Determinants of diarrhea, phenotypic and genomic characterization of Escherichia coli isolates from diarrheic and non-diarrheic patients attending public primary health facilities in Addis Ababa and Hossana, Ethiopia
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Date
2025-01
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Addis Ababa University
Abstract
Background: Diarrheal diseases are significant public health challenge in low- and middle-income countries. Escherichia coli (E. coli) is a common cause of diarrhea. It is genetically diverse bacterium, characterized by different strains that are classified into various pathotypes based on their virulence. Many strains of E. coli carry mobile genetic elements (MGEs) that encode antimicrobial resistance genes (ARGs) and virulence factors, thus improving their adaptability and survival. The genetic diversity and antimicrobial resistance characteristics of E. coli in Ethiopia remain poorly studied despite their important role in human diseases.
Objectives: to assess determinants of diarrhea, investigate antimicrobial resistance characteristics of E. coli isolates, and assess the genetic diversity of E. coli isolated from stool samples of patients attending public primary healthcare facilities.
Methods: This study was conducted from December 2021 to September 2022 at health facilities in Addis Ababa and Hossana. We used a health facility based matched case-control and cross-sectional study designs. Participants, cases (366 patients with diarrhea) and controls (366 patients without diarrhea) were recruited by convenience sampling technique for assessing determinants of diarrhea, and conditional logistic regression model was used in Stata version 14.0 to examine the determinants of diarrheal diseases. Conventional culture techniques and biochemical tests were used for the isolation and preliminary identification of E. coli isolates. A total of 260 E. coli isolates, confirmed by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) mass spectrometry, were used for further analysis. Phenotypic antimicrobial resistance was determined using the disc diffusion method according to the guidelines of the European Committee on Antimicrobial
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Susceptibility Testing (EUCAST). Real-time PCR tests were used to identify the diarrheagenic E. coli (DEC) pathotypes. Selected E. coli isolates were then subjected to whole genome sequencing (WGS) on the Illumina platform and bioinformatic analysis was performed to characterize the E. coli isolates.
Results: Statistical analysis revealed that being partially vaccinated (AOR: 2.70; 95%CI: 1.2, 5.9), use of tap water (AOR: 2.20; 95%CI: 1.1, 4.4), use of protected well/spring water for drinking (AOR: 13.90; 95%CI: 3.7, 51.5), overcrowded sleeping arrangement (AOR: 1.55; 95%CI: 1.2, 1.8), having contact with animal feces/food (AOR: 15.10; 95%CI: 4.2, 53.6), the cleaning frequency of water-fetching materials (i.e. cleaned sometimes (AOR: 2.40; 95%CI: 1.2, 4.5) and rarely (AOR: 3.03; 95%CI: 1.2, 7.4)), and use of open pit latrine (AOR: 5.61; 95%CI: 1.5, 21.0) were significantly associated with increased odds of having diarrhea. The overall detection rate of DEC-specific virulence-associated genes (VAGs) was 11.5% among the E. coli isolates tested. Four common pathotypes were identified, with enteropathogenic E. coli (EPEC) being the predominant pathotype. A hybrid pathotype carrying virulence factors from necrotoxic E. coli (NTEC2) and Shiga toxin-producing E. coli (STEC) strains was detected using whole-genome sequence (WGS) analysis. The studied isolates were distributed across seven phylogenetic groups (A, B1, B2, C, D, E and G). The predominant (49%) E. coli isolates belonged to the phylogenetic group B1. The DEC isolates were observed in four of the identified phylogenetic groups. The isolates belonged to 36 different sequence types (STs) and 42 different serotypes, of which 8 isolates (15.7%) were categorized as ‘rough,’ including five DEC isolates from the pathotypes STEC, EPEC and enterotoxigenic E. coli (ETEC). The isolates carried a total of 131 VAGs, including DEC specific genes (eae, aggR, eltIAB-8, eltIAB-11, estah-STa3, stx1, stx2). Overall 54.2% of E coli isolates were resistant to at least one antimicrobial agent tested. The highest resistance (52.7%) was observed for ampicillin. Out of all isolates, 19.2% were multidrug-resistant (MDR), and 8.5% were extended-spectrum -lactamase (ESBL) producers. Our results indicated that 82.4% of the sequenced E. coli isolates harbored 46 different ARGs that confer resistance to different antimicrobials. The blaTEM-1B gene was the most commonly detected -lactamase-encoding
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gene in both DEC and non-DEC isolates. No resistance to any of the tested antimicrobial was detected in STEC isolates and no ARGs were detected in these isolates. Seventy percent of the isolates resistant to third-generation cephalosporins and 78.9% of the phenotypically confirmed ESBL-producing E. coli isolates carried the blaCTX-M-15 gene. Mutations in the gyrA, parC, and/or parE genes were observed in 31.4% of the tested isolates. E. coli isolates from all phylogenetic groups contained various ARGs, with the highest frequencies being in phylogroups A and B1. In contrast, E. coli isolates from phylogenetic group B2 contained only four ARGs. Except for three isolates, all tested E. coli isolates were found to carry between one and eleven plasmids. The IncF group of plasmids with different replicon types and variants was the predominant plasmid group. Additionally, genetic determinants associated with conjugative transfer were identified in 56.9% of the isolates. The ISEc1 and IS609 were the most frequently detected IS elements in the tested E. coli isolates. Transposons such as Tn2, Tn7, Tn5403, Tn6024, and Tn6082 were detected in E. coli isolates tested.
Conclusion: The occurrence of diarrheal diseases was influenced by various factors, including the vaccination status of children, drinking water sources, crowded sleeping arrangements, exposure to animal feces or food, occasional or infrequent cleaning of water collection items, and the absence of toilet facilities or reliance on open latrines. Considerable genetic diversity was observed among E. coli isolates. The isolates had a broad spectrum of antimicrobial resistance and a high proportion of self-transferable conjugative elements, indicating a significant potential for horizontal gene transfer and the spread of clinically important virulence and antibiotic resistance traits.
Recommendations: Implementation of strategies to control the infection and spread of this health threat is essential. Enhanced surveillance covering a large geographical area will help to address this growing public health challenge.
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Keywords
Diarrhea, Escherichia coli, antimicrobial resistance, genetic diversity, phylogeny, antimicrobial resistance genes, virulence-associated genes, mobile genetic elements, phylogenetic groups, ESBL, whole-genome sequencing, Ethiopia