Chromosomal Integration of Huge and Complex bla NDM-Carrying Genetic Elements in Enterobacteriaceae

In this study, a detailed genetic dissection of the huge and complex bla NDM-carrying genetic elements and their related mobile genetic elements was performed in Enterobacteriaceae. An extensive comparison was applied to 12 chromosomal genetic elements, including six sequenced in this study and the other six from GenBank. These 12 genetic elements were divided into five groups: a novel IME Tn6588; two related IMEs Tn6523 (SGI1) and Tn6589; four related ICEs Tn6512 (R391), Tn6575 (ICEPvuChnBC22), Tn6576, and Tn6577; Tn7 and its derivatives Tn6726 and 40.7-kb Tn7-related element; and two related IMEs Tn6591 (GIsul2) and Tn6590. At least 51 resistance genes, involved in resistance to 18 different categories of antibiotics and heavy metals, were found in these 12 genetic elements. Notably, Tn6576 carried another ICE Tn6582. In particular, the six bla NDM-carrying genetic elements Tn6588, Tn6589, Tn6575, Tn6576, Tn6726, and 40.7-kb Tn7-related element contained large accessory multidrug resistance (MDR) regions, each of which had a very complex mosaic structure that comprised intact or residual mobile genetic elements including insertion sequences, unit or composite transposons, integrons, and putative resistance units. Core bla NDM genetic environments manifested as four different Tn125 derivatives and, notably, two or more copies of relevant Tn125 derivatives were found in each of Tn6576, Tn6588, Tn6589, and 40.7-kb Tn7-related element. The huge and complex bla NDM-carrying genetic elements were assembled from complex transposition and homolog recombination. Firstly identified were eight novel mobile elements, including three ICEs Tn6576, Tn6577, and Tn6582, two IMEs, Tn6588 and Tn6589, two composite transposons Tn6580a and Tn6580b, and one integron In1718.


INTRODUCTION
New Delhi metallo-b-lactamase (NDM) is able to hydrolyze nearly all b-lactams except aztreonam and thus mediates resistance to penicillins, cephalosporins, and carbapenems (Yong et al., 2009). It is hypothesized that the bla NDM gene is originally integrated into the Acinetobacter chromosome from an unknown environmental species and then captured by two copies of ISAba125, giving rise to ISAba125-composite transposon Tn125 (Poirel et al., 2012). With the transposition of Tn125, bla NDM is disseminated among Acinetobacter, Enterobacteriaceae and Pseudomonas species; Tn125 and its bla NDM -carrying derivatives, with various truncations and deletions, can be found in the accessory resistance regions of bacterial plasmids or chromosomes (Wu et al., 2019). There are reports of chromosomal location of bla N D M in Enterobacteriaceae species including Escherichia coli (Pfeifer et al., 2011;Poirel et al., 2011;Shen et al., 2017;Reynolds et al., 2019), Providencia stuartii , Proteus mirabilis (Girlich et al., 2015), Klebsiella pneumoniae (Sakamoto et al., 2018), and Proteus vulgaris (Kong et al., 2020), but few of them are subjective to detailed genetic dissection of bla NDM -carrying accessory resistance regions (Girlich et al., 2015;Sakamoto et al., 2018;Reynolds et al., 2019;Kong et al., 2020).
Integrative and conjugative elements (ICEs) and integrative and mobilizable elements (IMEs) (Bellanger et al., 2014;Delavat et al., 2017;Botelho and Schulenburg, 2021) are two different types of mobile genetic elements which are frequently integrated into bacterial chromosome, contributing to dissemination of resistance genes. ICEs have the ability to transfer between cells because of their self-encoded conjugation function. It is typically composed of attL (attachment site at the left end), int (integrase), xis (excisionase), rlx (relaxase), oriT (origin of conjugative replication), cpl (coupling protein), a P (TivB)-or F (TivF)type T4SS machinery (mating pair formation), and attR (attachment site at the right end). IMEs are not selftransmissible, and they achieve the intercellular mobility with the help of other conjugative elements that encode proteins involved in complete conjugation function. IMEs typically have attL, int, rlx, oriT, and attR, but contained no conjugal transfer genes. Tn7 is a unit transposon with the ability to integrate into bacterial chromosomes and plasmids, and it encodes five core transposition determinants TnsA and TnsB (transposases), TnsC (regulator), and TnsD and TnsE (DNA-binding proteins), as well as three TnsB-binding sites and four TnsB-binding sites at its left and right ends, respectively (Peters, 2014).
In this work, whole-genome sequencing identified four bla NDM-1/-3 -carrying genetic elements plus two additional genetic elements harboring other resistance genes in the chromosomes of four isolates of Providencia rettgeri, Proteus mirabilis, and K. pneumoniae. An extension sequence comparison was then applied to a collection of 12 chromosomal genetic elements (including the above six ones sequenced in this work) that could be grouped into ICEs, IMEs, and Tn7 unit transposon and its derivatives. Data presented here gave a detailed genetic dissection of the huge and complex bla NDM -carrying genetic elements and their related mobile genetic elements in multiple Enterobacteriaceae species.

Bacterial Strains
The four chromosomal bla NDM -carrying isolates (Table S1) were screened from more than two hundred bla NDM -carrying Enterobacteriaceae isolates routinely collected from China hospitals and livestock farms. Providencia rettgeri 1701091 and Proteus mirabilis 1701092 (Table S1) were recovered in 2017 from the chicken intestinal contents in two different China livestock farms. K. pneumoniae QD23 and Providencia rettgeri 51003 were recovered from the urine specimens of two different patients with nosocomial infections in two Chinese public hospitals in 2015 and 2017, respectively. Bacterial species identification was performed using genome sequence-based average nucleotide identity analysis (http://www.ezbiocloud. net/tools/ani) (Richter and Rossello-Moŕa, 2009).

Sequencing and Sequence Assembly
Bacterial genomic DNA was isolated using the UltraClean Microbial Kit (Qiagen, NW, Germany) and sequenced from a sheared DNA library with average size of 15 kb (ranged from 10 to 20 kb) on a PacBio RSII sequencer (Pacific Biosciences, CA, USA), as well as a paired-end library with an average insert size of 350 bp (ranged from 150 to 600 kb) on a HiSeq sequencer (Illumina, CA, USA). The paired-end short Illumina reads were used to correct the long PacBio reads utilizing proovread (Hackl et al., 2014), and then the corrected PacBio reads were assembled de novo utilizing SMARTdenovo (https://github.com/ ruanjue/smartdenovo).

Conjugal Transfer
Conjugal transfer experiments were carried out with rifampinresistant Escherichia coli EC600 or sodium azide-resistant E. coli J53 being used as a recipient, and the 1701092 or QD23 isolate as a donor. Three milliliters of overnight cultures of each of donor and recipient bacteria were mixed together, harvested and resuspended in 80 ml of Brain Heart Infusion (BHI) broth (BD Biosciences). The mixture was spotted on a 1 cm 2 hydrophilic nylon membrane filter with a 0.45 µm pore size (Millipore) that was placed on BHI agar (BD Biosciences) plate and then incubated for mating at 37°C for 12 to 18 h. Bacteria were washed from filter membrane and spotted on Muller-Hinton (MH) agar (BD Biosciences) plates, for selecting an E. coli transconjugant carrying bla NDM or carrying tetA(C). Then 200 mg/L sodium azide (for J53) or 1,000 mg/L rifampin (for EC600), together with 4 mg/L imipenem (for bla NDM ) or 8 mg/L tetracycline [for tetA(C)] was used for transconjugant selection.

PCR Identification
All the wild-type and transconjugant strains was subjected to PCR amplification followed by amplicon sequencing, for determining the sequences of bacterial 16S rRNA genes (Frank et al., 2008), the presence of key markers such as bla NDM , tetA (C), int, and parM, and also the location/boundary of mobile genetic elements such as Tn6588, Tn6589, Tn6576, Tn6577, and Tn6590 (data not shown).

Phenotypic Assays
Activity of Ambler class A/B/D carbapenemases in bacterial cell extracts was determined by a modified CarbaNP test (Feng et al., 2016). Bacterial antimicrobial susceptibility was tested by BioMeŕieux VITEK 2 and interpreted as per the Clinical and Laboratory Standards Institute (CLSI) guidelines (CLSI, 2020).

Nucleotide Sequence Accession Numbers
The complete chromosome sequences of the 1701091, 1701092, QD23, and 51003 isolates were submitted to GenBank under accession numbers CP042860, CP042857, CP042858, and CP042861 respectively.

Genome Sequencing for Dissection of Chromosomal bla NDM -Carrying Genetic Elements
The complete genome sequences of four bla NDM -carrying isolates Providencia rettgeri 1701091, Proteus mirabilis 1701092, K. pneumoniae QD23, and Providencia rettgeri 51003 were determined in this work through high-throughput genome sequencing. A total of six chromosome-borne accessory resistance regions were identified: bla NDM-1/-3 -carrying Tn6588, Tn6589, Tn6576, and 40.7-kb Tn7-related element from strains 1701091, 1701092, QD23, and 51003, respectively; tetA(C)-and bla CTX-M-14 -carrying Tn6577 were from strain 1701092; and strAB-carrying Tn6590 was from strain 51003. A detailed sequence comparison was applied to a collection of 12 chromosomal genetic elements, which included the above mentioned six genetic elements sequenced in this study, together with six additional ones from GenBank (four reference/prototype ones Tn6523, Tn6512, Tn7, and Tn6591, and two bla NDM -carrying ones Tn6575 and Tn6726). These 12 genetic elements could be further divided into five distinct groups: a novel IME Tn6588; two related IMEs Tn6523 and Tn6589; four related ICEs Tn6512, Tn6575, Tn6576 and Tn6577; Tn7 and its two derivatives Tn6726 and 40.7-kb Tn7-related element; and two related IMEs Tn6591 and Tn6590 (Table 1). Six (Tn6588, Tn6589, Tn6575, Tn6576, Tn6726, and 40.7-kb Tn7-related element) of them harbored bla NDM . At least 51 resistance genes, involved in resistance to 18 different categories of antibiotics and heavy metals, were identified in these 12 elements ( Figure 1 and Table S2).

Two Related IMEs Tn6591 and Tn6590
The 15.5-kb IME Tn6591 (GIsul2) (Wei et al., 2003), initially found in Shigella flexneri 2457T, was integrated into the chromosomal gene guaA (glutamine-hydrolyzing GMP synthase) and had a 12.6-kb backbone (containing attL, int, oriT and attR) with insertion of a single accessory module ISCR2-sul2 unit ( Figure 11). Tn6590 (15.6 kb in length) was integrated at the same chromosomal site, and Tn6590 differed from Tn6591 by only truncation of ISCR2-sul2 unit due to insertion of strAB-carrying DTn5393c.

Plasmids of the Four Strains Sequenced in This Study
Proteus mirabilis 1701092 carried no plasmids, and all accessory resistance regions (Tn6577 and Tn6589) were located in the chromosome. Besides chromosome-borne accessory resistance regions, an IncFII plasmid p701091-FII (carrying no resistance genes), an IncI plasmid pQD23-CTXM [harboring bla CTX-M-104 and erm(B)], and an IncFII plasmid p51003-FII (containing bla TEM-1B and bla CTX-M-3 ) together with an Col3M plasmid p51003-qnrD (having qnrD) were identified in Providencia spp. 1701091, K. pneumoniae QD23, and Providencia spp. 51003, respectively. Coexistance of a large array of resistance genes in both chromosome and plasmids of a single bacterial isolate makes it tends to become extensively resistant.

Transferability and Antimicrobial Susceptibility
This work identified three ICEs Tn6577, Tn6582 and Tn6576 in total, all of which had essential conjugal transfer genes. Notably, Tn6582 was located within Tn6576. As for conjugation experiments, Tn6577 was transferred from the wild-type isolate (susceptible to rifampin) into E. coli EC600, generating the transconjugant Tn6577-TETA(C)-EC600; Tn6582 could be transferred from the wild-type isolate (non-susceptible to rifampin but susceptible to sodium azide) in E. coli J53 to obtain Tn6582-NDM-J53, but repeated conjugation attempts failed to transfer Tn6576 into E. coli J53. Tn6577-TETA(C)-EC600 was highly resistant to tetracycline and ceftriaxone owing to presence of tetA(C) and bla CTX-M-14 . Tn6582-NDM-J53 was highly resistant to ceftriaxone and imipenem resulted from production of NDM enzyme (data not shown). The Ambler class B carbapenemase activity was detected in Tn6582-NDM-J53 and its wild-type isolate.

DISCUSSION
Since the bla NDM gene was initially identified in India in 2009 (Yong et al., 2009), it spread rapidly all over the world (Dortet et al., 2014). Although bla NDM was initially discovered in a plasmid of K. pneumoniae (Yong et al., 2009), the chromosomal location of bla NDM in Enterobacteriaceae species has been reported in recent years (Pfeifer et al., 2011;Poirel et al., 2011;Girlich et al., 2015;Shen et al., 2017;Sakamoto et al., 2018;Reynolds et al., 2019;Kong et al., 2020). There were few reports related to a detailed genetic dissection of different kinds of bla N D M -carrying accessory resistance regions in the chromosomes (Girlich et al., 2015;Sakamoto et al., 2018;Reynolds et al., 2019;Kong et al., 2020), but none of them had a systematic summary for these bla NDM -carrying mobile genetic elements.
Data presented here involved a total of six chromosomal bla NDM -carrying genetic elements Tn6575, Tn6726, Tn6588, Tn6589, Tn6576, and 40.7-kb Tn7-related element, and the last four were sequenced in this work. These six genetic elements belonged to three different categories: ICEs (Tn6575 and Tn6576), IMEs (Tn6588 and Tn6589), and two derivatives (Tn6726 and 40.7-kb Tn7-related element) of Tn7 unit transposon. Notably, Tn6576 carried another ICE Tn6582. These ICEs and IMEs would have the intercellular selfmobility as they carried essential conjugal transfer genes (Bellanger et al., 2014;Botelho and Schulenburg, 2021).
Tn6726 would have the intracellular mobility as it had a complete core transposition module tnsABCDE, while 40.7-kb Tn7-related element would loss its mobility due to lesion in tnsABCDE.
Tn6512-related ICEs were frequently reported in Vibrio, Proteus, and Shewanella (Burrus et al., 2006;Nonaka et al., 2012;Lei et al., 2016;Fang et al., 2018). Tn6575 and Tn6576 were the only two bla NDM -carrying Tn6512-related ICEs (last accessed 15 December 2019). Tn6523-related IMEs were frequently reported in Salmonella and Proteus mirabilis (Hall, 2010;Siebor and Neuwirth, 2013;Sung et al., 2017). This study presented Tn6589, the first bla NDM -carrying Tn6523-related IME. Tn7, and its derivatives had the ability to integrate into FIGURE 7 | Comparison of Tn6580a from Tn6577 and Tn6580b from Tn6576, and related regions. Genes are denoted by arrows. Genes, mobile elements, and other features are colored based on their functional classification. Shading denotes regions of homology (nucleotide identity ≥95%). Numbers in brackets indicate nucleotide positions within the chromosomes of strains 1701092 and QD23. The accession numbers of Tn6503a (Feng et al., 2015) and Tn6309  used as reference are KP987215 and KX710094, respectively. bacterial plasmids and chromosomes (Peters, 2014). There were several reports of Tn7 derivatives located in bacterial chromosomes (Chen et al., 2018;Chen et al., 2019). To date, Tn6726 and 40.7-kb Tn7-related element were the only two bla NDM -carrying Tn7 derivatives integrated into chromosomes. Different to 40.7-kb Tn7-related element, Tn6726 carried a series of backbone genes of IncHI3 plasmid, which means that bla NDM together with its surrounding genetic environment in Tn6726 might be originated from a IncHI3 plasmid. In summary, Tn6512-related ICEs, Tn6523-related IMEs, and Tn7 derivatives recently began to be a reservoir of bla NDM genes in Enterobacteriaceae. Each of these six bla NDM -carrying genetic elements had large accessory resistance regions: i) Tn6575, Tn6588, Tn6589, and 40.7kb Tn7-related element; each had a single MDR region, 85.0 kb, 90.1 kb, 66.4 kb, and 33.8 kb in length, respectively; ii) Tn6726 contained a 162.6-kb ISKpn26-composite transposon Tn6728 integrated with a 40.9-kb MDR region; and iii) Tn6576 harbored a 406.4-kb ICE Tn6582 (containing two distinct MDR-1 and MDR-2 regions, 38.9 kb and 43.7 kb in length, respectively), and additionally a 55.2-kb ISPpu12-composite Tn6580b that as a whole could be considered as a MDR region. Each of these large MDR regions had a very complex mosaic structure, which was composed of intact or residue mobile genetic elements including ISs, unit or composite transposons, integrons and putative resistance units, and likely assembled from complex transposition and homologous recombination.
Multiple copies of bla NDM located in a single plasmid or chromosome were reported in previous studies (Jovcićet al., 2013;Shen et al., 2017;Feng et al., 2018), and all these bla NDM genes were around ISCR1. Similarly, Tn6576, Tn6588, Tn6589, and 40.7-kb Tn7-related element in this study also contained ISCR1around bla NDM genes. It was confirmed that ISCR1 captured adjacent genes (frequently including antibiotic resistance genes) at the end of its initiation of replication (oriIS) through rolling-circle transposition (Toleman et al., 2006). Our sequencing data suggested that ISCR1 might experience multiple rounds of capturing bla NDM and further integrating bla NDM into the integrons, resulting in the presence of multiple copies of ISCR1-accociated bla NDM genes in a single genetic element.
There were eight novel (firstly identified in this study) mobile genetic elements, including three ICEs Tn6576, Tn6577, and Tn6582, two IMEs Tn6588 and Tn6589, two composite transposons Tn6580a and Tn6580b, and one integron In1718. Additional 12 genetic elements (IME: Tn6590; composite FIGURE 9 | Organization of MDR region from Tn6728, and comparison to related regions. Genes are denoted by arrows. Genes, mobile elements, and other features are colored based on their functional classification. Shading denotes regions of homology (nucleotide identity ≥95%). Numbers in brackets indicate nucleotide positions within the chromosome of strain KP64. The accession numbers of Tn1548 (Galimand et al., 2005), Tn6909, and ISAba14-aphA6-ISAba14 unit used as reference are AF550415, CP032168, and CP046406, respectively.   transposons: Tn6578, Tn6581a, Tn6581b, Tn6581c, and Tn6728; unit transposons: Tn6726, Tn6727, Tn6909, and Tn6911; IS: ISPvu1; and 40.7-kb Tn7-related element) were newly designated (firstly designated in this study, but with previously determined sequences). The four previously designated ICEs/IMEs SGI1, R391, ICEPvuChnBC22, and GIsul2 were renamed as standard Tn designations Tn6523, Tn6512, Tn6575, and Tn6591, respectively. All the putative resistance units presented in this work were annotated and collected in a custom and yet unpublished database.

CONCLUSION
This study dealt with an extensive sequence comparison of 12 chromosomal genetic elements, including six bla NDM -carrying ones. All these bla NDM -carrying genetic elements had huge and complex MDR regions. The core bla NDM genetic environments manifested as four different Tn125 derivatives. Notably, two or more copies of bla NDM were found in each of the four genetic elements. Egiht novel mobile elements were firstly identified, including three ICEs Tn6576, Tn6577, and Tn6582, two IMEs Tn6588 and Tn6589, two composite transposons Tn6580a and Tn6580b, and one integron In1718. This study would provide a deeper genetic insight into the chromosomal integration of bla NDM -carrying genetic elements in Enterobacteriaceae.

DATA AVAILABILITY STATEMENT
The datasets generated for this study can be found in the complete chromosomal nucleotide sequences of 1701091, 1701092, QD23 and 51003, which were submitted to GenBank under accession numbers CP042860, CP042857, CP042858 and CP042861, respectively.

ETHICS STATEMENT
This study uses the bacterial isolates obtained from the Chinese livestock farm and public hospitals as listed in Table S1. The local legislation did not require the study to be reviewed or approved by an ethics committee, because the bacterial isolates involved in this study was part of the routine laboratory procedures. The research involving biohazards and all related procedures were approved by the Biosafety Committee of the Beijing Institute of Microbiology and Epidemiology.

AUTHOR CONTRIBUTIONS
DZ and HY conceived the study and designed experimental procedures. XL, YJ, and FC performed the experiments. XL, XJ, and LZ analyzed the data. LH, DW and YS contributed to reagents and materials. XL and ZY wrote the original draft. DZ and HY reviewed the manuscript. All authors contributed to the article and approved the submitted version.

FUNDING
This work was supported by the National Science and Technology Major Project (2018ZX10733402) of China, and the Foundation of the Public Welfare Program of Natural Science Foundation (LGF19H200006) of Zhejiang.