Prevalence, virulence factors and drug resistance of enteroaggregative Escherichia coli isolated from children under five years old in Kiambu County Hospital

Enteroaggregative Escherichia coli (EAEC) is recognized as the leading bacterial pathogen in children hospitalized for acute diarrheal illness reflecting a significant cost in health resources. However, asymptomatic carriage of EAEC is also high making it difficult to distinguish the pathogenic and the non-pathogenic strains of EAEC. Establishing a marker which corresponds with diarrheal illness would be of significant value in routine detection of pathogenic EAEC and its differentiation from nonpathogenic EAEC. The aim of this study was to determine the prevalence and identify the putative virulence factors of EAEC between children admitted with diarrhea and in agematched asymptomatic children under five years old. In this study, 354 fecal samples from diarrheal patients and 354 from asymptomatic controls collected during a casecontrol study in Kiambu County, Kenya, were analyzed. Microbial culture and biochemical analysis were carried out for confirmatory identification of Escherichia coli (E. coli). At least five different colonies from each sample identified as E. coli were then examined by Multiplex-PCR for determination of EAEC and for the detection of the various putative virulence genes. Enteroaggregative Escherichia coli was detected in 60/354 (16.95%) diarrheal patients and 60/354 (16.95%) asymptomatic controls. Genes that encode a transcriptional regulator and E. coli heat-stable enterotoxin 1, aggR and astA, respectively, were detected significantly more in patients than in controls (P=0.0271 and 0.0443, respectively). Usher, aggregative adherence fimbriae (AAF) III/IV assembly unit, agg3/4C, was not detected in any EAEC strains from patients while 93.2% of the EAEC strains from asymptomatic controls were found positive for the gene. The most frequent AAF pilin gene was that of AAF/I encoded by aggA (10% and 20% in patients and asymptomatic controls respectively) followed by those of AAF/IV (agg4A), AAF/II (aafA) and AAF III (agg3A) respectively. Other virulence associated genes including pet, orf61, sepA and eilA were not detected in any of the EAEC from patients while their prevalence in asymptomatic controls was 3.3%, 59.3%, 17% and 86%, respectively. In typical EAEC astA, aatA, agg4A and aggA were detected more in patients than in asymptomatic controls. However, no statistical difference was observed in their distribution between patients and asymptomatic controls on typical EAEC. In atypical EAEC, astA was detected significantly more in patients than in asymptomatic controls (P=0.0059, OR =4.17). Several different combinations of the virulence markers were found among the EAEC isolates in this study. High resistance rate was observed in amoxicillin, trimethoprim/sulfamethoxazole and tetracycline. Among the penicillin, sulfonamide and tetracycline groups, most of the EAEC pathogens showed multi-drug resistant patterns. Data from this study showed a high prevalence of EAEC in diarrheal patients in the study area. However asymptomatic carriage of EAEC is equally high. The study also reveals a high degree of variability of genotypic markers in EAEC isolated from Kiambu County. These results underscore the potential usefulness of the aggR and astA genes as possible virulence markers for detection and differentiation of virulent strains of EAEC from avirulent strains. This study also indicates that a certain subset of EAEC is unrelated to diarrhea, for which agg3/4C may be a marker. Overall, the markers reported here can find great application in routine detection of EAEC.