Escherichia coli flora and diarrhea in Nicaraguan children

Abstract

A combination of a phenotyping method (PhP typing) for the identification of clonal groups, and a genotyping method (PCR) for the identification of virulent strains of E. coli were used in order to obtain insight into diarrheal disease morbidity and epidemiology of diarrheagenic E. coli strains in children in León, Nicaragua. Fecal samples from 381 children aged 0-5 years suffering from diarrheal disease and 145 healthy children of the same age were analyzed. Besides, a cohort of 20 healthy infants aged 0-12 months were followed by repeated sampling (8 samples per infant). All samples were analyzed by a multiplex PCR (EAEC, ETEC; EPEC, EHEC, EIEC markers) on the primary streak and from the 282 positive samples 2,164 E. coli isolates were analyzed by single-colony PCR, and from all samples, eight E. coli isolates were phenotyped; totally 4,753 E. coli isolates were analyzed.

Our main results are:

1) Diarrheagenic E. coli (DEC) positive samples were found at high rates in infants aged 3-60 months (Paper I), but were rare in infants below 3 months of age (Paper IV). Thus, colonization by DECs seem to start early in life and increase with age.

2) The phenotypic diversities among all isolates from diarrheal cases and from healthy children were equal and high, thus giving indication that no large outbreak of DEC diarrhea occurred during the time period studied (Paper II).

3) EAEC and EPEC could not be correlated to diarrhea, whereas ETEC and EHEC were significantly more common in diarrheal cases (Papers I, II, and III).

4) ETEC estA and EHEC were only found in diarrheal cases, and isolates positive for these DEC types belonged to a few PhP types that could represent pathogenic clones (Paper III).

5) DEC positive isolates seldom dominated the fecal E. coli flora, but for all DEC types, except for EAEC, a high proportion of DEC positive isolates in the fecal E. coli flora seemed to be correlated to diarrhea. Thus, a low proportion of DEC in the fecal E. coli flora may not be of significance for development of diarrhea, but may represent occasional carriage of virulence gene(s) by normal flora strains (Paper III).

6) Newborns were found to rapidly exchange their E. coli strains during their first year of life. Colonization by single DEC positive isolates was rarely correlated to diarrheal disease (Paper IV).

Our findings suggest that there are certain stable types or clones of diarrheagenic E. coli that can circulate in a population, and have the possibility to colonize the intestine and to dominate the E. coli flora, thereby causing diarrhea. These are the true DECs. However, the virulence genes are promiscuous and may spread to the E. coli bacteria of the normal flora, whereupon these scarce gene markers are detected by the sensitive PCR test in samples from both healthy and diseased individuals, without necessarily having a pathogenic role.

Thus, the obtained information on the distribution of DEC pathotypes and their diversities may be used to optimize the use of today’s diagnostic tools, which in turn might affect treatment approaches and vaccine development strategies.