Characterization of
Shigella flexneri
serotype 6 strains from geographically diverse low- and middle-income countries
Authors: Gabor Caitlin E. , Chong Charlotte E. , Lemme-Dumit Jose M. , Hazen Tracy H. , Baker Kate S. , Kotloff Karen L. , Kasumba Irene N. , Tennant Sharon M. , Badji Henry , Hossain M. Jahangir , Omore Richard , Ochieng Benjamin , Awuor Alex O. , Ogwel Billy , Juma Jane , Barry Eileen M. , Rasko David A. 
Published: December 2024

Shigella flexneri
serotype 6 (
Sf
6) is one of the most common serotypes recovered from surveillance studies of moderate to severe diarrhea. Despite the clinical significance of
Sf
6, this serotype is understudied. In this work, we have performed both serotype-specific genomic and phenotypic comparisons of
Sf
6 isolates to one another and non-
S
.
flexneri
serotypes. Comparative genomic analyses identified significant nucleotide homology between
Sf
6 strains (
n
= 325), despite a broad range of collection timeframes and geographic locations. We identified
Sf
6 specific factors, including a potential novel
Shigella
virulence factor (type II secretion system). Additionally, we identified established
Shigella
virulence genes (
ospG
) and metabolic genes (
rutABCDEFGR
) that were absent in
Sf
6 strains while present in the majority of 728 non-
Sf
6 strains. Complete sequencing of 11 clinical
Sf
6 strains, demonstrated that the
Sf
6 virulence plasmid (pINV) is ~38 kb smaller than the average non-
Sf
6 pINV (~228 kb). Comparisons of
S. flexneri
species level antibiotic susceptibility highlighted that clinical
Sf
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
Sf
6 identified reduced intracellular invasion and cytokine induction from HT-29 cells, as well as reduced Ipa protein effector secretion, compared with
S. flexneri
serotype 2a strain 2457T. Together these data highlight conserved and unique serotype-specific genotypic and phenotypic features for
Sf
6. This level of conservation has not been noted for other
S. flexneri
serotypes and is promising for vaccine and diagnostic assays to provide global
Sf
6-specific coverage.

IMPORTANCE

Shigellosis 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
Shigella
vaccine and the observed rise in antimicrobial-resistant
Shigella
spp. highlights the urgency for effective preventative and interventional strategies. The inclusion of
S. flexneri
serotype 6 (
Sf
6) is a necessary component of a multivalent vaccine strategies based on its clinical and epidemiological importance. Given the genomic diversity of
Sf
6 compared with other
S. flexneri
serotypes and
Sf
6 unique O-antigen core structure, serotype-specific characterization of
Sf
6 is a critical step to inform
Shigella
-directed vaccine and alternative therapeutic designs. Herein, we identified conserved genomic content among a large collection of temporally and geographically diverse
Sf
6 clinical isolates and characterized genotypic and phenotypic properties that separate
Sf
6 from non-
Sf
6
S. flexneri
serotypes.