Epidemiological and molecular approach to determine factors associated with diarrheagenic E. coli in food for children under five in Mozambique

Abstract

Epidemiological and molecular approach to determine factors associated with diarrheagenic E. coli in food for children under five in Mozambique Diarrhoeagenic Escherichia coli (DEC) is associated with diarrhoea and is responsible for around 200,000 deaths worldwide annually, with children under five being the most affected. Children under five from Mozambique are at constant risk for developing diarrhoea due to factors. These include lack of improved drinking water access or sanitation and poor literacy of caregivers, coupled with the fact that their immune systems are under development. In 2018, approximately 500,000 cases of diarrhoea in Mozambique were caused by foodborne pathogens. Recent studies have shown that DEC strains are among the pathogens that cause enteric infection. This study aims to determine sources (food and water) and factors associated with foodborne pathogens causing diarrhoea in children under five years old in rural and urban areas of Maputo province, Mozambique. A total of three hundred children under five years of age with diarrhoea were selected in two Health Care Centres of Maputo province in Mozambique as study cases. The caregivers of the children completed a semi-structured questionnaire. This allowed obtaining information related to demographics, housing conditions, food consumed a week before the children’s diarrhoeagenic episodes, presence of domestic animals, and the general health of the child. Faecal (n =300), food (n = 167) and water (n = 100) samples were collected for diarrhoeagenic bacterial identification. Escherichia coli strains were identified from food and water samples using the Bacteriological Analytical Manual (BAM) protocol developed by the U.S. Food and Drug Administration (FDA). A technical reference guide produced by the Ministério da Saúde de Moçambique (Mozambique’s Ministry of Health) was also used. This analysis was conducted at the Hygiene and Food Technology Laboratory of Eduardo Mondlane University. On the other hand, E. coli strains in faecal samples were identified based on colony morphology characteristics and biochemical tests, performed at the National Institute of Health. The confirmed E. coli strains were whole genome sequenced at the Agricultural Research Council, Pretoria, South Africa. The fastq files of the identified DEC strains were molecularly characterised, and their relatedness was evaluated using the EnteroBase platform. Explanatory variables associated with DEC in children and DEC/Salmonella in food were analysed using chi-square tests and binomial logistic regression. Antimicrobial resistance (AMR) gene profiles were explored through genome analysis using hierarchical clustering, Pearson correlation, and network analysis in R. Only Enteropathogenic E. coli (EPEC) pathotypes (2.0%) were detected in faecal samples of children involved in this study, representing a low prevalence compared to the overall burden of childhood diarrhoea in Mozambique (9.0%). Feeding children with yoghurt was seen as a protective factor against diarrhoea by EPEC. Food and drinking water consumed by the children under five with diarrhoea from Maputo were contaminated with enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC) and EPEC strains, with a prevalence of 13.0%. Salmonella spp. were also detected, with a prevalence of 2.2%. Feeding children with infant formula, fruit puree, ready-to-eat meals, and bottled water was associated with DEC. There was an abundance of DEC strains belonging to sequence types (STs) ST40 (40.0%), ST206 (20.0%), and ST301 (20.0%) and serotypes O109:H21 (40.0%), O88:H5 (20.0%) and O80:H2. These strains harboured antimicrobial resistance genes conferring resistance to aminoglycosides, fosfomycin, polymyxins, beta-lactam, fluoroquinolone, trimethoprim, tetracycline and sulphonamides. Phylogenetic analysis revealed the relatedness of the DEC strains detected in food, drinking water and childrens’s faecal samples. This suggests that food and drinking water are the potential sources of DEC contributing to diarrhoea in the children under five years old in Maputo. With the present study, we conclude that diarrhoeagenic pathogens were present in faecal samples from children under five with diarrhoea (EPEC) and in food and water they consume (ETEC, EIEC, EPEC and Salmonella spp.). Foods were the main factors associated with these pathogens (yoghurt, infant formula, fruit puree, ready-to-eat meals, and bottled water). The DEC strains belonged to ST and serotypes known to cause diarrhoea, and they carry AMR genes that confer resistance to antimicrobials that are important for clinical practices. Food and drinking water are the potential sources of DEC in children under five years involved in this study. The results of this study provide valuable data on the contribution of food and water to diarrhoea in children, addressing a gap in existing research that has primarily focused on detecting diarrhoeagenic pathogens in faecal samples. In addition, the study demonstrates that DEC strains present in food and water consumed by children in Maputo harbour AMR genes, underscoring the importance of preventing contamination by these pathogens. So, the results of this study highlight the need for education on proper food preparation, storage, and water treatment practices, as well as the adoption of a One Health approach to monitor, prevent, and control the spread of antimicrobial resistance across sectors. The study also underscores the need for further research on caregivers’ attitudes and practices related to food preparation. It also calls for investigation of additional sources contributing to diarrhoea in children under five years old.