-bearing genetic elements, we utilized rifampicin-resistant E. coli C600 and sodium azide-resistant E. coli J53 as the recipients for the conjugation assay. The liquid mating method was employed for this experiment. To begin with, overnight cultures of the original isolates and recipient strains were grown in Luria-Bertani (LB) broth. The cultures were then adjusted to an optical density of 0.6 at 600 nm. A 5-μL portion of each culture was diluted 1:200 in fresh LB broth and incubated at 37 °C with gentle shaking for a period of 4 h. Subsequently, the conjugation mixtures were diluted tenfold and plated on selective agar plates to quantify the recipients (rifampicin/sodium azide) as well as the transconjugants (rifampicin plus meropenem/sodium azide plus meropenem). The conjugation frequency was determined by calculating the ratio of transconjugants obtained per input recipient cell [42]. Finally, PCR analysis was carried out to conclusively verify that the transconjugants were indeed derived from the recipient E. coli strains C600 and J53.

WGS of the flanking region of bla NDM

We utilized the FastPure Bacteria DNA Isolation Mini Kit (Vazyme, Nanjing, China) to extract the genomes of 51 strains resistant to meropenem. The extracted DNA's concentration and purity underwent assessment through NanoDrop 2000 and gel electrophoresis, with final precise concentration confirmation using the QubitTM 4.0 fluorometer (Invitrogen, CA, USA). Subsequently, Illumina HiSeq 2500 was employed for short-read sequencing of the extracted DNA, generating paired-end reads measuring 2 × 150-bp. Following this, we subjected the collected raw reads, with a minimum coverage of 100-fold, to trimming using Trimmomatic v.0.36 [43]. De novo assembly was then conducted using SPAdes v.3.13.1 [44]. Based on diverse resistant phenotypes and phylogenetic analysis, we identified seven representative strains for further sequencing using Nanopore MinION [45], which provided single-molecule long reads. We achieved the complete genome by employing hybrid assembly of both short and long reads via Unicycler v.0.4.8 [46]. The putative coding sequences flanking the blaNDM genes were annotated by Rapid Annotations using Subsystems Technology (https://rast.nmpdr.org/). To analyze the plasmid type, ARGs, virulence genes, and mobile genetic elements of the blaNDM-positive isolates, we utilized ABRicate (https://github.com/tseemann/abricate). Additionally, we generated a circular genome comparison map using BLAST Ring Image Generator [47] while EasyFig [48] was implemented for the line alignment of core genetic structures.

Phylogenetic analysis of bla NDM-bearing isolates from various origins

The assembled genomes of blaNDM-positive strains, with complete isolation information, were retrieved and then downloaded from the National Center for Biotechnology Information (NCBI; as of 1 September, 2022) (https://www.ncbi.nlm.nih.gov/pathogens). Draft genome sequences were re-annotated using Prokka v.1.11 [49], and core genomes were extracted and aligned using Roary v.3.6.1 [50] before being subjected to phylogenetic tree construction using FastTree v.1.4.3 [51]. The phylogenetic tree was visualized and embellished with the corresponding features of each isolate using iTOL (https://itol.embl.de/), and multilocus sequence typing (MLST) was performed according to PubMLST (https://pubmlst.org/). To estimate the phylogenetic groups (A, B1, B2, D, E, and F) of the E. coli isolates, the assembled genome was uploaded to ClermonTyping based on the concept of in vitro PCR assays [52]. Single nucleotide polymorphisms (SNPs) were analyzed using snp-dists v.0.7.0 to detect pairwise SNP distances. Resistance genes and virulence genes were visualized using TBtools [53]. WGS data generated from this study have been deposited in GenBank and under BioProject accession no. PRJNA877800.

Comparative susceptibility analysis of different bla NDM variants to β-lactam antibiotics

To assess and compare the susceptibility of different blaNDM variants to different β-lactam antibiotics, 849-bp-long DNA fragments comprising the complete blaNDM gene and homology arm were amplified using primers (Table S1), ligated into pET-28a(+) using ClonExpress II One Step Cloning Kit (Vazyme, Nanjing, China), and expressed in E. coli BL21(DE3). Transformants were selected on LB agar plates containing 2 mg/L meropenem and 100 mg/L kanamycin, and confirmed by Sanger sequencing with T7 primer (Table S1). Antimicrobial susceptibility was tested using different β-lactam antibiotics (meropenem, imipenem, aztreonam, ticarcillin, ceftazidime, and ceftiofur) on transconjugants and transformants to compare the susceptibilities of different blaNDM variants [36].

Availability of data and materials

WGS data generated from this study have been deposited in GenBank and under BioProject accession no. PRJNA877800.

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Funding

This work was supported by the Sichuan Science and Technology Program (2022ZDZX0017), the National Natural Science Foundation of China (32161133005), the China Postdoctoral Science Foundation (2022T150555) and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

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Authors and Affiliations

  1. Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, College of Veterinary Medicine, Yangzhou University, Yangzhou, Jiangsu, China

    Yi Yin, Kai Peng, Yan Li, Wenhui Zhang, Yanyun Gao, Xinran Sun, Zhiqiang Wang & Ruichao Li

  2. Institute of Comparative Medicine, Yangzhou University, Yangzhou, Jiangsu, China

    Zhiqiang Wang & Ruichao Li

  3. Department of Food Science and Nutrition, Faculty of Science, The Hong Kong Polytechnic University, Kowloon, Hong Kong, China

    Sheng Chen

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  1. Yi Yin
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Contributions

Y.Y.: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Software, Visualization, Writing – original draft. K.P.: Conceptualization, Writing – review & editing. Y.L.: Writing – review & editing. WZ.: Writing – review & editing. Y.G.: Sample collection, Writing – review & editing. X.S.: Sample collection, Writing – review & editing. S.C.: Supervision, Writing – review & editing. Z.W.: Supervise this project equally, Writing – review & editing. R.L.: Supervision, Project administration, Writing – review & editing.

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Correspondence to Zhiqiang Wang or Ruichao Li.

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Supplementary Information

Additional file 1:

 Table S1. Primers used in this study. Table S2. The prevalence of blaNDM-positive samples in LPMs A and B. Table S3. Number of blaNDM-positive samples from LPMs and their prevalence among different regions in Jiangsu. Table S4. Origins and numbers of blaNDM-positive strains in LPMs. Table S5. Species and numbers of blaNDM-positive strains in LPMs. Table S6. Antibiotic susceptibility testing (MICs, mg/L) of 336 blaNDM-positive strains. Table S7. Basic information of 51 blaNDM-positive strains sequenced by Illumina/Nanopore sequencing. Table S8. Basic information of blaNDM-positive strains used for phylogenetic analysis in Fig. 2. Table S9. The SNPs distribution of blaNDM-positive strains used for phylogenetic analysis in Fig. 2. Table S10. Basic information of blaNDM-positive strains used for phylogenetic analysis in Fig. 3. Table S11. The SNPs distribution of blaNDM-positive strains used for phylogenetic analysis in Figure S2. Table S12. Characteristic of blaNDM-bearing plasmids in different bacterial species from LPMs. Fig. S1. The heat map of resistance genes and virulence genes of 51 blaNDM-positive isolates in this study. The darker the blue, the higher the similarity. Fig. S2. Phylogenetic analysis for 54 blaNDM-positive K. pneumoniae strains. Phylogenetic maximum-likelihood tree generated using iTOL software based of SNP analysis performed using the snp-dists tool (see Table S10 and S11 in the supplemental material). A total of five clades (green, blue, purple, yellow, and pink) were identified. Strains expressed by red are from this study.

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Yin, Y., Peng, K., Li, Y. et al. Transmission patterns of multiple strains producing New Delhi metallo-β-lactamase variants among animals and the environment in live poultry markets. One Health Adv. 2, 12 (2024). https://doi.org/10.1186/s44280-024-00050-2

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