• Genetic diversity of locus of enterocyte effacement genes of enteropathogenic Escherichia coli isolated from Peruvian children.

      Contreras, C. A.; Ochoa, T. J.; Ruiz, J.; Lacher, D. W.; Durand, D.; DebRoy, C.; Lanata, C. F.; Cleary, T. G. (Society for General Microbiology, 2014-03-19)
      The aim of this study was to determine the frequency and allele associations of locus of enterocyte effacement encoded esp and tir genes among 181 enteropathogenic Escherichia coli (EPEC) strains (90 diarrhoea-associated and 91 controls) isolated from Peruvian children under 18 months of age. We analysed espA, espB, espD and tir alleles by PCR-RFLP. EPEC strains were isolated with higher frequency from healthy controls (91/424, 21.7 %) than from diarrhoeal samples (90/936, 9.6 %) (P,0.001); 28.9% of diarrhoeal and 17.6% of control samples were typical EPEC (tEPEC). The distribution of espA alleles (alpha, beta, beta2 and gamma) and espD alleles (alpha, beta, gamma and a new variant, espD-N1) between tEPEC and atypical EPEC (aEPEC) was significantly different (P,0.05). espD-alpha was more common among acute episodes (P,0.05). espB typing resulted in five alleles (alpha, beta, gamma and two new suballeles, espB-alpha2 and espB-alpha3), while tir-beta and tir-gamma2 were the most common intimin receptor subtypes. Seventy-two combinations of espA, espB, espD and tir alleles were found; the most prevalent combination was espA-beta, espB-beta, espD-beta, tir-beta (34/181 strains), which was more frequent among tEPEC strains (P,0.05). Our findings indicate that there is a high degree of heterogeneity among EPEC strains isolated from Peruvian children and that aEPEC and tEPEC variants cluster.
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    • Genotypic and Phenotypic Characterization of Enterotoxigenic Escherichia coli Strains Isolated from Peruvian Children

      Rivera, F. P.; Ochoa, T. J.; Maves, R. C.; Bernal, M.; Medina, A. M.; Meza, R.; Barletta, F.; Mercado, E.; Ecker, L.; Gil, A. I.; Hall, E. R.; Huicho, L.; Lanata, C. F. (American Society for Microbiology, 2014-03-19)
      Enterotoxigenic Escherichia coli (ETEC) is a major cause of childhood diarrhea. The present study sought to determine the prevalence and distribution of toxin types, colonization factors (CFs), and antimicrobial susceptibility of ETEC strains isolated from Peruvian children. We analyzed ETEC strains isolated from Peruvian children between 2 and 24 months of age in a passive surveillance study. Five E. coli colonies per patient were studied by multiplex real-time PCR to identify ETEC virulence factors. ETEC-associated toxins were confirmed using a GM1-based enzyme-linked immunosorbent assay. Confirmed strains were tested for CFs by dot blot assay using 21 monoclonal antibodies. We analyzed 1,129 samples from children with diarrhea and 744 control children and found ETEC in 5.3% and 4.3%, respectively. ETEC was more frequently isolated from children >12 months of age than from children <12 months of age (P < 0.001). Fifty-two percent of ETEC isolates from children with diarrhea and 72% of isolates from controls were heat-labile enterotoxin (LT) positive and heat-stable enterotoxin (ST) negative; 25% and 19%, respectively, were LT negative and ST positive; and 23% and 9%, respectively, were LT positive and ST positive. CFs were identified in 64% of diarrheal samples and 37% of control samples (P < 0.05). The most common CFs were CS6 (14% and 7%, respectively), CS12 (12% and 4%, respectively), and CS1 (9% and 4%, respectively). ST-producing ETEC strains caused more severe diarrhea than non-ST-producing ETEC strains. The strains were most frequently resistant to ampicillin (71%) and co-trimoxazole (61%). ETEC was thus found to be more prevalent in older infants. LT was the most common toxin type; 64% of strains had an identified CF. These data are relevant in estimating the burden of disease due to ETEC and the potential coverage of children in Peru by investigational vaccines.
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    • Phylogenetic relationships of Shiga toxin-producing Escherichia coli isolated from Peruvian children

      Contreras, C. A.; Ochoa, T. J.; Ruiz, J.; Lacher, D. W.; Rivera, F. P.; Saenz, Y.; Chea-Woo, E.; Zavaleta, N.; Gil, A. I.; Lanata, C. F. (Society for General Microbiology (SGM), 2014-06-10)
      The aim of this study was to determine the prevalence, virulence factors (stx, eae, ehxA and astA) and phylogenetic relationships [PFGE and multilocus sequence typing (MLST)] of Shiga toxinproducing Escherichia coli (STEC) strains isolated from four previous cohort studies in 2212 Peruvian children aged ,36 months. STEC prevalence was 0.4% (14/3219) in diarrhoeal and 0.6% (15/2695) in control samples. None of the infected children developed haemolytic uraemic syndrome (HUS) or other complications of STEC. stx1 was present in 83% of strains, stx2 in 17 %, eae in 72 %, ehxA in 59% and astA in 14 %. The most common serotype was O26: H11 (14%) and the most common seropathotype was B (45 %). The strains belonged mainly to phylogenetic group B1 (52 %). The distinct combinations of alleles across the seven MLST loci were used to define 13 sequence types among 19 STEC strains. PFGE typing of 20 STEC strains resulted in 19 pulsed-field patterns. Comparison of the patterns revealed 11 clusters (I–XI), each usually including strains belonging to different serotypes; one exception was cluster VI, which gathered exclusively seven strains of seropathotype B, clonal group enterohaemorrhagic E. coli (EHEC) 2 and phylogenetic group B1. In summary, STEC prevalence was low in Peruvian children with diarrhoea in the community setting. The strains were phylogenetically diverse and associated with mild infections. However, additional studies are needed in children with bloody diarrhoea and HUS.
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