ES RESTAURADA LA SALVACIÓN PARA LOS MUERTOS

Alignment of the UK-ST313 genomes to the lineage 2 representative strain D23580 showed that some UK-ST313 strains carry prophages with mosaic homology to BTP1 and BTP5 (Figure 4.6). To reveal the true extent of homology between the BTP1 and BTP5-like prophages detected, prophages were manually extracted from a subset of 12 UK-ST313 draft genome assemblies that were representative of the phylogenetic diversity of all UK-ST313s. Draft genome assemblies consist of a number of short assembled fragments (contigs), which increase in length and frequency depending on the quality of the genome assembly. To extract prophages sequences, the contigs containing the phage attR and attL sites (proximal sites to either end of the prophage) were identified, and the sequence between these sites was determined to be a putative prophage. In some cases, it was not possible to extract prophage sequences, as the BTP1 or BTP5 attachment sites were separated across contigs. However, in most cases, the BTP1 or BTP5 attachment site was either intact on a single contig (indicating no prophage), or separated by a length corresponding to a putative prophage on a single contig (indicating the presence of a prophage occupying the attachment site). Additionally, to control for the possibility that the BTP1 and BTP5- like prophages in the UK-ST313 strains might have distinct attachment sites to BTP1 and BTP5, large contigs (>50 kb) were extracted which contained homology to BTP1 or BTP5, and contained core genome sequence (non-phage). Using this approach, 3 BTP1 or BTP5 like prophages were identified in a total of 10 of the 12 UK-ST313 genomes.

Two UK-ST313 strains, U15 and U8 contain a prophage with 71% identity to BTP1 at the nucleotide level (Figure 4.11A). This prophage, denoted BTP1UK-1_{, contains} almost identical structural genes to BTP1, but differs in the immunity, tail and gtr operon regions. The attachment site of BTP1UK-1 _{was the same as that of BTP1 (data} not shown).

Two UK-ST313 strains, U12 and U5 contain a prophage with 48.5% identity to BTP1 at the nucleotide level (Figure 4.11B). This prophage, denoted BTP1UK-2_{, contains} many of the same structural genes as BTP1, but has no similarity to the tailspike and gtr operon of BTP1. Interestingly, prophage BTPUK-2 _{carriesa repressor region with} considerable identity to the repressor region of BTP1 including the ST313-td gene, but contains a different cI repressor and cro allele to BTP1. Prophage BTP1UK-2 _was found to be > 99% identical to a temperature phage called SE1. Phage SE1 was

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originally isolated from a strain of S. Enteritidis, and mediates generalised transduction in S. Typhimurium (Llagostera et al., 1986). The attachment site of the prophage BTP1UK-2 _{(SE1) was the same as BTP1, as the core chromosomal sequence} bordering the prophage on this contig corresponds to the attachment site of the BTP1 prophage in D23580.

Finally, a prophage with 57% identity to BTP5 at the nucleotide level was found in 6 UK-ST313 strains (U11, U16, U7, U9, U10, U4) (Figure 4.12). This prophage, denoted BTP5UK-1_{, encoded identical capsid, tail and lysis genes to BTP5, but did not encode} genes homologous to the replication, immunity or integrase genes of BTP5. The BTP5 attachment site was intact in strains containing prophage BTP5UK-1_{. The attachment} site of the prophage was found to be between the CpxQ sRNA and the fieF gene (STMMW_40261), though neither element was interrupted by the integration of the prophage (data not shown).

The presence of the novel BTP1-like and BTP5-like prophages discovered in the UK- ST313 genomes did not correlate with the phylogeny (Figure 4.13) i.e. prophages were not associated with particular phylogenetic clusters, showing that the presence of these prophage is not under high selection pressure in the UK-ST313 niche. For example, BTP5UK-1 _{was present in UK-ST313 isolates spanning the diversity of the} tree, which may suggest that, though there is considerable genetic diversity amongst the UK-ST313 isolates in this study, they exist within the same ecological niche, and are therefore exposed to the same phage population.

151 Figure 4.11 Novel BTP1-like prophages identified in the genomes of UK-ST313 isolates. A. Prophage BTP1UK-1 _{was identified in UK-ST313 strains U15 and U8 and} shares 71% identity to prophage BTP1. B. Prophage BTP1UK-2 _{was identified in UK-} ST313 strains U12 and U5 and shared 48.5% identity with BTP1. Prophage BTP1UK-2 was found to be more than 99% identical to phage SE1 derived from Salmonella Enteritidis (inset). Comparative genomic analysis was conducted as described in 2.12.5.

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Figure 4.12 A novel BTP5-like prophage identified in the genomes of UK- ST313 isolates. A. Prophage BTP5UK-1 _{was identified in UK-ST313 strains U11,} U16, U7, U9, U10 and U4. BTP5UK-1 _{was 57% identical to BTP5. B. Prophage} BTP5UK-1 _{occupies a distinct attachment site to BTP5, between the CpxQ sRNA and} the fieE gene. Comparative genomic analysis was conducted as described in 2.12.5.

153 Figure 4.12 The phylogenetic distribution of

the BTP1-like and BTP-5 like prophages identified in the UK-ST313 genomes. The BTP1 & BTP5 attachment site are represented as grey and black un-filled squares. Presence of a prophage (indicated by a filled coloured circle), in an attachment site is indicated by the position of the circle inside the square. Circles outside of the attachment site boxes represent prophages occupying attachment sites distinct from that of BTP1 and BTP5. Prophages were identified by comparative genomic analysis as described in 2.12.5.

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4.8 Discussion

Recent reports of iNTS disease have been associated with Salmonella Typhimurium ST313 in sSA (Kingsley et al., 2009; Okoro et al., 2015) and suggested that ST313 was geographically restricted to sSA. This prompted investigation into the presence of ST313 amongst S. Typhimurium isolates from the UK.

It was discovered that 2.7% of Salmonella Typhimurium isolates referred to Public Health England are of MLST type ST313 and that this sequence type is heterogeneous in terms of clinical presentation, genomic characteristics and epidemiology. The UK-isolated ST313 strains are predominantly fully antimicrobial- susceptible and cause gastroenteritis. This was also the case with the recently described ST313 strains from Brazil (Almeida et al., 2017), raising the possibility that the Brazilian isolates are more closely related to the UK-ST313 reported here than to African lineage 2.

Significant correlation was identified between travel to Africa and infection with the previously described African-associated, ST313 lineage 2. The amount of diversity between the lineage 2 ST313 isolates from the UK (Figure 4.4) was not consistent with an immediate, common source of exposure. Instead, it is more probable that these isolates were linked to travel to Africa, a hypothesis that was supported by epidemiological data that linked the isolates to African travel (Table 4.1).

This study revealed novel diversity within ST313, which was previously restricted to two African lineages that had exhibited recent clonal expansion (Okoro et al., 2012). The African lineages are placed into an evolutionary context by showing that lineage 1 and 2 do not form a monophyletic group within ST313, which is suggestive of two separate introductions of ST313 into sSA. African lineages 1 and 2 diverged from their MRCA with UK-lineages around 1796 and 1903 respectively (4.3). These findings reflect the limitations of classifying bacterial pathogens simply on the basis of sequence type and show that in the post-genomic era, the resolution offered by MLST may not be sufficient to describe epidemiologically relevant population structures of Salmonella.

It has been estimated that 9.2% of cases of Salmonellosis in the EU can be attributed to international travel and therefore sequencing Salmonella isolated in Europe can provide valuable information regarding the global diversity of Salmonella associated with human disease (Guerin et al., 2007; Pires et al., 2014). The genome of one UK- isolated lineage 2 isolate associated with travel to Kenya, U60, contained sequences

155 with high nucleotide similarity to a recently described IncHI2 plasmid, pKST313, that was carried by ceftriaxone-resistant ST313 isolates from Kenya (Kariuki et al., 2015). Until now the blaCTX-M-15 gene has only been found to be plasmid-associated in Salmonella. A blaCTX-M-15 gene carried on the chromosome was discovered in UK- isolated lineage 2 strain U60, causing disruption of the ompD locus which has two implications. Firstly, chromosomal integration ensures stability of ESBL-resistance even in the absence of the plasmid. Secondly, ompD encodes an outer membrane porin of S. Typhimurium that is highly immunogenic (Gil-Cruz et al., 2009) and absent from S. Typhi. Accordingly, the disruption of ompD could enhance the reported “stealth” phenotype of ST313 lineage 2 infection (Carden et al., 2015). Notably, OmpD has been proposed as a vaccine target for iNTS (Ferreira et al., 2015) and the absence of OmpD from African ST313 populations could have implications for future iNTS vaccine development.

People infected with ST313 lineage 2 in the UK were significantly more likely to suffer from invasive disease than patients infected with UK-ST313 isolates, though the HIV / immunosuppression-status was not known for these patients. This observation provided an excellent opportunity to use comparative genomics to put genetic findings that have been linked the pathology of lineage 2 ST313 into the context of closely related, gastrointestinal-associated strains. The only genetic characteristics found to be common to both lineages 1 and 2 and absent from the UK-ST313 genomes were the BTP1 and BTP5 prophages and the plasmid-associated MDR loci. The two African lineages do not share a common ancestor that carried either prophage, suggesting independent acquisition of BTP1 and BTP5 by ST313 lineage 1 and 2. Whilst the MDR loci of lineage 1 and 2 confer similar patterns of AMR, they are genetically distinct indicating independent origins. The maintenance of the prophage and plasmid-encoded accessory genome in two distinct ST313 lineages in Africa implies a strong selection pressure that caused convergent evolution of the two African lineages. In contrast, there was evidence for an assortment of distinct prophage repertoires in the UK-ST313 isolates, indicating an absence of selection for specific mobile elements.

Aside from the addition of mobile genetic elements and virulence factors, genome degradation by the accumulation of pseudogenes and deletion events accompanies adaption to a more invasive lifestyle (Georgiades and Raoult, 2011; Nuccio and Bäumler, 2014). Initial analysis of the African ST313 representative strain D23580 genome identified 23 pseudogenes compared to the 6 present in ST19 strain SL1344

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(Kingsley et al., 2013). However, the majority of the genome degradation found in lineage 2 strain D23580 was conserved in UK representative strain U2. The only pseudogenes associated with characterized genes that were found to be specific to African lineage 2 ST313 were the SPI2-secreted effector gene sseI, lipid A modification gene lpxO and macrolide efflux pump gene macB, each of which could potentially play a role in infection dynamics (Andersen et al., 2015; Carden et al., 2017; Gibbons et al., 2005).

A number of the in vitro phenotypes that have been reported for lineage 2 ST313, which could contribute to a host-adapted lifestyle (Carden et al., 2015; Ramachandran et al., 2015; Singletary et al., 2016), were examined in the UK-ST313 to look for an African lineage-specific phenotype. Swimming motility was highly variable amongst the strains tested, and UK-ST313 isolates behaved identically to African lineage 2 isolates in the catalase and RDAR morphotype assays. No African lineage-specific phenotypic characteristics were detected, suggesting that reduced motility, defective catalase activity and loss of RDAR formation are not directly linked to iNTS disease. A key contributing factor to iNTS disease is host immunosuppression (Feasey et al., 2012), and one limitation of this retrospective study was that the underlying health status of the patients was unknown. This study does highlight the extraordinary epidemiological insights that routine genomic surveillance of pathogens by public health agencies can offer and the ability to understand the pathogenesis of novel pathovars.

This chapter has described previously unknown diversity in the ST313 sequence type that gives an insight into the convergent evolution towards niche specialization that has occurred in African ST313 lineages 1 and 2. The routine genomic surveillance of pathogens described here is now being adopted internationally and will bring an unprecedented ability to monitor emerging threats, such as the arisal of extended- spectrum beta-lactamase resistance. More generally, whole-genome sequencing of clinical isolates represents a new window with which to view the epidemiology and microbiology of infectious diseases.

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Chapter 5

Investigating novel prophages

BTP1 & BTP5 at the

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5.1 Introduction

The BTP1 and BTP5 prophages are a defining feature of African S. Typhimurium ST313 isolates belonging to both lineages, and ST313 genomes lacking either or both of the prophages are rare (Okoro et al., 2015). Findings described in Chapter 4 suggested that the BTP1 and BTP5 prophages were acquired independently by the two African lineages of S. Typhimurium ST313, suggesting a strong selection pressure for this particular prophage complement in the sub-Saharan African niche, and representing convergent evolution of the two lineages in Africa (Chapter 4.5). What is the selection pressure that drove the acquisition and is driving the conservation of these prophages? The aim of this chapter is to put the prophages characterised in Chapter 3 into context within the genome of African S. Typhimurium ST313, and to explore how the novel prophages may contribute to the biology of the lysogen, at the transcriptomic level.

5.1.1 Acknowledgement of the specific contribution of collaborators to the results described in Chapter 5.

The work described in this chapter is unpublished. I acknowledge the following contribution of collaborators to the results described in this chapter. Unless specified below, all work was completed by the Author.

Rocío Canals & Disa Hammarlöf University of Liverpool, UK

Collection of all D23580 WT RNA-seq & dRNA-seq samples

(Canals et al., 2018; Hammarlöf et al., 2017) Will Rowe

University of Liverpool, UK

Generation of differential gene expression values (CPMs) for prophage mutant RNA- seq experiments (described in 2.12.4)