Chromosomal integration of blaCTX-M genes in diverse Escherichia coli isolates recovered from river water in Japan

Highlights • E. coli isolates with blaCTX-M genes from river water were characterized.• Fourteen E. coli genomes were completed.• Many of the blaCTX-M genes were located on the chromosome.• The genetic contexts and integration sites of blaCTX-M genes were highly diverse.


Introduction
Occurrence of antibiotic-resistant bacteria (ARB) in surface waters is a public health concern. Among ARB, extended-spectrum-β-lactamase (ESBL)-producing Escherichia coli (ESBLEC) represent a major threat to public health, because ESBLEC can be important causes of communityonset infections for which treatment options are limited (Pitout, 2010;Pitout and Laupland, 2008). ESBL enzymes are divided into several families (e.g., CTX-M and ESBL variants of TEM and SHV), among which CTX-M enzymes have been recognized as the most common ESBL type (Castanheira et al., 2021). Previous studies have reported the predominance of CTX-M ESBLs among environmental E. coli. For example, CTX-M-14(-like) was prevalent among ESBLEC in surface waters in China, Japan, and Korea, and CTX-M-15 was prevalent among ESBLEC in surface waters in France, Germany, and Switzerland (Falgenhauer et al., 2021;Girlich et al., 2020;Jang et al., 2013;Liu et al., 2018;Miyagi and Hirai, 2019;Zurfluh et al., 2013).
ESBL genes are often found on plasmids, which facilitates their horizontal spread (Castanheira et al., 2021). However, some previous studies also detected ESBL genes on chromosomes and suggested that integration of ESBL genes into chromosomes could contribute to the stabilization and maintenance of ESBL genes in the absence of antibiotic selection pressure (Hirai et al., 2013;Nair et al., 2021;Rodriguez et al., 2014). Thus, information on the location (chromosome or plasmid) of ESBL genes is important to predict the mobility and stability of these genes. Although, previous studies have reported the occurrence of ESBLEC in environmental waters, limited information exists regarding the locations and genetic contexts of ESBL genes in environmental ESBLEC (Falgenhauer et al., 2021;Girlich et al., 2020;Liu et al., 2018). Here, we completely sequenced the genomes of CTX-M-producing E. coli isolates recovered from river water in Japan, aiming to elucidate the locations and genetic contexts of the bla CTX-M genes in environmental ESBLEC. The genetic contexts of chromosomal bla CTX-M genes in the genomes of environmental E. coli retrieved from the NCBI Reference Sequence Database (RefSeq) were also characterized.

Bacterial isolates
We previously collected 531 E. coli isolates from the Yamato River in Japan between 2011 and 2013 and detected 18 isolates with bla CTX-M Fig. 1. Genetic contexts of chromosomal bla CTX-M genes. Red arrows indicate antibiotic resistance genes, gray arrows indicate mobile elements, and white arrows indicate other genes. The blue shaded box indicates the regions with 100% nucleotide identity. The entire resistance regions of KKa019 and KOr019 are shown in Fig. 2. 5-bp TSD are shown next to each TPU. hp: hypothetical protein.
genes (Gomi et al., 2014;Gomi et al., 2017). Briefly, we collected a total of 27 water samples from 10 sites in the Yamato River in August and November of 2011, February of 2012, and October of 2013 (note that water samples were not collected from all sampling sites on each sampling occasion). The samples were processed using the membrane filter method with XM-G agar (Nissui, Tokyo, Japan) to obtain E. coli isolates. In total, 531 E. coli isolates were obtained, and a subset of isolates (n = 155) was selected and subjected to Illumina short-read sequencing. Among the sequenced isolates, we detected 18 isolates that carried bla CTX-M genes. These 18 isolates were analyzed as described below.

Antibiotic susceptibility testing
Antibiotic susceptibility was assessed by microdilution using the dry plate Eiken assay (Eiken, Tokyo, Japan) as described previously (Gomi et al., 2017) (see Table S1 for the antimicrobial agents used). The results were interpreted according to CLSI criteria (CLSI, 2020) and EUCAST epidemiological cutoff (ECOFF) values (http://www.eucast.org). ESBL production was confirmed following the CLSI guidelines (CLSI, 2020). A detailed description of antibiotic susceptibility testing is provided in the Supplementary Materials and methods.

Genome sequencing and assembly
DNA was extracted from each isolate using a DNeasy PowerSoil Pro Kit (Qiagen, Hilden, Germany). The short-read sequencing library was prepared using the Illumina DNA Prep (Illumina, San Diego, CA), and the library was sequenced on an Illumina NovaSeq 6000 system with 250-bp paired-end sequencing (although we obtained Illumina reads for the isolates in our previous study (Gomi et al., 2017), the Illumina reads had low depth/uneven coverage and hence we sequenced the isolates again with Illumina technology in the present study). The long-read sequencing library was prepared using the SQK-LSK109 kit (Oxford Nanopore Technologies, Oxford, UK), and the prepared library was sequenced on the MinION with a FLO-MIN106 flow cell.

Characterization of environmental E. coli genomes with bla CTX-M genes in RefSeq
RefSeq E. coli genomes with an assembly level of "complete genome" (n = 1903) were downloaded in January 2022. Metadata information, such as collection date and isolation source, was extracted from the GenBank files using an in-house Python script, and genomes of E. coli from environmental waters were retrieved (n = 53). Antibiotic resistance genes were detected using ABRicate (https://github.com/tseemann/abricate), and genomes carrying bla CTX-M on the chromosome were analyzed as described above.

Accession numbers
The complete genomes and sequence read files were deposited in GenBank and the NCBI SRA under BioProject PRJNA800231 (also see Table S2 for the accession number of each replicon for the completed genomes and Table S1 for SRA accession numbers of the remaining genomes).
Of the 18 isolates, the genomes of 14 could be completed. These genomes carried 0 to 8 plasmids (see Table S2 for details). The genomes of the remaining four isolates could not be completed, probably due to the presence of long repeats in the genomes. The location and genetic contexts of the bla CTX-M genes were determined for the 14 isolates with completed genomes. Of these 14, nine carried bla CTX-M genes on chromosomes and five carried bla CTX-M genes on plasmids.

Genetic contexts of chromosomal bla CTX-M genes
In eight of the nine isolates which carried bla CTX-M genes on chromosomes, the bla CTX-M genes were associated with ISEcp1 and carried on transposition units (TPU) of different sizes (2,855 bp to 11,093 bp) (Fig. 1). These TPU were flanked by AT-rich 5-bp target site duplications (TSD). None of the TPU were identical, and the insertion sites of the TPU were different in all cases, indicating that different transposition events contributed to the chromosomal integration of each TPU. Notably, the TPU in KFu023 contained multiple resistance genes, including bla CTX-M-genes of different origins (Partridge et al., 2018). In most cases, these TPU were directly inserted into chromosomes. However, in KKa019, the TPU was part of a 35,625 bp multiresistance region bracketed by two copies of IS26 (annotated as IS15DI in the ISFinder database), and the whole multiresistance region was flanked by 8-bp TSD (CGTTGCCG) in the chromosome (Fig. 2a). KTa008 carried two copies of bla CTX-M-14 , which were separated by ~1,169 kbp in the chromosome. Analysis of the genetic contexts of these bla CTX-M-14 genes indicated transposition of an already chromosomally-located TPU and an adjacent region into another chromosomal location (Fig. S1). This type of 'recurrent' transposition was previously reported in clinical ESBLEC, though the TPU and insertion sites were different (Hamamoto and Hirai, 2019).
The above results indicate that analysis of sequences immediately upstream and downstream of bla CTX-M (e.g., identification of ISEcp1 upstream and orf477 downstream of bla CTX-M ) is not sufficient to track the movement and evolution of bla CTX-M , since TPU can be very long (sometimes > 10,000 bp) or can be inserted into a large multiresistance region. This can be overcome, for example, by determining the complete genomes as shown in the present study.
We searched for E. coli genomes in the public database which carry  the same TPU in the same chromosomal location as those identified in the present study. BLASTN searches were performed against the nucleotide collection (nr/nt) database in January 2022 using the TPU and surrounding sequences identified in the present study as query sequences. Three of the nine TPU/insertion site combinations were found in the database, while the remaining six were not found, potentially corresponding to novel integration events (Table S3). In KOr019, bla CTX-M-2 was associated with ISCR1 and situated within a complex class 1 integron, which is congruent with previous studies (Canton et al., 2012;Partridge et al., 2018). This complex class 1 integron was closely related to a recently reported transposon Tn7337 (Fig. 2b) (Coppola et al., 2022). KOr019 carried the IRt (inverted repeat at tni end)-IS6100-IRt structure, but this structure was truncated by IS26 in Tn7337. The 17,747 bp structure from IRi (inverted repeat at intI1 end) to IRt was inserted into the chromosome of KOr019, creating 5-bp TSD of AAATG. This insertion seems to have been mediated by Tni proteins provided in trans, since the tni genes in the complex class 1 integron were absent (Partridge et al., 2018).

Genetic contexts of bla CTX-M genes on plasmids
The five completed plasmids with bla CTX-M genes belonged to IncI1 (n = 4) and IncB/O/K/Z (n = 1) (Fig. 3a). IncI1 plasmids were typed as four different STs, namely ST3, ST16, ST113, and ST167, by IncI1 pMLST (Carattoli et al., 2014). TPU were inserted into plasmid backbones in the IncI1/ST3, IncI1/ST16, and IncI1/ST167 plasmids. Interestingly, the IncI1/ST16 and IncI1/ST167 plasmids carried the same TPU (except for insertion of IS1 in the IncI1/ST16 plasmid) inserted into the same location. IncI1/ST167 is a single-locus variant of IncI1/ST16, which indicates the TPU was inserted into the plasmid backbone before the ST diversification. In the IncI1/ST113 plasmid, an IS26-ΔIS10R-bla CTX-M-8 -IS26 structure was inserted into the plasmid. BLASTN analysis identified IncI1 plasmids belonging to the same IncI1-STs and carrying the same TPU/IS26 structures inserted into the same locations from different countries (e.g., MH847453 from France for bla CTX-M-1 -carrrying IncI1/ST3, CP056650 from the UK for bla CTX-M-55 -carrrying IncI1/ST16, KY964068 from Portugal for bla CTX-M-8 -carrrying IncI1/ST113, CP021208 from China for bla CTX-M-55 -carrrying IncI1/ST167), which indicates circulation of epidemic CTX-M IncI1 plasmids in geographically distant countries. Comparison of IncI1 plasmids revealed the replication region, the leading region, and the transfer regions in the IncI1 reference plasmid R64 were well conserved in all IncI1 plasmids detected in this study, while many genes in the stability region were lacking (Fig. 3b). In the IncB/O/K/Z plasmid, bla CTX-M-14 was linked to ΔTn1721 and a class 1 integron. Similar genetic contexts were previously reported (e.g., JF701188).

Chromosomal location of bla CTX-M in the RefSeq assemblies
Among the 53 RefSeq environmental E. coli genomes, 20 carried bla CTX-M . Sixteen carried bla CTX-M on plasmids and four carried bla CTX-M on chromosomes ( Table S4). None of the 20 RefSeq genomes with bla CTX-M belonged to ST131, again indicating a potential difference in clonal composition of ESBLEC in environmental settings and clinical settings. The four genomes with chromosomal bla CTX-M belonged to ST38 (n = 2), ST130 (n = 1) and ST648 (n = 1) and originated from various countries (Japan, New Zealand, Norway, and Switzerland), which indicates that diverse E. coli isolates carrying chromosomal bla CTX-M are also present in environmental waters in other countries. In all four genomes, bla CTX-M was associated with TPU ranging from 2,971 bp to 13,552 bp. A TPU containing multiple resistance genes besides bla CTX-M and a TPU inserted into a multiresistance region were detected, again highlighting the importance of these mechanisms in the emergence of multidrug resistant E. coli in the environment (see Fig. S2 for the analysis of the genetic contexts of bla CTX-M in these genomes).
ST38, which was detected in two RefSeq genomes containing chromosomal bla CTX-M , was also detected in two isolates with chromosomal bla CTX-M sequenced in this study. Interestingly, ST38 was also found to be prevalent in some previous studies reporting chromosomal bla CTX-M (Guenther et al., 2017;Guiral et al., 2011;Rodriguez et al., 2014). The reason for the prevalence of ST38 among E. coli with chromosomal bla CTX-M is unknown. However, multiple integration events seem to have been involved in the acquisition of chromosomal bla CTX-M in ST38 (see Fig. 1, Fig. 2a, and Fig. S2), which potentially indicates ST38 has genetic mechanisms that can promote chromosomal integration of bla CTX-M .

Study limitations
This study has some limitations. The E. coli isolates analyzed in the present study were collected between 2011 and 2013 (i.e., ~10 years ago). Additionally, a limited number of complete genomes were determined (n = 14), and the isolates were collected regionally, not nationwide. However, we also included RefSeq environmental E. coli genomes from various countries with various collection dates in our analysis, which might mitigate these limitations.

Conclusions
Here, we demonstrated that the genetic contexts of bla CTX-M genes in environmental E. coli are highly diverse in terms of the associated mobile elements (various TPU, ISCR1, and IS26) and integration sites. This study also revealed that the chromosomal locations of bla CTX-M genes were unexpectedly frequent in environmental ESBLEC in Japan. Chromosomal integration of bla CTX-M might allow E. coli to stably maintain bla CTX-M in environmental waters, though further studies are needed to confirm this.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.