Characterisation of enteric pathogens detected in day care centers in the Vhembe District, Limpopo Province, South Africa

Abstract

Background: Child daycare centres (DCCs) host a variety of pathogenic enteric microorganisms which may be found in various areas within the compound. These enteric pathogens are known to be the most common causes of childhood diarrhoea worldwide in children under the age of 5 years, leading to a high mortality rate in developing countries. The main objective of this study was therefore to characterise selected enteric pathogens detected in DCCs in the Vhembe region, Limpopo Province, South Africa. This study is among the first in South Africa to simultaneously investigate bacterial and viral enteric pathogens across multiple reservoirs, including children, daycare workers, environmental surfaces, toys and stored drinking water, using molecular and phylogenetic approaches. Method: This study was carried out in two randomly selected daycare centres (one rural and one peri-urban DCC) in the Vhembe region between 2019 and 2021. A total of 166 samples were randomly collected from handwash samples of daycare workers (N=26) and children (N=39), swabs from the daycare environment (N=45), stool samples from the children’s diapers (N=38), stored drinking water containers (N=8), and children’s toys (N=10). For bacterial analysis, the Colilert® Quanti-Tray® 2000 was used to isolate Escherichia coli (E. coli) from all the samples collected. Stool samples were directly processed for extraction using a method based on a 96-well semi-automated extraction method. The membrane filtration technique was used for the isolation of Salmonella and Shigella. Stool samples were cultured on Salmonella-Shigella agar. Total nucleic acid extraction of presumptive E. coli, isolates were done using a 96-well extraction method. Extraction of Salmonella and Shigella was done using the Qiagen extraction kit. Extracted DNA was used for multiplex polymerase chain reaction (m-PCR) using specific primer sets. Positive samples were randomly selected for sequencing. For viral analysis, Norovirus (NoV), Rotavirus (RV) and Sapovirus (SaV) were identified using the membrane filtration technique, while RNA from stool samples was extracted using a 96-well semi-automated extraction method. Detection of these selected viruses was performed using the Seegene Allplex™ Gastrointestinal Full Panel Assay. Positive samples for NoV, RV and SaV were amplified using One-Step Ahead Reverse Transcription Polymerase Chain Reaction (One-Step RT-PCR) followed by Sanger sequencing. Phylogenetic analysis was performed using MEGA X. Results: The prevalence of bacteria was 38% (63/166) for E. coli, while no Salmonella or Shigella were detected. The predominant E. coli pathotype was EAEC, which was successfully sequenced (12 from the rural and 7 from the peri-urban DCC), and the isolates showed that they shared a common ancestor with strains circulating globally and causing outbreaks of diarrhoea in children under 5 years of age. The prevalence of viruses was RV 13% (22/166), NoV 7% (12/166; NoV GI, 2/166 and NoV GII, 10/166), and SaV 3% (5/166). Genotyping of RV showed that all positive samples were RV A, while NoV sequencing identified norovirus GII.1, although GII.4 remains the predominant strain circulating globally. No statistical association was observed between clinical symptoms and age among children handwash samples (p = 0.328), or between symptoms and the number of positive cases among participants (p = 0.161). However, an association between DCC and diarrhoea prevalence was observed (p=0.0394). Single bacterial and viral pathogen detections were observed in both the rural and peri-urban DCCs. Bacteria-bacteria and bacteria-viral co-detections were also observed in both DCCs. Conclusion: The high prevalence of diarrhoea-associated E. coli pathotypes, NoV (GI and GII), RV A and SaV circulating in the DCCs is of concern, as these pathogens are associated with high mortality among children under the age of 5 years. Furthermore, phylogenetic analysis indicated that the detected E. coli strains identified in both DCCs were closely related, suggesting possible transmission within the daycare environment. The detection of enteric pathogens in children, staff, environmental surfaces, toys and stored drinking water highlights the interconnected human-environment interface and underscores the importance of a One Health approach to understanding and controlling enteric disease transmission. The results from this study will provide a basis for further investigations into transmission pathways and will inform local health authorities. In addition, the findings provide important baseline data for strengthening molecular surveillance of enteric pathogens in South African daycare centres and similar settings across Africa, contributing to evidence-based public health policies, improved infection prevention and control strategies, and the development of routine surveillance systems for childhood diarrhoeal diseases. The study also supports future longitudinal studies, implementation of genomic surveillance, antimicrobial susceptibility testing and integrated environmental monitoring to improve outbreak detection, preparedness and response.