Characterization of a P1-bacteriophage-like plasmid (phage-plasmid) harbouring bla CTX-M-15 in Salmonella enterica serovar Typhi

Antimicrobial-resistance (AMR) genes can be transferred between microbial cells via horizontal gene transfer (HGT), which involves mobile and integrative elements such as plasmids, bacteriophages, transposons, integrons and pathogenicity islands. Bacteriophages are found in abundance in the microbial world, but their role in virulence and AMR has not fully been elucidated in the Enterobacterales . With short-read sequencing paving the way to systematic high-throughput AMR gene detection, long-read sequencing technologies now enable us to establish how such genes are structurally connected into meaningful genomic units, raising questions about how they might cooperate to achieve their biological function. Here, we describe a novel ~98 kbp circular P1-bacteriophage-like plasmid termed ph681355 isolated from a clinical Salmonella enterica serovar Typhi isolate. It carries bla CTX-M-15, an IncY plasmid replicon (repY gene) and the ISEcP1 mobile element and is, to our knowledge, the first reported P1-bacteriophage-like plasmid (phage-plasmid) in S . enterica Typhi. We compared ph681355 to two previously described phage-plasmids, pSJ46 from S . enterica serovar Indiana and pMCR-1-P3 from Escherichia coli , and found high nucleotide similarity across the backbone. However, we saw low ph681355 backbone similarity to plasmid p60006 associated with the extensively drug-resistant S . enterica Typhi outbreak isolate in Pakistan, providing evidence of an alternative route for bla CTX-M-15 transmission. Our discovery highlights the importance of utilizing long-read sequencing in interrogating bacterial genomic architecture to fully understand AMR mechanisms and their clinical relevance. It also raises questions regarding how widespread bacteriophage-mediated HGT might be, suggesting that the resulting genomic plasticity might be higher than previously thought.


INTRODUCTION
Salmonella enterica subspecies enterica serovar Typhi is the causative agent of typhoid fever and is associated with an estimated 11 million infections and 116 000 deaths globally each year [1]. The majority of this disease burden is concentrated in South Asia and other low to middle income countries (LMICs) [1].
Growing rates and spread of antimicrobial resistance (AMR) pose a threat to the effective empirical treatment and control of typhoid fever. Third-generation cephalosporins (extended spectrum β-lactams) are frequently used in the treatment of typhoid fever. The emergence and spread of extensively drug-resistant (XDR) S. enterica Typhi in Pakistan, and now globally, is a publichealth concern as it has left azithromycin (one of the last oral antibiotics available) and meropenem (carbapenem) as the only options available for treatment [2][3][4][5][6][7][8] (https://emergency.cdc.gov/han/2021/han00439.asp).
Resistance to cephalosporins is principally mediated by acquisition of a plasmid carrying extended-spectrum β-lactamase (ESBL) genes, the most prevalent of which are the CTX-M type ESBLs [9]. Recently bla CTX-M- 15 and bla CTX-M-55 present on a transmissible ~84 kbp IncY incompatibility group plasmid (p60006) were shown to be associated with extended-spectrum β-lactam resistance in XDR outbreak S. enterica Typhi isolates from Pakistan [2,3]. We are also now observing chromosomal integration of ESBL genes and the loss of the IncY plasmid in these XDR S. enterica Typhi isolates, demonstrating evolution of the AMR drug region throughout the outbreak [3].
Mobile and integrative genetic elements, including plasmids, bacteriophages, transposons, integrons and pathogenic islands, are important vehicles of horizontal gene transfer (HGT) enabling transmission of genetic information between bacteria [10]. Plasmids are considered the most common and important genetic element to spread ESBLs between bacterial strains, but studies have shown the mobilization or transfer of AMR genes by bacteriophages in various bacterial species, including Escherichia coli and non-typhoidal Salmonella (NTS) [11][12][13][14][15], and reported a mcr-1 gene in E. coli and a bla CTX-M-27 gene in NTS present on bacteriophage-like IncY elements (phage-plasmid) that were 97 and 104 kbp in size, respectively. In another report [13], a 115 kbp circular P1-like bacteriophage harbouring a bla SHV-12 element in E. coli was characterized. P1-like bacteriophages are known to replicate in their host as independent low copy number plasmid-like elements [13].
Recent advances in sequencing technologies, especially long-read sequencing, now enable us to identify and characterize novel bacteriophage/plasmid-like elements, as well as look at their genetic diversity [16][17][18]. Long-read sequencing has also enabled the ability to characterize the genetic architecture of individual mobile integrative elements harbouring AMR determinants [19][20][21][22][23], which is essential in order to characterize the precise biological mechanisms underpinning HGT mechanisms.
In this study, we describe a circular P1-bacteriophage-like plasmid (phage-plasmid) harbouring a bla CTX-M-15 gene and IncY plasmid replicon (repY) isolated from a clinical S. enterica Typhi isolate from a traveller returning to the UK from Iraq. To the best of our knowledge, this is the first time such a genomic element, and the implications of its presence towards AMR acquisition and maintenance, have been described for S. enterica Typhi. That is especially relevant due to the current heavy burden of the disease, and the pathogenic potential that S. enterica Typhi strains carrying AMR might present in the future.

Strain selection and details
A laboratory-confirmed S. enterica Typhi isolate termed 681 355 was referred to the Gastrointestinal Bacterial Reference Unit (GBRU), UK Health Security Agency (UKHSA) [formally Public Health England (PHE)] in January 2019. This isolate was from a traveller returning to the UK from Iraq. Epidemiological information and phylogenetic analysis of this isolate have previously

Impact Statement
Whole-genome sequencing has revolutionized the way we identify and characterize antimicrobial-resistance (AMR) genes and regions/elements in pathogens. Illumina sequencing coupled with Nanopore sequencing and careful data curation allows the mining of pathogen genomes to detect, characterize and track novel mobile elements involved in AMR transmission. In this study, we have identified a circular P1-bacteriophage-like plasmid (termed phage-plasmid) harbouring a bla CTX-M-15 gene conferring extended-spectrum β-lactamase resistance in Salmonella enterica serovar Typhi. It is the first time, to our knowledge, that such a DNA element has been described in this organism. There is increasing evidence from the literature to show that the horizontal spread of AMR genes mediated by bacteriophages and bacteriophage-like plasmid elements is much more common than previously envisioned. This current study shows the potential ability of using Nanopore sequencing for the detection and characterization of these elements, highlighting the importance of including long-read sequence data for the screening and surveillance of mechanisms involved in AMR transmission. Understanding AMR carriage and transmission patterns provides information to support appropriate clinical management and inform implementation of public-health control measures.
been described by Godbole et al. [24]. Ethical approval for the detection of gastrointestinal bacterial pathogens from faecal specimens, or the identification, characterization and typing of cultures of gastrointestinal pathogens, submitted to GBRU is not required as it is covered by UKHSA's surveillance mandate.

Antimicrobial-susceptibility testing
Antimicrobial-susceptibility testing was performed on this isolate as described by Chattaway et al. [25]. Minimum inhibitory concentrations (MICs) were determined by agar dilution using Mueller-Hinton agar for the standard panel of antibiotics recommended for Salmonella spp. by the European Committee on Antimicrobial Susceptibility Testing (EUCAST); breakpoints and screening concentration criteria were used for interpretation of results as described by EUCAST (2020; https://www.eucast.org/).

DNA extraction, library preparation, Illumina sequencing and data processing
Genomic DNA was extracted from S. enterica Typhi culture using the QIAsymphony system (Qiagen). The sequencing library was prepared using the Nextera XP kit (Illumina) for sequencing on the HiSeq 2500 instrument (Illumina), run with the fast protocol. fastq reads were processed using Trimmomatic v0.27 [26] to remove bases with a Phred score of <30 from the leading and trailing ends, with reads <50 bp after quality trimming discarded.

Genotyping and in silico AMR typing
Sequence type (ST) and serovar were determined from reads using most (v1.0) as described by Tewolde et al. [27] and eBURST group (eBG) as described by Achtman et al. [28].

DNA extraction, library preparation, Nanopore sequencing and data processing
High-molecular mass DNA was extracted from S. enterica Typhi isolate 681 355 using the Fire Monkey HMW DNA extraction kit (RevoluGen) and DNA concentration was determined via Qubit (Thermofisher Scientific), as previously described [23]. Library preparation was performed using the rapid barcoding kit (SQK-RBK004) (Oxford Nanopore Technologies). The prepared library was loaded onto a FLO-MIN106 R9.4.1 flow cell (Oxford Nanopore Technologies) and sequenced using the MinION system (Oxford Nanopore Technologies) for 72 h.
Data produced in a raw FAST5 format was basecalled using Guppy v3.2.6 Fast model (Oxford Nanopore Technologies) into fastq format. Read de-multiplexing, quality control, trimming and filtering were completed as described elsewhere [35] with the only modification being bases=490 Mbp, to generate approximately 100× coverage of a Salmonella genome (approximately 4.9 Mbp).

In silico plasmid typing and comparison of ph681355 and replicon to publicly available sequences
The plasmid replicon was identified for each non-chromosomal contig within the final assembly using PlasmidFinder v2.1 [43] with the Enterobacteriaceae, minimum identity=90 % and minimum coverage=90 % parameters set. brig [44] was used to compare ph681355 to the bla CTX-M-27 S. enterica serovar Indiana Chinese SJ46 phage-plasmid (GenBank accession no. NC_031129), E. coli bacteriophage P1 (accession no. AF234172), the mcr-1 E. coli phage-plasmid (accession no. KX880944) and plasmid p60006 (accession no. LT906492) isolated from a Pakistan XDR S. enterica Typhi outbreak isolate that also harboured bla CTX-M-15 . Parameters used included -perc_identity=90 and -e value=1×10-10. The coding sequences (CDSs) were annotated using Prokka v1.13 [42] as stated in the previous section, with AF234172 acting as a reference for CDS and gene annotation. The repY genes from the above plasmids were compared to the repY gene from ph681355 also using blastn [45].

Detection and characterization of ph681355 structural variation
To determine whether multiple isoforms were present within the Nanopore reads, the Nanopore fastq reads for the sample were aligned to the finalized assembly using Minimap2 v2.17 [46] and SAMtools v0.7.17 [30]. The alignment was visualized using Integrative Genomics Viewer (igv) v2.12.3 [47] and the breakpoints of each isoform were identified. Once breakpoints were identified relative to each isoform, those positions were used with SAMtools v0.7.17 [30] to isolate reads that aligned and spanned across both ends of each breakpoint (i.e. spanned the homologous region in question). Any reads that aligned across a given set of breakpoints had to share the same size as it existed in the fastq file, and not be clipped within the alignment, to be considered. From here, the relative proportions of reads aligning to each isoform were calculated.

Data deposition
Illumina and Nanopore fastq files and polished assembly for S. enterica Typhi isolate 681 355 are available from the NCBI under BioProject PRJNA248792. The SRA (sequence read archive) accession numbers are as follows: Illumina fastq -SRR8554071; Nanopore fastq -SRR16296518. The GenBank accession numbers are CP083411 for the chromosome and CP083412 for ph681355 (phage-plasmid).

Sample 681355 genome statistics and genotyping
Isolate 681 355 was confirmed to be S. enterica Typhi ST1, a member of serovar Typhi eBURST group 13 (eBG13). Previous phylogenetic analysis confirmed that this strain sat within the dominant global H58 haplotype, but it did not cluster with the recent XDR S. enterica Typhi outbreak strains in Pakistan [2,24]. Nanopore sequencing and processing produced a final genome of two contigs, one chromosome (4 782 729 kbp) and one of 98 174 kbp (ph681355).
There was only a 4.9 % sequence similarity between ~98 kbp phage-plasmid ph681355 and the ~84 kbp plasmid p60006 (LT906492), although the repY plasmid replicon gene and ISEcpl-bla CTX-M-15 -tnpA resistance cassette were present in both (Fig. 1). Our analysis shows that the ESBL resistance is carried by the same 3.5 kbp ISEcpl-bla CTX-M-15 -tnpA resistance gene cassette but that different mechanisms are involved in the transmission of ESBL resistance.

Detection and characterization of structural variation on ph681355
When confirming the validity of the ph681355 contig, it was noted that some Nanopore reads were clipped in the alignment at the same loci as the mobile genetic element in which the bla CTX-M-15 gene is located (Fig. 2). Artificial (in silico) removal of the bla CTX-M-15 mobile genetic element also showed Nanopore reads aligning across this region with the absence of clipping (Fig. 2). This suggests that there are two isoforms of the ph681355 within the single Nanopore read set, one set (approximately 40 % of reads) confirming the presence of the mobile genetic element within ph681355 and a second set (approximately 60 %) aligning correctly (with no clipping) suggesting the absence of the mobile genetic element from ph681355.

DISCUSSION
The transmission of ESBL resistance is increasing in Enterobacterales mainly due to the presence of bla CTX-M and bla SHV class genes [3, 13-15, 48, 49], and highlighted by the recent XDR S. enterica Typhi outbreak isolates in Pakistan that have subsequently spread globally [2,3,25,50]. The bla CTX-M-15 gene responsible for ESBL resistance was present on a ~84 kbp IncY plasmid (p60006). Nair et al. [3] showed the rapid evolution of bla CTX-M-15 resistance mechanisms with the loss of the plasmid and the integration of bla CTX-M-15 into various regions of the chromosome of XDR S. enterica Typhi isolates.
In this study, a S. enterica Typhi isolate (681355) from a patient returning from Iraq had a bla CTX-M-15 gene present on an extrachromosomal element harbouring a plasmid replication repY gene. Phylogenetic analysis conducted by Godbole et al. [24] showed that this Iraqi S. enterica Typhi strain belonged to the global H58 haplotype but did not cluster with the XDR S. enterica Typhi outbreak Pakistani isolates [2].
Multiple modes for the rapid transmission of AMR and virulence genes involving mobile genetic elements have been widely described [51,52]. More recently, studies have used long-read sequence data to demonstrate the involvement of P1-and P7-like bacteriophage elements in AMR gene transfer [13][14][15]. These bacteriophage-like elements (phage-plasmid) have both the bacteriophage-like lytic replication (repL) and plasmid replication (rep) genes, which we observed in ph681355 [13][14][15] (Fig. 1).
However, a phage-plasmid had only been described once in S. enterica serovar Indiana [14] and, hence, to our knowledge ph681355 is the first one described in S. enterica Typhi. ph681355 is a chimeric molecule that consists of a bacteriophage backbone, a plasmid replicon (rep gene), a drug-resistance region(s)/gene(s) (e.g. a ISEcpl-bla CTX-M-15 -tnpA gene cassette) and a lysogenized P1 bacteriophage sequence [49] that have resulted from recombination of integrative elements on plasmids and prophage (chromosomally integrated, lysogenized bacteriophages) genomes.
Our findings provide supporting increasing evidence of the role played by viral vectors in the vertical and horizontal transfer of AMR genes and mobile elements between bacteria [13][14][15]49]. Phage-plasmids of sizes between 90 and 120 kbp have been described in other Enterobacterales, such as Citrobacter, Enterobacter and Pantoea [17], and it has been suggested that the repY replicon in both plasmids and phage-plasmids is associated with strains harbouring ESBL genes [13,53]. However, the bla CTX-M-27 gene in the S. enterica serovar Indiana isolate [14] was present on a 104 kbp repA-like phage-plasmid and a bla CTX-M-15 gene was present on a 94 kbp IncF1A phage-plasmid in Klebsiella pneumoniae [49]. Phage-plasmids of different compatibility groups can be involved in the transmission of additional resistance determinants such as colistin (mcr-1) [15].
Nanopore sequencing provided extra context, revealing two isoforms of the same phage-plasmid structure, with and without a bla CTX-M-15 mobile genetic element (Fig. 2). Our observation suggests an ongoing process, whereby AMR genes can be dynamically acquired and lost depending on the evolutionary pressures surrounding the phage-plasmid and its host.
We are beginning to detect previously undescribed elements of AMR transmission, such as phage-plasmids, due to our ability to assemble complete genomes via long-read sequencing. Understanding the structure of such genomic elements is essential to fully elucidate the biological mechanisms of AMR and their clinical relevance. For instance, with long-read sequencing data we are now able to detect structural variants within the reads of a single bacterial culture [54]. This is showcased by our observation that in a proportion of the culture the bla CTX-M-15 resistance cassette is missing from the phage-plasmid (Fig. 2).
However, technical difficulties arise from genome assembly, which at times requires manual curation to ensure the validity of these assemblies for downstream analyses [37,55,56]. The same is true for accurate prophage and phage-plasmid detection and annotation directly from assemblies. Even from the abundant short-read sequence data (e.g. Illumina) and rapidly increasing long-read data, prophages and phage-plasmids sequences can often be missed [56,57]. This is exacerbated by the number of in silico bacteriophage detection tools, which produce differing results from methodologies varying from reference-based detection through to machine-learning approaches [56,[58][59][60][61][62][63].
In conclusion, this study demonstrates the use of modern sequencing and data curation techniques in the successful detection, characterization and tracking of novel mobile elements involved in AMR transmission. We show the potential role of phageplasmids in the capture and spread of ESBL resistance genes in S. enterica Typhi, in agreement with an increasing body of evidence showing the importance of horizontal spread of AMR genes mediated by bacteriophages and phage-plasmids [13]. Our findings also demonstrate the utility of long-read sequencing for non-typeable plasmids and phage-plasmids in detection and surveillance procedures once adequate sequencing capabilities become available. Future work will include the screening of gastrointestinal bacterial pathogens at UKHSA for the presence of repL and other relevant genes to detect the involvement of phage-plasmids in the transmission of AMR genes.

Conflicts of interest
The authors declare there are no conflicts of interest.