Some serovars of
Salmonella
are not or rare found to cause salmonellosis in human. In our clinic-based surveillance, three rare
Salmonella
4,5,12:a:- strains were recovered from three patients with diarrhea. To explore their genetic and epidemiological characteristics and pathogenesis, we conducted whole-genome sequencing,
in vitro
invasion assays in mammalian cells, and
in vivo
virulence assays in an animal model. The three isolates had indistinguishable molecular patterns and similar genome sequences, and clustered together with an isolate from edible fish traded among countries. The isolates had biochemical reactions identical with those of
Salmonella
subspecies
enterica
but belonged to subspecies
salamae
according to genome phylogeny, revealing a new serovar,
S. enterica
subsp. II serovar 4,5,12:a:-. The strains contained multiple virulence genes, elicited temporary bacteremia and enteritidis and caused cell damage in the mouse liver and cecum. This study provides evidence that this new
Salmonella salamae
serovar can infect humans and cause clusters of cases, and whole-genome sequencing detection and surveillance of
Salmonella
can help to accurately define
Salmonella
classification and clonality, improve diagnosis, facilitate outbreak detection and aid in the source tracing of salmonellosis epidemics.