Background: Among Shigella species, Shigella boydii has always displayed a smaller role to the overall Shigella burden, frequently placed at third in epidemiological studies and described as restricted to Southeast Asia. Here we characterize an S. boydii isolated from an epidemiological study enrolling 1,339 Brazilian children from the Amazon region, in which Shigella species solely was the fourth cause of bacterial diarrhea. S. boydii strain 183 was isolated from rotavirus co-infected children with acute diarrhea. Here we aimed to characterize this strain regarding virulence and, immune response in a pulmonary model. Methods: An in vitro HEp-2 epithelial cell invasion assay was used to compare the invasive phenotype of S. boydii strain 183 with clinical and highly virulent S. flexneri strain, both isolated from Brazilian children. A murine pulmonary model was performed to assess lung damage by histopathological analysis. mRNA expression of immune response key genes was retrieved by multiplex real-time PCR and correlations were obtained by network analysis. Broad genome analysis was performed to confirm S. boydii identity and define its virulence profile. Results: S. boydii strain 183 showed fewer invasion rates in vitro and tissue damage in vivo as compared to virulent S. flexneri 201. When compared to a survival challenge in mice, S. boydii had 100% survival against 10% of virulent S. flexneri. Overall, mRNA immune gene expression suggests a protective response against S. boydii strains 183, in contrast to the inflammatory response induced by the virulent S. flexneri strain 201. Network analysis with S. boydii strain 183 displayed IFN-γ protagonism, contrasting with the correlations centralized on TNF-α by the virulent S. flexneri strain 201. The genome showed a lack of effector proteins and enterotoxins in S. boydii strain 183, and sequencing analysis of Ipa invasins revealed mutations at functional sites. This avirulent S. boydii strain 183 presents the Type II Secretion System, T6SS, in addition to T3SS. Conclusions: In addition to causing no disease, S. boydii strain 183 lacks effector proteins and enterotoxins. The presence of T6SS additional secretion system could provide an advantage to establish this strain among commensal bacteria.

In the present study, 470 children less than 72 months of age and presenting acute diarrhea were examined to identify associated enteropathogenic agents. Viruses were the pathogens most frequently found in stools of infants with diarrhea, including 111 cases of rotavirus (23.6% of the total diarrhea cases) and 30 cases of adenovirus (6.3%). The second group was diarrheogenic Escherichia coli (86 cases, 18.2%), followed by Salmonella sp (44 cases, 9.3%) and Shigella sp (24 cases, 5.1%). Using the PCR technique to differentiate the pathogenic categories of E. coli, it was possible to identify 29 cases (6.1%) of enteropathogenic E. coli (EPEC). Of these, 10 (2.1%) were typical EPEC and 19 (4.0%) atypical EPEC. In addition, there were 26 cases (5.5%) of enteroaggregative E. coli, 21 cases (4.4%) of enterotoxigenic E. coli, 7 cases (1.4%) of enteroinvasive E. coli (EIEC), and 3 cases (0.6%) of enterohemorrhagic E. coli. When comparing the frequencies of diarrheogenic E. coli, EPEC was the only category for which significant differences were found between diarrhea and control groups. A low frequency of EIEC was found, thus EIEC cannot be considered to be a potential etiology agent of diarrhea. Simultaneous infections with two pathogens were found in 39 diarrhea cases but not in controls, suggesting associations among potential enteropathogens in the etiology of diarrhea. The frequent association of diarrheogenic E. coli strains was significantly higher than the probability of their random association, suggesting the presence of facilitating factor(s).