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Genomic surveillance detects Salmonella enterica serovar Paratyphi A harbouring blaCTX-M-15 from a traveller returning from Bangladesh

Whole genome sequencing (WGS) has been used routinely by Public Health England (PHE) for identification, surveillance and monitoring of resistance determinants in referred Salmonella isolates since 2015. We report the first identified case of extended-spectrum-β-lactamase (ESBL) Salmonella enterica serovar Paratyphi A (S. Paratyphi A) isolated from a traveller returning to England from Bangladesh in November 2017. The isolate (440915) was resistant to ciprofloxacin and harboured both the mobile element ISEcp9 –blaCTX-M-15-hp-tnpA and blaTEM-191, associated with ESBL production. Phenotypic resistance was subsequently confirmed by Antimicrobial Susceptibility Testing (AST). S. Paratyphi A 440915 harboured an IncI1 plasmid previously reported to encode ESBL elements in Enterobacteriaceae and recently described in a S. Typhi isolate from Bangladesh. Results from this study indicate the importance of monitoring imported drug resistance for typhoidal salmonellae as ceftriaxone is the first line antibiotic treatment for complicated enteric fever in England. We conclude that WGS provides a rapid, accurate method for surveillance of drug resistance genes in Salmonella, leading to the first reported case of ESBL producing S. Paratyphi A and continues to inform the national treatment guidelines for management of enteric fever.


Introduction
Enteric fever is a systemic infection caused by the human adapted pathogens; Salmonella enterica serovar Typhi (S. Typhi) and S. enterica serovar Paratyphi (S. Paratyphi) A, B and C. Enteric fever continues to carry a burden of morbidity and mortality with an estimated~28 million cases reported globally in 2000 [1]. S. Paratyphi A is the second most common cause of enteric fever after S. Typhi with approximately one S. Paratyphi A infection occurring for every four S. Typhi infections [1]. PLOS  The greatest burden of illness is experienced by infants, children and adolescents in Southcentral and Southeast Asia [1]. As described for typhoid fever (caused by S. Typhi), paratyphoid fever is rare in industrialised countries although known to occur among travellers to endemic parts of the world [2] [3]. In 2015, 18 EU/EEA countries reported a total of 845 confirmed cases, a notification rate of 0.23 cases per 100 000 population [4]. Between April 2015 and September 2018, Public Health England (PHE) reported an average of 115 cases in England and Wales of Paratyphoid A fever per year. The number of cases has been constant during this time period with a majority of cases acquired abroad; 54% were from travellers returning to the United Kingdom (UK) from the Indian subcontinent (https://www.gov.uk/ government/publications/typhoid-and-paratyphoid-laboratory-confirmed-cases-in-englandwales-and-northern-ireland).
Antibiotic resistance in S. Paratyphi A is an emerging public health problem. Resistance to multiple first line antibiotics e.g. ampicillin, chloramphenicol and co-trimoxazole (multidrug resistance [MDR]) [5], nalidixic acid [6] and ciprofloxacin [7][8] has arisen in multiple countries since 2000. Alternative antimicrobial treatments, including third generation cephalosporins (ceftriaxone) or azithromycin, are increasingly used as first line therapies [9]. Recently, resistance to these newer drugs has been reported in enteric fever-causing salmonellae [10] [11] [12]. While these cases have largely been sporadic, a large-scale extensively drug resistant (XDR) S. Typhi outbreak began in Pakistan in 2016, harbouring resistance to third generation cephalosporins [13]. Resistance to ceftriaxone or other extended-spectrum ß-lactams is usually due to the production of extended-spectrum-ß-lactamases (ESBLs) of which bla CTX-M type ESBLs are one of the determinants for cephalosporin resistance in Salmonella [14]. Many bla CTX-M variants are described in the literature, with bla CTX-M-9 , bla CTX-M-14 and bla CTX-M-15 being the most commonly reported [15][16] [17]. bla CTX-M type ESBLs are usually encoded by transmissible plasmids [18], hence routine surveillance of resistance determinants is essential to understand when and where populations may be affected.
WGS has been used at the PHE Gastrointestinal Bacterial Reference Unit (GBRU) since April 2015 for routine identification, surveillance, and detection of outbreak transmission events and Antimicrobial Resistance (AMR) determinants [3][19] [20]. Through routine genomic surveillance in November 2017, we identified an ESBL-harbouring S. Paratyphi A strain isolated from a traveller returning to the UK from Bangladesh. Here we report the characterisation, location and composition of the region encoding ß-lactam resistance and suggest the possible transmission mechanism of this ESBL resistant S. Paratyphi A isolate imported into the UK.

Case history
Enteric fever is a notifiable disease in the UK. Information was retrospectively collected from an enhanced surveillance questionnaire collected by the public health team from the case and hospital case notes.

Bacterial isolate and phenotypic identification
A stool specimen submitted to the hospital in September 2017 was subjected to EntericBio, a rapid panbacterial PCR assay screening, and found to be Salmonella spp. PCR positive. The stool was cultured for Salmonella and the isolate was referred to GBRU for confirmation in November 2017. It was grown on selective media, MacConkey and chromogenic agar, to rule out contamination from other Enterobacteriaceae. A single colony was selected for inoculation into broth for WGS DNA extraction and grown in Mueller-Hinton agar for antimicrobial susceptibility testing. 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 covered by PHE's surveillance mandate.

Antimicrobial susceptibility testing
Minimal inhibitory concentration (MICs) of the isolate were determined by agar dilution using Mueller-Hinton agar for the standard panel of antibiotics recommend by EUCAST. EUCAST breakpoints and screening concentration criteria were used for interpretation [21]. Confirmation of azithromycin MIC was performed by Etest VR (bioMerieux, France). Temocillin and cefoxitin were included in the panel to aid detection of OXA-48-like carbapenemases and AmpC production, respectively. ESBL detection was confirmed using aztreonam, cefotaxime/cefotaxime + clavulanic acid (4ug/mL, ceftazidime/ceftazidime + clavulanic acid (4ug/ mL), cefepime/cefepime+clavulanic acid (4ug/mL).

Whole genome sequencing and analysis
DNA extraction of the Salmonella isolate was carried out using a modified protocol of the Qiasymphony DSP DNA Midi Kit (Qiagen) as described in Nair et al. 2016 [22]. In brief, 0.7 mL of overnight Salmonella broth culture was harvested. Bacterial cells were pre-lysed in 220 μL of ATL buffer (Qiagen) and 20 μL of Proteinase K (Qiagen), and incubated with shaking for 30 min at 56˚C. Four microlitres of RNase at 100 mg/mL (Qiagen) was added to the lysed cells, which were then re-incubated for a further 15 min at 37˚C. DNA from the treated cells was then extracted on the Qiasymphony SP platform (Qiagen) and eluted in 100 μL of water. Extracted DNA was fragmented and tagged for multiplexing with NEXTERA XT DNA Sample Preparation Kits, followed by paired-end sequencing on an Illumina HiSeq platform to produce 101 bp paired-end reads (Illumina, Cambridge, UK).
Resistance genes were determined using Genefinder, a customised algorithm that uses Bowtie 2 to map reads to a set of reference sequences and Samtools to generate an mpileup file [23], as previously described Day et al. 2018 [3]. Briefly, the data are parsed based on read coverage of the query sequence (100%), consensus base-call on variation (>85%) and the nucleotide identity (>90%) to determine the presence of the reference sequence or nucleotide variation within that sequence. β-Lactamase variants were determined with 100% identity using the reference sequences downloaded from the Lahey (www.lahey.org) or NCBI (https:// www.ncbi.nlm.nih.gov/pathogens/beta-lactamase-data-resources) β-lactamase data resources. Known acquired resistance genes and resistance-conferring mutations relevant to β-lactams, fluroquinolones, aminoglycosides, chloramphenicol, macrolides, sulphanomides, tetracyclines, trimethoprim, rifamycins and Fosfomycin were included in the analysis [24] [25].
Pairwise BLAST comparison of IncI1 plasmids harbouring ß-lactam resistance. Plasmids from S. Typhi (BioProject PRJEB21992) [30], S. Enteritidis (KT868530) [31] and S. Enteritidis (accession NC_018659) were selected for comparison using the following two criteria: (i) the presence of the same mobile element and resistance gene (ISEcp9 and (bla BLACTX-M-15 ) and (ii) the same IncI1 incompatibility group as the plasmid from isolate 440915 being investigated. FASTA files from all plasmids were compared and visualised using BRIG v0.95 [33]. The plasmid fasta was extracted for pPRJEB21992 from a plasmid SPAdes assembly performed on the genome sequence data from accession PRJEB21992 [34] (version 3.11.1, '-careful'). The orientation and position of specific genes was drawn using Easyfig v 2.1 [35].
Nucleotide sequence accession number. Short-read FASTQ sequence for the S. Paratyphi A 440915 plasmid described in this study has been deposited in the NCBI Sequence Read Archive under GenBank accession number MK238490 and BioProject PRJNA505238.
Phylogenetic analysis. To place isolate 440915 in context of the S. Paratyphi A population, SNP analysis was performed on the 439 isolates of S. Paratyphi A referred to PHE from 1st April 2014 to December 2017 (S1 Table). The PHE isolates were supplemented with 20 genome sequences from Zhou et al. 2018 [36] with representatives covering the 7 described lineages (A-G). Illumina reads were quality trimmed [37] with bases removed from the forward and trailing end with a PHRED score of less than 30. Reads were mapped to the S. Paratyphi A reference genome ATCC 9150 (Genbank accession CP000026.1) using BWA-MEM v.0.7.12 [38]. SNPs were identified using GATK v.2.6.5 [39] in unified genotyper mode. Core genome positions that had a highquality SNP (>90% consensus, minimum depth 10x, GQ> = 30) in at least one isolate were extracted using SnapperDB v0.2.5 [40] and processed through Gubbins v2.0.0 [41] to account for recombination events. RaxML v.8.1.17 [42] was used to derive the maximum likelihood phylogeny of the isolates using the GTRCAT substitution model with the automatic bootstrapping criteria 'autoMRE'. FASTQ reads from all sequences in this study can be found at the PHE Pathogens BioProject at the National Centre for Biotechnology Information (Accession PRJNA248792).

WGS process
The whole WGS process, from growing bacterial cells to interpreting sequence data for identification, AMR characterisation and high throughput single nucleotide polymorphism typing for surveillance requires approximately 5 days. A rapid turnaround time considering the amount of data obtained.

Results
A 44 year old male patient who had returned to England from a 6 week trip to Bangladesh in September 2017 was diagnosed with infective colitis. From a stool specimen, an enteric PCR was positive for Salmonella spp; the stool cultured a presumptive S. Paratyphi A that was resistant to quinolones, and sensitive to azithromycin. The isolate was sent to GBRU and confirmed as S. Paratyphi A (isolate 440915) in November 2017 by WGS. Further case details are given in Supplementary Data (S1 Data).
Our WGS analysis revealed S. Paratyphi A 440915 to be an ESBL-producing strain encoding both bla BLACTX-M-15 and a bla  . A point mutation conferring resistance to quinolones was detected in gyrA [83:S-F].ESBL production was phenotypically confirmed with cefotaxime clavulanic acid synergy. The ciprofloxacin MIC was 0.75 mg/L and the isolate was sensitive to azithromycin (8 mg/L).
Isolate 440915 was confirmed as S. Paratyphi A multi locus sequence type (ST)129, a member of serovar Paratyphi A eBURST group 11. S. Paratyphi A 440915 clusters in lineage A of the S. Paratyphi A population as defined by Zhou et al. 2014 [36]. Within lineage A, isolate 440915 belongs to a monophyletic clade with 29 other isolates from cases reported through GBRUs routine surveillance. Of these cases 16/29 (55%) reported recent travel to Bangladesh, 2/29 reported travel to Pakistan, 1/29 reported no travel and the remaining 10 cases had no travel information available. Within this clade all isolates harboured a single mutation in gyrA [83:S-F] conferring resistance to ciprofloxacin; no other resistant determinants were detected (Fig 3).

Discussion
This study reports the first ESBL-producing S. Paratyphi A isolated in the UK using PHE's WGS-based surveillance for AMR determinants [3][19]  [44]. It is unsurprising that isolate 440915 harboured bla CTX-M-15 on an IncI1 plasmid; these plasmids have been most frequently associated with bla CTX-M type ESBL carriage [45], are transmissible between enteric pathogens [46] and commonly found in the Enterobacteriaceae. IncI1 plasmids have also been shown to have no biological cost on the fitness of E. coli isolates harbouring them [47] which means they may be maintained even in the absence of selective antibiotic pressure. The high nucleotide identity between ISEcp9-bla CTX-M-15 -hp-tnpA in S. Typhi PRJEB21992 [30] and the current S. Paratyphi A 440915 isolate in this study makes it plausible to speculate that one transmitted to the other and hence supports the transmissible nature of ISEcp9 linked to ß-lactam resistance [48].
ESBL-carrying S. Paratyphi A and S. Typhi isolates were not seen globally or particularly in Europe until recently [10] [13][49] [50]. The ESBL S. Typhi isolate recently reported from Bangladesh was the second isolate since 2000 [30]. A global population structure study by Zhou et al. 2018 [36] classified S. Paratyphi A into seven lineages with lineages A and C being the most dominant. The phylogenetic analysis described here indicates that S. Paratyphi A 440915 is closely related to S. Paratyphi A belonging to lineage A, circulating in Bangladesh and the Indian subcontinent (Fig 3). We therefore postulate that the dissemination of ESBL resistance can be sustained by different mechanisms including the horizontal dissemination of this transferable IncI1 plasmid or transposition of the mobile element (ISEcp9bla CTX-M-15 -hp-tnpA) within this closely related lineage. We believe it is only a matter of time until the ISEcp9 bla CTX-M-15 -hp-tnpA mobile element (or a variation) inserts into a successful chromosomal background, as seen with the recent outbreak in Pakistan caused by S. Typhi H58 and an IncY plasmid [13].
This ESBL S. Paratyphi A 440915 isolate and recent sporadic and outbreak ESBL enteric fever cases reported in the UK, Germany and Pakistan [13][49] [50] indicate the need for active surveillance in the UK for cases returning from the Indian subcontinent. In the UK, the standard empirical treatment for complicated enteric fever from South Asia has continued to be third-generation cephalosporins. Diagnostic laboratories in England usually perform AST on faecal isolates of typhoidal salmonellae with antibiotics such as azithromycin and ciprofloxacin, using gradient assays like E-tests (bioMerieux, France). However, in order to avoid treatment failures, AST should be routinely performed accordingly to EUCAST guidelines on all presumptive isolates (faecal and invasive) of S. Paratyphi and S. Typhi specifically looking for ceftriaxone resistance. Even though WGS predicts AMR determinants it is not used for clinical management and hence ceftriaxone resistant strains are phenotypically tested to assess ESBL production [51] to tailor clinical treatment. Treatment should be tailored depending on the AST. The patient in this case fully recovered from the illness and had negative clearance specimens one year later despite not being treated with the appropriate antibiotics (refer to S1 Data). Although paratyphoid is a milder illness than typhoid fever, further research is required to monitor outcomes of ESBL producing strains including enhanced clinical surveillance to assess whether the clinical outcome includes relapses.

Conclusion
Extended spectrum ß-lactamase producing S. Paratyphi A has been identified in the UK from the Indian subcontinent through PHE's use of routine WGS. Routine WGS provides a rapid and accurate method for surveillance of drug resistance genes and can inform the national treatment guidelines for management of enteric fever. WGS data as obtained by GBRU also allows passive surveillance to monitor the spread of drug resistant S. Typhi and S. Paratyphi and to detect outbreaks, as well as to serve as a sentinel surveillance for drug resistant enteric fever agents circulating in different regions of the world where the disease is endemic.