Characterization of <i>Shigella flexneri</i> serotype 6 strains from geographically diverse low- and middle-income countries

<i>Shigella flexneri</i> serotype 6 (<i>Sf</i>6) is one of the most common serotypes recovered from surveillance studies of moderate to severe diarrhea. Despite the clinical significance of <i>Sf</i>6, this serotype is understudied. In this work, we have performed both serotype-specific genomic and phenotypic comparisons of <i>Sf</i>6 isolates to one another and non-<i>S</i>. <i>flexneri</i> serotypes. Comparative genomic analyses identified significant nucleotide homology between <i>Sf</i>6 strains (<i>n</i> = 325), despite a broad range of collection timeframes and geographic locations. We identified <i>Sf</i>6 specific factors, including a potential novel <i>Shigella</i> virulence factor (type II secretion system). Additionally, we identified established <i>Shigella</i> virulence genes (<i>ospG</i>) and metabolic genes (<i>rutABCDEFGR</i>) that were absent in <i>Sf</i>6 strains while present in the majority of 728 non-<i>Sf</i>6 strains. Complete sequencing of 11 clinical <i>Sf</i>6 strains, demonstrated that the <i>Sf</i>6 virulence plasmid (pINV) is ~38 kb smaller than the average non-<i>Sf</i>6 pINV (~228 kb). Comparisons of <i>S. flexneri</i> species level antibiotic susceptibility highlighted that clinical <i>Sf</i>6 isolates from Africa in the Global Enteric Multicenter Study (GEMS) and Vaccine Impact on Diarrhea in Africa (VIDA) study demonstrated geographic, serotype-specific susceptibility pattern. Phenotypic analyses of <i>Sf</i>6 identified reduced intracellular invasion and cytokine induction from HT-29 cells, as well as reduced Ipa protein effector secretion, compared with <i>S. flexneri</i> serotype 2a strain 2457T. Together these data highlight conserved and unique serotype-specific genotypic and phenotypic features for <i>Sf</i>6. This level of conservation has not been noted for other <i>S. flexneri</i> serotypes and is promising for vaccine and diagnostic assays to provide global <i>Sf</i>6-specific coverage.IMPORTANCEShigellosis is an ongoing global public health crisis with >270 million annual episodes among all age groups; however, the greatest disease burden is among children in low- and middle-income countries (LMIC). The lack of a licensed <i>Shigella</i> vaccine and the observed rise in antimicrobial-resistant <i>Shigella</i> spp. highlights the urgency for effective preventative and interventional strategies. The inclusion of <i>S. flexneri</i> serotype 6 (<i>Sf</i>6) is a necessary component of a multivalent vaccine strategies based on its clinical and epidemiological importance. Given the genomic diversity of <i>Sf</i>6 compared with other <i>S. flexneri</i> serotypes and <i>Sf</i>6 unique O-antigen core structure, serotype-specific characterization of <i>Sf</i>6 is a critical step to inform <i>Shigella</i>-directed vaccine and alternative therapeutic designs. Herein, we identified conserved genomic content among a large collection of temporally and geographically diverse <i>Sf</i>6 clinical isolates and characterized genotypic and phenotypic properties that separate <i>Sf</i>6 from non-<i>Sf</i>6 <i>S. flexneri</i> serotypes.