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    Characterization of NDM-5-producing Enterobacteriaceae isolates from retail grass carp(Ctenopharyngodon idella) and evidence of blaNDM-5-bearing IncHI2 plasmid transfer between ducks and fish

    2022-04-28 06:48:00LuChaoLvYaoYaoLuXunGaoWanYunHeMingYiGaoKaiBinMoJianHuaLiu
    Zoological Research 2022年2期

    Lu-Chao Lv, Yao-Yao Lu, Xun Gao, Wan-Yun He, Ming-Yi Gao, Kai-Bin Mo, Jian-Hua Liu,2,*

    1 College of Veterinary Medicine, Key Laboratory of Zoonosis of Ministry of Agricultural and Rural Affairs, National Risk Assessment Laboratory for Antimicrobial Resistant of Microorganisms in Animals, Guangdong Provincial Key Laboratory of Veterinary Pharmaceutics Development and Safety Evaluation, South China Agricultural University, Guangzhou, Guangdong 510642, China

    2 Guangdong Laboratory for Lingnan Modern Agriculture, Guangzhou, Guangdong 510642, China

    ABSTRACT

    Keywords: blaNDM-5; Enterobacteriaceae;Plasmid; Fish; Carbapenemase

    INTRODUCTION

    Carbapenemase-producingEnterobacteriaceae(CPE),especially New Delhi metal-β-lactamase (NDM) producers,have been increasingly reported worldwide and pose a significant challenge to public health (Wu et al., 2019).Since the discovery of NDM-1 in 2008 (Yong et al., 2009), NDM-producingEnterobacteriaceaehave spread globally.To date,41 NDM enzyme variants (NDM-1–NDM-41) (https://www.ncbi.nlm.nih.gov/pathogens/refgene/#NDM) have been identified, with theblaNDM-1andblaNDM-5genes being the most prevalent (Wu et al., 2019).

    In 2011,blaNDM-5was first reported in anE.colistrain isolated from a patient in the UK (Hornsey et al., 2011).Since then,blaNDM-5has been detected in more than 40 countries(https://www.ncbi.nlm.nih.gov/pathogens/microbigge/#blaNDM-5).NDM-5 is the most common NDM variant inE.coli,especially in China and Southeast Asia.AlthoughblaNDM-5is widespread due to diverse self-transferable plasmids such as IncX3 and IncF (FII, FIA, and FIB) (Wu et al., 2019), it is rarely reported in the IncHI2 plasmid, except in swine- and duckoriginE.colifrom Guangdong Province, China (Ma et al.,2021; Zhao et al., 2021b).

    In China, NDM-5-producingEnterobacteriaceaehave been widely detected in humans (Tian et al., 2020), farm animals(Ma et al., 2021), companion animals (Wang et al., 2021a),wild animals (Wang et al., 2017), retail meats (Zhang et al.,2019), and the environment (Zhao et al., 2021a), but rarely in aquatic products.Aquatic products are also considered important reservoirs and transmission vectors of resistant bacteria (Xu et al., 2020).Of note, the integrated duck-fish freshwater aquaculture system is very common in Guangdong, and antimicrobial resistant bacteria can be transmitted between ducks and fish (Shen et al., 2020).Grass carp (Ctenopharyngodon idella) is the most popular freshwater fish in aquaculture and is cultivated in 32 provinces in China.According to the China Fishery Statistical Yearbook 2020(https://data.cnki.net/trade/Yearbook/Single/N2021020168?z=Z009), the production of grass carp reached 5.5 million tons in 2019, accounting for 21.7% of the maximum annual production in freshwater fish.However, the occurrence of clinically important resistant bacteria, such as CPE, in grass carp has rarely been studied.Hence, we investigated the prevalence of CPE in intestinal samples of grass carp from wet markets in Guangzhou and characterizedblaNDM-positive isolates and plasmids to understand the transmission mechanism ofblaNDM5in aquatic products.

    MATERIALS AND METHODS

    Sample collection, bacterial isolation, and detection of blaNDM

    In January 2019, a total of 196 intestinal samples from grass carp were randomly collected from 24 wet markets located in seven districts of Guangzhou, China.We collected fish samples from different stalls, with three intestinal samples randomly collected from each sampling booth.Each sample was placed in a separate sterile sample bag and transported to the laboratory in a freezer box for processing within 12 h.The fish intestines were dissected with sterile surgical scissors, and 1 g of intestinal content was enriched in 2 mL of Luria-Bertani (LB) broth at 37 °C overnight with shaking.The overnight cultures were streaked onto MacConkey agar plates supplemented with 1 mg/L meropenem and incubated at 37 °C for 18–24 h.Enterobacteriaceaecolonies with different morphologies were selected from the plates to screen forblaNDM-,blaKPC-, andblaOXA-48-positive isolates using polymerase chain reaction (PCR) with specific primers as described previously (Poirel et al., 2011).

    Antimicrobial susceptibility testing

    According to the recommendations of the Clinical and Laboratory Standards Institute (2017), the minimal inhibitory concentrations (MICs) of 18 antimicrobials against NDM-positiveEnterobacteriaceaeisolates were determined using the agar dilution or broth microdilution (colistin and tigecycline)methods.Escherichia coliATCC 25 922 was used as the control.The MICs were interpreted according to the criteria of CLSI (M100-S30) and EUCAST (http://www.eucast.org).

    Conjugation experiments

    In this study, streptomycin-resistantE.coliC600 was used as the recipient, and eachblaNDM-positive isolate was used as the donor for conjugation by broth mating at 37 ℃ for 16–20 h.Transconjugants were selected on MacConkey agar plates supplemented with 3 000 mg/L streptomycin and 1 mg/L meropenem.Conjugation frequency was calculated following previously reported methods (Chen et al., 2007).

    Whole-genome sequencing and bioinformatics analysis

    Whole-genomic DNA of NDM-positive isolates was sequenced using the Illumina Hiseq X Ten and Oxford Nanopore MinIon platforms, and complete genomes were obtained by hybrid assembly using Unicycler v0.4.7 (Wick et al., 2017).MLST v2.19 (https://github.com/tseemann/mlst) was used to identify the sequence type (ST) of theblaNDM-positive strains.Plasmid replicons, antimicrobial resistance genes, and heavy metal resistance genes were analyzed using ABRicate v1.0(https://github.com/tseemann/abricate) with the PlasmidFinder(Carattoli et al., 2014), ResFinder (Zankari et al., 2012), and AMRFinderPlus databases (https://github.com/ncbi/amr),respectively.Plasmid double-locus sequence typing (pDLST)for IncHI2 plasmids was identified using pMLST v2.0(https://cge.cbs.dtu.dk/services/pMLST/).Insertion sequence(IS) elements were identified using ISfinder (https://isfinder.biotoul.fr/).Single nucleotide polymorphism (SNP)calling was performed using Snippy (https://github.com/tseemann/snippy).TheblaNDM-carrying plasmids were further compared and analyzed using the BLAST ring image generator (Alikhan et al., 2011).The genetic context ofblaNDMwas analyzed by GalileoTMAMR (http://galileoamr.arcbio.com/mara/), Gene Construction Kit v4.5 software (Textco BioSoftware, USA), and Easyfig v2.2.5 (http://mjsull.github.io/Easyfig/files.html).

    Nucleotide sequence accession numbers

    The complete genome sequences of sevenblaNDM-5-positiveEnterobacteriaceaewere deposited in GenBank under BioProject No.PRJNA636005.

    RESULTS

    Characterization of blaNDM-5-carrying isolates

    A total of seven (3.57%) unduplicated carbapenem-resistant isolates, including sixE.coliand oneC.freundii, were obtained from the seven intestinal samples of grass carp(Table 1).All seven isolates were identified asblaNDM-5-positive by PCR and sequencing, whileblaKPCandblaOXA-48were not detected.

    The sevenblaNDM-5-carrying isolates showed multidrugresistant phenotypes and harbored multiple resistance genes(Tables 1, 2).Molecular typing results showed that theC.freundiistrain belonged to ST557.Six of the NDM-5-positiveE.colistrains belonged to five different STs, namely ST48,ST57, ST101, ST155, and ST9124.The two ST48E.coliisolates (PY9F04M and PY9F07M) were recovered from the same market but from different sample booths (Table 1) and were related as they showed only 10 core-genome SNP(cgSNP) differences from each other (Schürch et al., 2018),although the resistance genes they carried were not the same(Table 1).

    Characterization of blaNDM-5-bearing plasmids

    The conjugation experiments indicated that the sevenblaNDM-5-carrying plasmids could be successfully transferred to the recipientE.coliC600 strain, and replicon typing results revealed that theblaNDM-5genes were located on IncX3 (n=5),IncHI2 (n=1), and IncHI2-IncF (n=1).The conjugation frequencies of the IncX3-type plasmids varied from ~10-4to 10-5cells/donor, while the conjugation frequencies of the IncHI2-type and IncHI2-IncF-type plasmids were ~10-6and~10-5cells/donor, respectively (Table 1).

    Table 1 Characterization of blaNDM-5-carrying Escherichia coli and Citrobacter freundii isolates

    Table 2 Antibiotic susceptibility of blaNDM-5-carrying isolates and their transconjugants

    The complete sequences of all sevenblaNDM-5-bearing plasmids were obtained using Illumina and Nanopore sequencing.The sequences of five IncX3 plasmids were similar to previously reportedblaNDM-5-bearing IncX3 plasmids,including plasmids pGDQ8D112M-NDM (GenBank Accession No.MK628734, duck, China), pNDM5_IncX3 (KU761328.1,Homo sapiens, China), pHNYX638-1 (MK033577, pork,China), and pHN7DH6 (MN276080, dog, China) (Figure 1A).

    Plasmid pHNBYF33-1, which belonged to IncHI2-ST3, was 238 926 bp in length with a GC content of 46.30% and carried 12 resistance genes.The BLASTn results indicated that plasmid pHNBYF33-1 exhibited high similarity (≥99.9%identity and ≥93.4% coverage) to fourblaNDM-5-carrying IncHI2 plasmids deposited in GenBank, i.e., pNDM33-1 (MN915011)(Zhao et al., 2021b), GFQ9D68 Contig5 (JAGFYC0100 00005), GDQ8D151 plasmid1 (JAGFYD010000002), and GDQ20D15 plasmid1 (JAGFYB010000003) (Figure 1B).Interestingly, all four plasmids were carried byE.colistrains recovered from ducks in Guangdong, China.

    Plasmid pHNTH9F11-1 (IncHI2-IncF) was 407 456 bp in size and had an average GC content of 48.03%.pHNTH9F11-1 harbored three different replicons, including IncHI2, IncFII,and IncFIB.BLASTn analysis showed that pHNTH9F11-1 was a cointegrate plasmid comprised of sequences of IncHI2(designated as pHNTH9F11-1_IncHI2), harboringblaNDM-5,and IncF24:A-:B1 (designated as pHNTH9F11-1_IncF)(Figure 1C).In addition, the hybrid plasmid pHNTH9F11-1 had 87% nucleotide sequence coverage of the IncHI2-IncFII plasmid pP2-3T (MG014722, swine, China).The sequence of plasmid pHNTH9F11-1_IncHI2 harboringblaNDM-5was similar(≥99.99% identity and 100% coverage) to that of theblaNDM-5-carrying plasmid pHNGD64-NDM (MW296099) from a swineE.colistrain (Ma et al., 2021).Plasmid pHNTH9F11-1_IncF exhibited similarity (≥99.99% identity and ≥90% coverage) to IncF24:A-:B- plasmid pPK8568-156kb (CP080127, chicken,Pakistan), carrying multiple heavy metal resistance genes(arsABCDandsitABCD) and a phage resistance system(BREX, bacteriophage exclusion system) (Goldfarb et al.,2015).Further analysis revealed that pHNTH9F11-1_IncF and pHNTH9F11-1_IncHI2 were bound by two identical ΔTn1721transposons (hp-tetR-tet(A)-eamA) with a length of 5 492 bp,suggesting that the cointegrate plasmid pHNTH9F11-1 was formed by homologous recombination of these two plasmids through ΔTn1721(Figure 1C).

    Genetic contexts of blaNDM-5 genes in IncX3 and IncHI2

    All fiveblaNDM-5-carrying IncX3 plasmids showed identical genetic contexts (i.e., IS3000-ΔISAba125-IS5-ΔISAba125-blaNDM-5-bleMBL-trpF-tat-IS26-ΔumuD-ISKox3) (Figure 2),similar to that of the classical IncX3 plasmid pHNYX658-1(Zhang et al., 2019).The genetic contexts ofblaNDM-5in pHNTH9F11-1_IncHI2 and pHNBYF33-1 (IncHI2) were similar to otherblaNDM-5-carrying IncHI2 plasmids in GenBank,including pNDM33-1, GDQ8D151 plasmid1, GFQ9D68 Contig5, and GDQ20D15 plasmid1 (Figure 2).In these four IncHI2-type plasmids, theblaNDM-5gene was identically embedded in a novel composite transposon (IS3000-ΔISAba125-IS5-ΔISAba125-blaNDM-5-bleMBL-trpF-tat-Δdct-IS26-ΔumuD-ΔISKox3-IS3000) inserted between IS1and IS10Rof the multidrug resistance region of the IncHI2 plasmid with 5 bp target site duplications (TSDs) (ACTTT).Previous research has found that excision of this transposon from the plasmid pNDM33-1 forms a circular intermediate (Zhao et al.,2021b).Here, we renamed this novel 13 918 bp longtransposon as Tn7051(https://transposon.lstmed.ac.uk/).Comparative analysis demonstrated that Tn7051shared 99.98% nucleotide sequence similarity (two SNP differences)with the genetic context ofblaNDM-5in the IncX3 plasmid pHNYX638-1 (MK033577, pork, China), except that ISKox3in Tn7051was truncated by a copy of IS3000, creating only 545 bp remains (ΔISKox3) (Figure 2).Interestingly, when comparing the Tn7051sequence, we found a Tn7051-like structure (14 482 bp) in three hybrid plasmids obtained from swineE.coliisolates in China (Yao et al., 2020), namely p4M9F (IncFIA-IncHI1A-IncHI1B, MN256759), p4M8F (IncHI1-IncY-IncFIA-IncFIB, MN256758), and p4M18F (IncHI1-IncYIncFIA-IncFIB, MN256757).In the Tn7051-like structure,ISKox3had more residues (1 108 bp) than that in Tn7051.Furthermore, the Tn7051-like structure exhibited 99.97%–100.00% nucleotide sequence identity (0–3 SNP differences) to the genetic context ofblaNDM-5in the IncX3 plasmid pHNYX638-1.Given that Tn7051and Tn7051-like transposons were similar to the genetic context ofblaNDM-5in the IncX3 plasmid pHNYX638-1, we speculated that both Tn7051and Tn7051-like transposons were likely derived from the IncX3 plasmid.

    Figure 1 Comparison of blaNDM-5-carrying plasmids

    Although the IncHI2 plasmids pHNBYF33-1 and pNDM33-1 shared the same Tn7051insertion site (Figure 2), compared with pNDM33-1, the plasmid pHNBYF33-1 lacked the ΔIS3000-ΔIS10-IS26-lnu(F)-aadA2-hp-IS26segment, which could be readily explained by a deletion event mediated by two copies of IS26located in the same orientation (Harmer & Hall, 2016).The genetic context ofblaNDM-5in the hybrid plasmid pHNTH9F11-1 was the same as that in plasmid pHNGD64-NDM, and was very similar to pNDM33-1, except for the lack of the IS26-ΔumuD-ΔISKox3-IS3000-ΔIS10-IS26-lnu(F)-aadA2-hp-IS26-hp-IS26-blaTEM-IS1Xunit (Figure 2).This may be due to the deletion of genes mediated by homologous recombination between two copies of IS26in the same direction (i.e., IS26in Tn7051and IS26upstream of ΔTnEc1),as IS26located upstream of ΔTnEc1had only an 8 bp TSD(CTTCTGGT) on one side (Figure 2).

    Proposed formation model of genetic contexts of blaNDM-5 in IncHI2 plasmids

    Based on detailed sequence analysis, the co-integration mechanism of IS26(Harmer & Hall, 2016), and the copy-outpaste-in mechanism of composite transposons (Piégu et al.,2015), we proposed a genetic environment formation model ofblaNDM-5in plasmids pHNBYF33-1 and pHNTH9F11-1, as shown in Figure 3.The assumed plasmid evolution process was as follows: IS3000was inserted into ISKox3of the IncX3 plasmid (Figure 3A), thus forming the IS3000-ΔISAba125-IS5-ΔISAba125-blaNDM-5-blaMBL-trpF-tat-Δdct-IS26-ΔumuDΔISkox3-IS3000-ΔISkox3unit (Figure 3B), hypothesized due to the absence of this unit in GenBank.The two sameorientated copies of IS3000, surroundingblaNDM-5, generated the circular intermediate Tn7051(Figure 3C), which was further inserted into the region between IS1and IS10of the IncHI2 plasmid (Figure 3D) with 5 bp TSDs (ACTTT), resulting in the formation of theblaNDM-5-carrying IncHI2 plasmids (i.e.,pNDM33-1, MN915011; GFQ9D68 Contig5, JAGFYC 010000005; GDQ8D151 plasmid1, JAGFYD010000002; and GDQ20D15 plasmid1, JAGFYB010000003) (Figure 3E).IS3000,located downstream of ISKox3, was truncated by IS26and,consequently, the IncHI2 plasmids evolved into the structure shown in Figure 3F (hypothesized due to the absence of a similar structure in GenBank).The IS26that previously truncated IS3000was recombined with the IS26adjacent toblaTEM-1(purple frame in Figure 3F), leading to the deletion of the ΔIS3000-ΔIS10-IS26-lnu(F)-aadA2-hp-IS26segment.As a result, the structure of the hypothetical plasmids (Figure 3F)entered theblaNDM-5-carrying plasmid pHNBYF33-1 (Figure 3G).Additionally, the two IS26elements in Figure 3F and Figure 3G (light green frame) integrated, resulting in the formation of theblaNDM-5-carrying plasmid pHNTH9F11-1(Figure 3H).Therefore, in summary, we speculated that Tn7051may contribute to the transfer ofblaNDM-5from the IncX3 plasmids to the IncHI2 plasmids, and the genetic contexts ofblaNDM-5on the IncHI2 plasmids in fish were likely derived from plasmids carried by ducks in Guangdong, China.

    Figure 3 Proposed formation mechanism of genetic environment of blaNDM-5 in plasmids pHNBYF33-1 and pHNTH9F11-1

    DISCUSSION

    As the most common CPE,blaNDM-positiveEnterobacteriaceaehave been isolated from seafood and aquatic environments in several countries (Das et al., 2019;K?ck et al., 2018).Moreover,blaNDM-positiveEnterobacteriaceaehave been detected in freshwater fish in Vietnam (Nakayama et al., 2022) and farmed fish in Egypt(Hamza et al., 2020).To the best of our knowledge, however,this is the first report ofblaNDMin freshwater fish from China.Of concern, as fish intestines are consumed in Guangdong,NDM-5-positiveEnterobacteriaceaein the intestines of retail fish products could spread to humans via the food chain.

    In China, IncX3 plasmids are the most common type of plasmid carryingblaNDM-5(Ma et al., 2020).NDM-5-producing IncX3 plasmids are widespread in environmental, animal, and clinical isolates (Ma et al., 2020), but are rarely reported inEnterobacteriaceaeof freshwater fish origin.The similar IncX3 plasmids found in this study further highlight the importance of the epidemic IncX3 plasmid in the spread of theblaNDM-5gene within the entire ecosystem.IncHI2/ST3 plasmids have been reported to mediate the transfer of various antibiotic resistance genes (ARGs), such asfosA3(Wang et al., 2020),floR(Cao et al., 2020),blaCTX-M(Lü et al., 2020), andmcr(Long et al.,2019; Zhi et al., 2016), as well as various NDM-type carbapenemase genes, such asblaNDM-1,blaNDM-9, andblaNDM-4(Liu et al., 2017; Oueslati et al., 2021).However,there are very few reports ofblaNDM-5-carrying IncHI2 (Ma et al., 2021; Zhao et al., 2021b).Consequently, we downloaded all available complete genomes (n=5 974; as of 1 September 2021) ofEnterobacteriaceaesubmitted to the NCBI assembly database (https://www.ncbi.nlm.nih.gov/assembly/) and found only fiveblaNDM-5-carrying IncHI2 plasmids (four from ducks and one from swine), all of which were from Guangdong,China.Although we could not trace the location of the grass carp farms and investigate the contamination source of theblaNDM-5-positiveEnterobacteriaceae, it is worth noting that the detection rate of theblaNDMgene in duck samples from Guangdong is high (>30%) (Wang et al., 2021b) and integrated duck-fish farming is very common in Guangdong(Shen et al., 2020).In the duck-fish farm model, duck feces are discharged without treatment, and a large number of ARGs or residual agents can directly contaminate the fish ponds, promoting the transmission of ARGs between ducks and fish.Thus, considering the high similarity of theblaNDM-5-bearing IncHI2 plasmids in the fish and ducks, and that theblaNDM-5-bearing IncHI2 plasmid is currently only found in Guangdong, we speculate that theblaNDM-5-bearing IncHI2 plasmids found inEnterobacteriaceaefrom retail fish may have been derived from duck feces-contaminated fish ponds in Guangdong.As such, greater attention should be paid to the transfer risk of antimicrobial resistant bacteria in integrated duck-fish farming.

    Here, pHNTH9F11-1 (IncHI2-IncF) was identified as a hybrid plasmid, formed by homologous recombination through ΔTn1721.In gram-negative bacteria, the fusion of plasmids mediated by insertion sequences, such as IS26, is rather universal, leading to a plasmid that can encode multiple resistance and hypervirulence genes, thereby posing a considerable threat to human health; for example, the cointegration event mediated by IS26between theblaNDM-5-bearing IncX3 plasmid andblaCMY-2-bearing IncA/C plasmid (Li et al., 2020).Moreover, the fusion of plasmids can expand the number of replicons and host range of plasmids, accelerating the dissemination of ARGs among various bacterial species(Dolejska et al., 2014; Wong et al., 2017).Of note, this fusion can also enable a non-conjugative plasmid to acquire conjugation ability, thereby facilitating the transmission of resistance genes, e.g., the recombination of non-conjugativemcr-1-carrying P7 phage-like plasmid pD72-mcr1 and conjugative F33:A-:B- plasmid pD72-F33 mediated by IS26,forming cointegrate plasmid pD72C with a conjugation frequency of 8×10-3cells/donor (He et al., 2019).Hence, the cointegrate plasmid pHNTH9F11-1 with multidrug resistance,heavy metal resistance, and phage resistance system (ability to resist invasion of bacteriophages) may provide an advantage for the host to survive in the environment.

    Composite transposons can mediate the jump of ARGs between different DNA molecules.The novel Tn7051and Tn7051-like transposons can both be moved by a copy-out-paste-in mechanism utilizing a double-stranded circular DNA intermediate (Yao et al., 2020; Zhao et al., 2021b), thereby contributing to the transfer of theblaNDM-5gene and expanding its transmission vectors.It has been widely reported thatblaNDM-5genes are mainly located on narrow-host-range plasmids (e.g., IncX3, IncF, and IncB/O) (Wu et al., 2019).However, the transfer ofblaNDM-5to the IncHI2 plasmid mediated by Tn7051and to the IncHI1-IncY-IncFIA-IncFIB plasmid mediated by Tn7051-like suggested that these transposons may further accelerate the horizontal spread of theblaNDM-5gene to various strains and plasmids, like Tn3000and Tn125, which mediate the between-plasmid jumps ofblaNDM-1and accelerate the transfer ofblaNDM-1in different strains (Acman et al., 2021).

    CONCLUSIONS

    This study revealed the emergence ofblaNDM-5inEnterobacteriaceaeof fish origin in China.To the best of our knowledge, this is the first report of theblaNDM-5gene, as well asblaNDM-5-bearing plasmids, in isolates from fish products in China.Our findings indicated thatblaNDM-5in the IncHI2 plasmids may originate from the IncX3 plasmid, transferred by the novel composite transposon Tn7051.Furthermore, theblaNDM-5-bearing IncHI2 plasmid may be transmitted from ducks, considering the common duck-fish freshwater aquaculture system in Guangdong.Based on the concept of“One health”, the surveillance of antibiotic resistance in aquatic products should be strengthened, and more measures should be taken to reduce the transfer of clinically important resistant bacteria, such as CPE, between food-producing animals and animal products.

    DATA AVAILABILITY

    The datasets in this study can be found in NCBI under BioProjectID PRJNA636005.The raw sequence data reported in this paper have been deposited in the Genome Sequence Archive (Genomics, Proteomics & Bioinformatics 2021) in the National Genomics Data Center (Nucleic Acids Res 2021),China National Center for Bioinformation/Beijing Institute of Genomics, Chinese Academy of Sciences (GSA:CRA005844), publicly accessible at https://ngdc.cncb.ac.cn/gsa.

    COMPETING INTERESTS

    The authors declare that they have no competing interests.

    AUTHORS’ CONTRIBUTIONS

    J.H.L.and L.C.L.conceived the research.X.G., Y.Y.L.,M.Y.G., K.B.M., W.Y.H., and L.C.L.collected the data.L.C.L.,J.H.L., Y.Y.L., X.G., and W.Y.H.analyzed and interpreted the data.Y.Y.L.and L.C.L.drafted the manuscript, J.H.L., W.Y.H.,and X.G.revised the report.All authors read and approved the final version of the manuscript.

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