S. sonnei 75/02 was isolated in 2002 from a sporadic case of shigellosis (Nógrády et al., 2013). Serotype of 75/02 was verified by slide agglutination using phase I and phase II Shigella sonnei specific hyperimmune sera.

The previously described drug resistance pattern of S. sonnei 75/02 (Nógrády et al., 2013) was verified by disc diffusion method, and we determined minimum inhibitory concentrations for each agent as follows. Lysogeny broth (LB) agar plates containing halving serial dilutions of ampicillin, chloramphenicol, streptomycin, and tetracycline were set up as described in Tóth et al. (2003). Serial dilution agar plates of trimethoprim and sulfomethoxazole were set up similarly with a range of concentration between 128 and 4 mg/l. In the case of sulfomethoxazole, Müller-Hinton agar was used. Five microliters drops from liquid LB cultures containing 10⁵ CFU/ml of 75/02 and E. coli MG1655 were spotted on the agar plates in triplicate. The lowest antibiotic concentration causing reduced growth was considered as the MIC.

Genomic DNA of strain 75/02 was isolated with GenElute Bacterial Genomic DNA Kit (Sigma-Aldrich, Cat.Num.: NA2100) according to the manufacturer's instructions. Sequencing library was generated using Illumina Nextera Mate Pair Kit (Cat.Num.: FC-132-1001) as per manufacturer's instructions. DNA sequencing was performed on an Illumina MiSeq machine using V2 sequencing chemistry. Mate-paired reads were processed following the manufacturer's recommendations (Data Processing of Nextera® Mate Paired Reads on Illumina Sequencing Platforms).

Plasmid DNA was isolated by alkaline lysis procedure (Bimboim and Doly, 1979) and separated by agarose gel electrophoresis.

De novo assembly was performed with CLC Genomics Workbench Tool (v8.5.1, CLC Bio). Contigs were arranged into scaffolds using SSPACE 3.0 (Boetzer et al., 2011). Gaps in scaffolds were closed with Spades v 3.1.1 (Bankevich et al., 2012) together with an in-house R script (unpublished results). Annotation of the genome was performed with the NCBI PGAAP annotation pipeline. Prophage regions were marked with PHAST (Zhou et al., 2011), virulence genes were identified with VirulenceFinder (Kleinheinz et al., 2014), resistance genes with ResFinder (Kleinheinz et al., 2014), and type III secreted effectors with the Effective database (Jehl et al., 2011). The PHAST hits were manually curated based on the genome annotation. The plasmid incompatibility testing primers and reference sequences proposed by Carattoli et al. (2005) were searched by Basic Local Alignment Search Tool (BLAST).

Based on the 97 highly informative SNPs originally selected by Sangal et al. (2013), 121 bp baits were designed (summarized in Supplementary Table 1) and subsequently used as BLAST query sequences to yield a 97-point genomic fingerprint for each analyzed Shigella genome. Genomes with incomplete genotype profiles were removed. Strains were placed in genotype groups based on their 97SNP profiles (Supplementary Table 2). From each of the 20 obtained profile groups, one representative strain was enrolled in the phylogenetic analysis (Table 1, Supplementary Table 3). Multiple alignment of the SNP tags was carried out in CLC Genomics Workbench Tool (v8.5.1, CLC Bio). Approximately-maximum-likelihood tree was created with FastTree (version 2.1.7 SSE3) using Shimodaira-Hasegawa test for local support value estimation. An additional in silico SNP genotyping round with a subset of 5 classifier SNPs (Mazi et al., 2015) were also carried out to compare S. sonnei 75/02 with the clones isolated in California, USA including the Stx positive ones responsible for the San Diego/San Joaquin outbreak (Kozyreva et al., 2016).

Finally, multi-locus sequence typing (MLST) was performed using the genes selected for the typing of for Escherichia coli and Shigella by Clermont et al. (2015) and Wirth et al. (2006).

Experiment meta-data from all available S. sonnei datasets were downloaded from the Sequence Read Archive (SRA). Experiments were filtered keeping only those projects, where the required minimum information was available (name of strain, year, and country of isolation). Redundant projects belonging to the same strains were removed. After filtering, 1131 informative S. sonnei NGS datasets were downloaded (summarized in Supplementary Table 4). A 3.2 kb long query sequence containing stxA and stxB genes was extracted from the genome of strain 75/02 and was used to test for the presence of Stx in the downloaded NGS datasets using BLASTN. In addition, a 3.5 kb query sequence encoding for a DNA polymerase III subunit was used as a positive control of the BLAST step.

The complete genome sequence of S. sonnei 75/02 reported in the present study was deposited in GenBank. Accession number for the chromosome is CP019689, while the seven identified plasmids are numbered from CP019690 to CP019696 (Table 2).

The whole size of the S. sonnei 75/02 genome is 5.2 Mb, of which 4,891,717 bp belongs to the main chromosome, and almost 300 kb corresponds to 7 plasmids of various sizes. The genome contains altogether 6,087 coding sequences (CDS), out of these 5,352 are real CDSs. The chromosome carries altogether 5,619 CDSs (with 4,986 real CDSs) as well as 96 tRNA and 22 rRNA genes. The general features of the 75/02 genome and plasmids are presented in Table 2.

The genome of S. sonnei 75/02 contains 7 plasmids with sizes ranging from 214 kb (pInv_75/02_1) to 2.7 kb. A summary of the main characteristics of the plasmids is given in Table 2. Plasmid pInv_75/02_1 (Figure 1) has an overall 99% sequence identity to S. sonnei FORC_11 pInv plasmid (pFORC11.1, GenBank CP010830.1). Plasmid p75/02_2 shows co-linearity only with five other plasmids in GenBank, of which only one is of S. sonnei origin, the others are from two strains of Salmonella serovar Diarizonae, and two E. coli plasmids, pHNDLDH19 harboring a bla-XTM gene and pHNSHP45 carrying colistin-resistance gene mcr-1 (Liu et al., 2016). Despite the co-linearity, p75/02_2 carries neither of these resistance genes. Plasmid p75/02_3 is most similar to a fragment of the 100 kb pIFM3804 isolated from E. coli strain B3804, which originally harbors a CTX-M resistance mechanism, that is missing from p75/02_3. Plasmid p75/02_4 is almost identical (over 99% sequence identity) to pESBL-117 of a uropathogenic E. coli (UPEC) strain (Brouwer et al., 2014; GenBank CP008734.1), carrying a bla_TEM−52 gene encoding a broad-spectrum beta-lactamase. Plasmid p75/02_5 is very similar (>99% sequence identity) to pBS512_5 carried by Shigella boydii strain CDC 3083-94 (GenBank CP001060.1), carrying no known virulence or antimicrobial resistance related genes. Plasmid p75/02_6 corresponds to a short fragment of plasmid A carried by S. sonnei 53G (GenBank HE616529.1). Plasmid p75/02_7 only has one complete coverage hit, which is located on the draft genome sequence of the EHEC strain FHI74 (GenBank LM996601). We aimed to reveal the incompatibility (Inc) groups, the primer sequences used for the identification of Inc groups of E. coli strains (Carattoli et al., 2005) could be found in the p75/02 plasmid sequences suggesting that p75/02_2 to p75/02_7 represent different Inc groups from plasmids of E. coli origin. However, pInv_75/02_1 harbors a close homolog of the repA gene from the Salmonella typhimurium pSLT plasmid (GenBank AE006471), which according to Carattoli et al. (2005) puts it into the F-II replicon type family (Table 2).

PHAST search identified 23 prophage regions (75/02pp1-23), of which 12 were labeled as “intact,” eight as “incomplete” and three were labeled as “questionable.” Except for 75/02pp18, which encodes the Stx1 phage, regions with high level of similarity are present in the four S. sonnei strains with complete genomes available: Ss046, 53G, FDAARGOS_90, and in FORC_11 (Genbank numbers NC_007384.1, NC_016822, CP014099, and CP010829, respectively). A summary of the prophage regions is given in Table 3, Figure 2.

S. sonnei 75/02 proved to be a phase I strain harboring a wide array of virulence genes found in most clinical S. sonnei isolates (reviewed by The et al., 2016).

The pInv plasmid (Figure 1) carries most of the key virulence determinants of Shigellae, including the gene clusters encoding a type III secretion system (TTSS) and various effectors. It also encompasses the Shigella O-antigen synthesis cluster, showing 99% average sequence identity to that of S. sonnei 53G characterized by Xu et al. (2002), as well as that harbored by S. sonnei FORC11 and Ss046. pInv_75/02_1 shows 99% sequence identity to that carried by S. sonnei FORC011 (GenBank NZ_CP010830.1), these plasmids harbor the Mxi-Spa region, which encodes the key virulence mechanisms of S. sonnei pathogenesis (reviewed by The et al., 2016). Plasmid pInv_75/02_1 also carries the gene encoding the Shigella enterotoxin type 2 (Shet2), matching the primers designed by Vargas et al. (1999), while the enterotoxin encoding senB gene is located on the chromosome, matching the protein sequence AAZ89288.1 carried by S. sonnei Ss046. The chromosome of S. sonnei 75/02 also carries five copies of the invasion plasmid antigen encoding gene ipaH. They are all associated with phage genes, and three of them are encoded in prophage regions identified with PHAST: 75/02pp3, 75/02pp13, 75/02pp14. S. sonnei 75/02 also carries a homolog of the gene encoding ShiA, which has a role in host inflammatory response and is part of a pathogenicity island in S. flexneri (Ingersoll et al., 2003). However, the flanking region of the ShiA-encoding gene is different in 75/02, but identical to those in other fully sequenced S. sonnei strains (FDAARGOS, FORC011, Ss046, and 53G). In S. sonnei strains the shiA gene seems to be seated within a short transposon-like region, which is part of IS21 in strain Ss046.

Similarly to S. sonnei Ss046 (Yang et al., 2005), strain 75/02 does not carry the pic gene, which was identified as an important mucinase with a role in invasion (reviewed in Anderson et al., 2016). Moreover, it only carries EspC, a per (plasmid encoded regulon) activated serine protease autotransporter protein toxin, which shows partial similarity to pic. However, gene sigA is present, encoding an immunoglobuline A protease, with also an important role in invasion (reviewed in The et al., 2016).

Besides the usual Shigella virulence genes, the most striking feature of S. sonnei 75/02 is that it harbors an inducible Stx1 converting phage (75/02pp18), which has been described in detail earlier (Tóth et al., 2016). The Shiga toxin production of the strain, and the transducibility of the Shigella Stx phage 75/02 were demonstrated. The lysogenized E. coli K-12 derivative strains produced Shiga toxin as well (Tóth et al., 2016).

Regarding the virulence of Stx producing Shigellae, in an earlier study Fontaine et al. (1988) demonstrated in S. dysenteriae that the invasive capacity of the pathogen is not influenced by the production of Stx, but on the other hand, the elimination of Stx decreased the strain's virulence in vivo.

The Stx phage 75/02 could also lysogenize a Shiga toxin negative S. sonnei strain (data not shown), similar observations were also published by Beutin et al. (1999). It is worth mentioning that within the sequence of the S. sonnei 75/02 prophage (KF766125.2) there is a tail fiber gene, which is 753 bp longer than its counterpart, ORF55 in the phage genome sequenced earlier, suggesting that the phage excision resulted in the truncation of this gene. The integration site of the phage proved to be the ynfG homolog gene cluster (Tóth et al., 2016), the same integration site, which was determined earlier for the Stx1 carrying phage POCJ-13 characterized from a S. flexneri strain (Gray et al., 2014). The peculiarity of this integration site that it is present undisrupted in the known S. sonnei strains with complete genomes (Ss046, 53G, FDAARGOS_90, and FORC_11).

We wanted to know if any of the previously sequenced S. sonnei strains contained Stx encoding genes, therefore screened altogether 1131 entries downloaded from the NCBI Short Read Archive (Supplementary Table 4). We found that besides S. sonnei 75/02, stx genes were only detectable in a subset of the San Diego/San Joaquin outbreak isolates characterized by Kozyreva et al. (2016). Given the differences in the Stx phage genomes (75/02 STX and Ss-VASD) already highlighted by their study, we can conclude that the strains behind the California outbreak represent a clearly distinct clone from 75/02 even though both contain Stx.

S. sonnei 75/02 proved to be resistant to streptomycin, ampicillin, sulfomethoxazol/trimethoprim and tetracycline as verified by disc diffusion method. In the current study, we determined the MIC-values and investigated the genetic basis of the observed MDR phenotype using ResFinder.

In all cases the MIC-values verified the previously observed multiresistance pattern, and the strain also showed resistance to chloramphenicol. A summary of results is given in Table 4.

According to the ResFinder results, the genome of 75/02 harbors several beta-lactamases on the chromosome, and p75/02_3 carries a bla_TEM−52 gene, encoding and extended spectrum beta-lactamase, identical to the one carried by plasmid pESBL-117 of a uropathogenic E. coli (UPEC) strain (GenBank CP008734.1). Either of these genes could serve as a basis of ampicillin resistance. We could verify the presence of the gene aadA1 (spectinomycin 9-O-adenyltransferase), which is responsible for streptomycin resistance, and we identified the dfrA1 gene encoding trimethoprim resistance. None of the sulfonamide (sul), tetracycline (tet), or chloramphenicol (cat) resistance encoding genes were present in the genome, but we identified an acridine efflux pump gene cluster, acrAB, which encodes a general drug efflux mechanism (Chen et al., 2007) with a possible role in the antimicrobial resistance displayed by the strain.

To reveal the phylogenetic position of S. sonnei 75/02, several comparison strategies were used. According to the core genome SNP-based phylogeny established by Holt et al. (2012), in silico SNP genotyping of 439 S. sonnei genome sequences were carried out. This phylogeny has the capability of distinguishing four (I-IV) separate phylogenetic groups. Group I and IV represent the more ancient European clones, while group II and III are the more recently emerged global clones. Group III is the most abundantly represented and diverse in the current databases, and it is divided into subgroups IIIa, IIIb, and IIIc.

Based on 97 SNP positions, analyzed genomes could be sorted to 20 different genotype groups. One representative sequence from each genotype group was taken to build the phylogenetic tree presented in Figure 3. The phylogeny based on 97 SNPs placed strain 75/02 closest to strains of the MDR global group (IIIc).

Systematic screening revealed that, in addition to isolate 75/02, Stx can only be found in a subset of San Diego/San Joaquin S. sonnei isolates. Therefore, a separate phylogenetic tree was also constructed comparing S. sonnei 75/02 with those strains isolated in California, USA (n = 67), reported by Kozyreva et al. (2016). Since the majority of the them failed to yield complete 97 SNP genotype profiles, only a limited subset of probes –as proposed by Mazi et al. (2015) –were used for this genotyping analysis. As expected, all isolates could be placed in one of the main lineages (I, II, IIIa, IIIb, or IIIc). Phylogenetic tree created by the representative sequences is presented in Figure 4. Again, S. sonnei 75/02 is placed in the IIIc lineage, together with all San Diego/San Joaquin isolates including all Stx positive California clones. In addition, this lineage contains historical isolates C96 and C98 from Lineage Global III. The remaining historical Linage Global III isolates (C81, C85, C92, C99) was classified as Class IIIa. All San Francisco strains, together with C42 were recognized as Class IIIb. Class I contains isolate C97 which was reported as the only Linage I member California isolate. Similarly, Class II is composed of the very same genomes (C80, C82, C88) as was Lineage II.

According to the MLST scheme of Institute Pasteur (Clermont et al., 2015), S. sonnei 75/02 represents sequence type ST563. In the Bacterial Isolate Genome Sequence Database (BigSDB) curated by Institute Pasteur, ST563 is represented by one S. sonnei strain. When typed according to the MLST scheme curated by the Warwick Medical School (Wirth et al., 2006), 75/02 represents the ST152 complex type. The Warwick database contains six isolates from ST152, five of them are S. sonnei, and the remaining one is S. flexneri.

Albeit our phylogenetic tree is generally congruent with the earlier SNP-based classifications (Holt et al., 2012; Sangal et al., 2013), we also notice that the number of observed genotype variations (n = 20) is rather small taking into account that 439 S. sonnei genomes were tested with a set of 97 in silico SNP baits. This low level of diversity is in harmony with the results of Holt et al. (2012) suggesting the occurrence of a bottleneck in the evolution of S. sonnei cca. 400–500 years ago.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.