Quantitative real-time polymerase chain reaction for enteropathogenic Escherichia coli: a tool for investigation of asymptomatic versus symptomatic infections

Abstract

Background: Enteropathogenic Escherichia coli (EPEC) strains are pediatric pathogens commonly isolated from both healthy and sick children with diarrhea in areas of endemicity. The aim of this study was to compare the bacterial load of EPEC isolated from stool samples from children with and without diarrhea to determine whether bacterial load might be a useful tool for further study of this phenomenon.

Methods: EPEC was detected by polymerase chain reaction (PCR) of colonies isolated on MacConkey plates from 53 diarrheal and 90 healthy children aged <2 years. DNA was isolated from stool samples by cetyltrimethylammonium bromide extraction. To standardize quantification by quantitative real-time PCR (qRT-PCR), the correlation between fluorescence threshold cycle and copy number of the intimin gene of EPEC E2348/69 was determined.

Results: The detection limit of qRT-PCR was 5 bacteria/mg stool. The geometric mean load in diarrhea was 299 bacteria/mg (95% confidence interval [CI], 77-1164 bacteria/mg), compared with 29 bacteria/mg (95% CI, 10-87 bacteria/mg) in control subjects (P = .016). Bacterial load was significantly higher in children with diarrhea than in control subjects among children <12 months of age (178 vs 5 bacteria/mg; P = .006) and among children with EPEC as the sole pathogen (463 vs 24 bacteria/mg; P = .006).

Conclusions: EPEC load measured by qRT-PCR is higher in diarrheal than in healthy children. qRT-PCR may be useful to study the relationship between disease and colonization in settings of endemicity.

Figure 1.

Quantitative real-time polymerase chain reaction (RT-PCR) standardization for enteropathogenic Escherichia coli (EPEC). A, RT-PCR results from representative experiments using DNA from a pure culture of EPEC E2348/69. Fluorescence from the PCR products is plotted against the corresponding number of copies of intimin (eaeA) gene, corresponding to 10¹ to 10⁶ bacilli, to obtain the threshold cycle (C_T). B, Standard curve for the RT-PCR analysis was done from the same stock of DNA diluted 10-fold. We plotted C_T against the log of the number of eaeA copies; the reaction efficiency was >97.3%. C, The melting temperature for the eaeA gene was 83.8 ± 0.23°C; curves are superimposed for the different DNA concentrations used in the analysis. D, Agarose gel (2%): (1–8) PCR products (248 pb) corresponding to the 10-fold dilutions (10⁸ to 10¹ bacilli); C, No template control; (M) 100-bp molecular weight ladder.

Figure 2.

Comparison of enteropathogenic Escherichia coli (EPEC) load among diarrhea and control samples. A, Diarrhea samples (gray bars) and samples from healthy control subjects (white bars). EPEC load in stool samples from children with (n = 53) and without (n = 90) diarrhea; *P = .016. B, EPEC load in children <12 months of age (diarrhea, n = 26; control, n = 31) and children 12–24 months of age (diarrhea, n = 27; control, n = 59); **P = .006. C, EPEC load in coinfections (diarrhea, n = 9; control, n = 13) and single-pathogen infection (diarrhea, n = 44; control, n = 77); ***P = .006.