Contents lists available at ScienceDirect International Journal of Medical Microbiology journal homepage: www.elsevier.com/locate/ijmm Virulence-related genes are associated with clinical and nutritional outcomes of Shigella/Enteroinvasive Escherichia coli pathotype infection in children from Brazilian semiarid region: A community case-control study Mariana Bona ⁎ , Pedro Henrique Medeiros, Ana Karolina Santos, Thiago Freitas, Mara Prata, Herlice Veras, Marília Amaral, Daniel Oliveira, Alexandre Havt, Aldo Ângelo Lima Institute of Biomedicine for Brazilian Semiarid, Federal University of Ceará, 1315 Coronel Nunes de Melo, 60430-270, Fortaleza, Brazil ARTICLE INFO Keywords: Shigella/EIEC pathotype Virulence genes Child diarrhea ABSTRACT Shigella/Enteroinvasive Escherichia coli (EIEC) pathotype is a major enteropathogen associated with diarrhea and malnutrition in children from developing countries. This study aimed to correlate Shigella/EIEC virulence-related genes (VRGs) with clinical symptoms, nutritional status and coenteropathogens in children from the Brazilian semiarid region. We designed a case-control study of community diarrhea in six cities of the Brazil semiarid region with 1200 children aging 2–36 months. Standardized questionnaire was applied for collecting socio- demographic, nutritional status and clinical information of the children. DNA samples were extracted from stools and diagnosed for Shigella/EIEC using PCR-based approaches. Positive samples were tested for 28 VRGs using four multiplex PCRs. Intestinal inflammation was determined by measuring fecal myeloperoxidase (MPO). Shigella/EIEC pathotype was detected in 5% of the children and was significantly associated with diarrhea. The genes sen (encoding Shigella enterotoxin 2), ipgB2, ipgB1 (both encoding type 3 secretion system-T3SS effectors that modulate actin filament), and ospF (encoding a T3SS effector involved in suppression of host responses) were further associated with diarrhea in Shigella/EIEC positive children. Among children presenting diarrhea, virA gene (encoding a T3SS effector that promotes microtubule destabilization) was associated with fever, while virB (encoding a major transcriptional activator) was associated with low height-for-age z-score. In addition, these VRGs were associated with increased fecal MPO, and coinfection with Salmonella spp. was associated with increased abdominal pain. These data reinforce the impact of Shigella/EIEC on diarrhea in children from Brazilian semiarid region and highlighted the contributions of specific virulence genes for its pathobiology. 1. Introduction Bacteria of the Shigella genus are a leading cause of infectious diarrhea worldwide, particularly in children from developing countries (Liu et al., 2016). Enteroinvasive Escherichia coli (EIEC) is a diarrhea- genic E. coli (DEC) that shares similar biochemical and pathogenesis features with Shigella (Maurelli, 2013). Although these pathogens are classically classified into two different genera based on historical and clinical reasons, several studies that have employed phylogenetic and genomic analyses do not support this distinction (Pupo et al., 2000; Lan et al., 2001; Escobar-Páramo et al., 2003; Lan et al., 2004; Peng et al., 2009; Pettengill et al., 2016). A recent phylogenomic study using a large number of diverse Shigella and EIEC genomes showed that Shigella serogroups belong to the same lineage, which contradicts the classification of the Shigella genus into species/serogroups (Pettengill et al., 2016). Genomic studies that attempted to identify specific genes for both Shigella and EIEC were not able to find any reliable markers. In light of their pathogenesis and genetic similarities, Shigella and EIEC have been suggested to be grouped into a single pathovar Shigella/EIEC (Kaper et al., 2004; Lan et al., 2004; Pettengill et al., 2016). The pathogenesis of the Shigella/EIEC pathotype is based on its ca- pacity to reach and invade colonic epithelial cells, leading to in- tracellular multiplication and spreading to adjacent cells with con- sequent cell death. The major genes that facilitate invasion and spread of Shigella/EIEC into human macrophages and enterocytes are encoded by a large virulence plasmid (Belotserkovsky and Sansonetti, 2018). This virulence plasmid contains the conserved 30 kb mix-spa locus, https://doi.org/10.1016/j.ijmm.2019.01.003 Received 31 August 2018; Received in revised form 1 January 2019; Accepted 30 January 2019 ⁎ Corresponding author.Present address: Clinical Research Unit and Institute of Biomedicine, R. Cel. Nunes de Melo, N 1315, Rodolfo Teófilo, Fortaleza, CE, 60.430- 270, Brazil. E-mail address: marianadbona@gmail.com (M. Bona). International Journal of Medical Microbiology xxx (xxxx) xxx–xxx 1438-4221/ © 2019 Elsevier GmbH. All rights reserved. Please cite this article as: Bona, M., International Journal of Medical Microbiology, https://doi.org/10.1016/j.ijmm.2019.01.003