Table 1.

Summary of the most frequently used methods for Shigella identification

Technique		Target	Ability to distinguish between Shigella and E. coli	References
Phenotypic	Biochemical tests	The enzymatic activity and the ability to ferment some sugars	Inability to distinguish between Shigella spp. and other genera, Hafnia, Providencia, atypical E. coli, and non-lactose fermenting or anaerogenic E. coli	[34]
	Serology	The O antigen present in the surface of bacteria	Occurrence of intra- and inter-species cross-reactions, occasional provisional Shigella serovars, serotype conversion, and untypable strains, not illustrative of phylogenetic relationships	[35]
	MALDI-TOF MS	The spectra of ribosomal proteins signature	Inability of conventional MALDI-TOF MS assays to discriminate between Shigella and E. coli. Advanced software or customized reference library could distinguish inactive and other non-lactose-fermenting E. coli from Shigella spp.	[36–38]
Molecular	PCR techniques	ipaH, and lacY uidA, and lacY	Differentiation between Shigella (ipaH + , lacY −), EIEC (ipaH + , lacY +), non-invasive E. coli (ipaH − , lacY +); however, recent data demonstrate low performance Differentiation between Shigella (uidA + , lacY −), and E. coli, including EIEC (uidA + , lacY +); however, recent data demonstrate low performance	[27, 39, 40]
	Single locus sequence-based	16S rRNA gene	Inability to distinguish E. coli from Shigella spp. due to the high sequence similarity (> 99%)	[41]
		rpoB gene	Inability to distinguish E. coli from Shigella spp. due to the high sequence similarity (> 99%)	[42]
		gyrB gene	Capability to differentiate between E. coli and Shigella spp., but further large validations are needed	[43]
	WGS	K-mers	Accurately differentiate E. coli from Shigella to the species level, but some EIEC isolates can be misidentified as S. flexneri or S. boydii	[44]
		SNP markers	Accurately differentiate EIEC from Shigella spp. with typing ability among strains of the same species	[27]
		Average of nucleotide identity	Most of the ANI-based delineation tools are unable to delineate Shigella spp. from E. coli. Notably, the GGDC web tool with the ANI-f1 formula showed some power	[45]
		Cg-MLST: conserved loci shared between Shigella and E. coli species	The cgMLST-based clustering differentiates between E. coli and Shigella spp. with high-resolution capabilities	[46]