Fulton P. Rivera

Genotypic and Phenotypic Characterization of Enterotoxigenic Escherichia coli Strains Isolated from Peruvian Children

ABSTRACT 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.

Frecuencia y patotipos de Escherichia coli diarrogénica en niños peruanos con y sin diarrea

UNLABELLED INTRODUCTION; Diarrheagenic E. coli (DEC) are a major cause of diarrhea in children in developing countries. However, they are not part of routine diagnosis in clinical laboratories. OBJECTIVES To determine the DEC prevalence in Peruvian children and to describe the genetic variability of these strains. MATERIALS AND METHODS A total of 8 003 E. coli strains previously isolated from eight different studies of diarrhea in children, mainly from peri-urban areas of Lima, were analyzed. Diagnosis of DEC was done with Multiplex real-time PCR using genes for each of the 6 DEC groups. Conventional PCR was performed for the detection of additional virulence genes. RESULTS Globally, the mean prevalence in diarrhea samples (n=4,243) was: enteroaggregative E. coli (EAEC) 9.9%, enteropathogenic E. coli (EPEC) 8.5%, enterotoxigenic E. coli (ETEC) 6.9%, diffusely adherent E. coli (DAEC) 4.8%, Shiga toxin-producing E. coli (STEC) 0.8% and enteroinvasive E. coli (EIEC) 0.6%. The relative frequency of each pathogen varies according to the age and the type of study. The main pathotypes in control samples (n=3,760) were EPEC (10.9%) and EAEC (10.4%). An important variability in the virulence genes frequency and molecular resistance mechanisms for each pathotype was found, without differences between diarrhea and control groups. CONCLUSIONS DEC are a major cause of diarrhea in Peruvian children. These pathogens are highly heterogeneous. Additional studies are required to determine the prevalence in rural areas of Peru and in severe diarrhea cases.

Diarrheagenic Escherichia coli in Human Immunodeficiency Virus (HIV) Pediatric Patients in Lima, Perú

We conducted a prospective study in three hospitals in Lima in human immunodeficiency virus (HIV) children to determine the frequency of diarrheagenic Escherichia coli. Five E. coli colonies/patients were studied by a multiplex real-time polymerase chain reaction to identify the six currently recognized groups of diarrhea-associated E. coli. We have analyzed 70 HIV-associated diarrheal and 70 control samples from HIV-infected children without diarrhea. Among the diarrheal episodes 19% were persistent, 3% dysenteric, and 33% were associated with moderate or severe dehydration. The diarrheagenic E. coli were the most commonly isolated pathogens in diarrhea (19%) and control samples (26%) (P = 0.42), including enteroaggregative (6% versus 10%), enteropathogenic (6% versus 10%), and enterotoxigenic E. coli (4% versus 3%), respectively. The HIV-infected children with diarrhea had the worse age-related immunosuppression, higher viral loads, and were on highly active antiretroviral treatment (HAART) less often than HIV-infected children without diarrhea. Diarrheagenic E. coli were highly resistant to ampicillin (74%) and cotrimoxazole (70%).

Phylogenetic relationships of Shiga toxin-producing Escherichia coli isolated from Peruvian children

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 toxin-producing 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.

Genotypic Characterization of Enterotoxigenic Escherichia coli Strains Causing Traveler's Diarrhea

ABSTRACT This study aims to characterize the presence of virulence factors of enterotoxigenic Escherichia coli (ETEC) causing travelers diarrhea. Among 52 ETEC isolates, the most common toxin type was STh, and the most frequent colonization factors (CFs) were CS21, CS6, and CS3. On the other hand, the nonclassical virulence factors EAST1 and EatA were frequently present.

Norovirus prevalence in 'pathogen negative' gastroenteritis in children from periurban areas in Lima, Peru.

Norovirus was detected in 17.4% of 224 diarrhoeal samples from children younger than 24 months of age in Lima, in whom all common pathogens had been excluded (pathogen negative). Norovirus was identified more frequently in children older than 12 months of age than in younger children (34% vs 8%, P<0.001). Among norovirus-positive samples, genogroup II was the predominant group (92%). Compared with rotavirus, norovirus episodes tended to be of shorter duration and less severe. The role of norovirus as a cause of diarrhoea and the ascertainment of its severity in developing countries needs further confirmation by future epidemiological studies.

Bovine Lactoferrin Decreases Cholera-Toxin-Induced Intestinal Fluid Accumulation in Mice by Ganglioside Interaction

Secretory diarrhea caused by cholera toxin (CT) is initiated by binding of CT’s B subunit (CTB) to GM1-ganglioside on the surface of intestinal cells. Lactoferrin, a breast milk glycoprotein, has shown protective effect against several enteropathogens. The aims of this study were to determine the effect of bovine-lactoferrin (bLF) on CT-induced intestinal fluid accumulation in mice, and the interaction between bLF and CT/CTB with the GM1-ganglioside receptor. Fluid accumulation induced by CT was evaluated in the mouse ileal loop model using 56 BALB/c mice, with and without bLF added before, after or at the same time of CT administration. The effect of bLF in the interaction of CT and CTB with GM1-ganglioside was evaluated by a GM1-enzyme-linked immunosorbent assay. bLF decreased CT-induced fluid accumulation in the ileal loop of mice. The greatest effect was when bLF was added before CT (median, 0.066 vs. 0.166 g/cm, with and without bLF respectively, p<0.01). We conclude that bLF decreases binding of CT and CTB to GM1-ganglioside, suggesting that bLF suppresses CT-induced fluid accumulation by blocking the binding of CTB to GM1-ganglioside. bLF may be effective as adjunctive therapy for treatment of cholera diarrhea.

Prevention of renal damage caused by Shiga toxin type 2: Action of Miglustat on human endothelial and epithelial cells

Typical hemolytic uremic syndrome (HUS) is responsible for acute and chronic renal failure in children younger than 5 years old in Argentina. Renal damages have been associated with Shiga toxin type 1 and/or 2 (Stx1, Stx2) produced by Escherichia coli O157:H7, although strains expressing Stx2 are highly prevalent in Argentina. Human glomerular endothelial cells (HGEC) and proximal tubule epithelial cells are very Stx-sensitive since they express high levels of Stx receptor (Gb3). Nowadays, there is no available therapy to protect patients from acute toxin-mediated cellular injury. New strategies have been developed based on the Gb3 biosynthesis inhibition through blocking the enzyme glucosylceramide (GL1) synthase. We assayed the action of a GL1 inhibitor (Miglustat: MG), on the prevention of the renal damage induced by Stx2. HGEC primary cultures and HK-2 cell line were pre-treated with MG and then incubated with Stx2. HK- 2 and HGEC express Gb3 and MG was able to decrease the levels of this receptor. As a consequence, both types of cells were protected from Stx2 cytotoxicity and morphology damage. MG was able to avoid Stx2 effects in human renal cells and could be a feasible strategy to protect kidney tissues from the cytotoxic effects of Stx2 in vivo.

High Prevalence and Increased Severity of Norovirus Mixed Infections Among Children 12–24 Months of Age Living in the Suburban Areas of Lima, Peru

In an active diarrhea surveillance study of children aged 12-24 months in Lima, Peru, norovirus was the most common pathogen identified. The percentage of mixed (bacterial and noroviral) infections was significantly higher among norovirus-positive samples (53%) than among norovirus-negative samples (12%). The combination of norovirus with the most common bacterial pathogens was associated with increased clinical severity over that of either single-pathogen norovirus or single-pathogen bacterial infections.

Comparative analysis of antimicrobial resistance in enterotoxigenic Escherichia coli isolates from two paediatric cohort studies in Lima, Peru

BACKGROUND Antibiotic resistance is increasing worldwide, being of special concern in low- and middle-income countries. The aim of this study was to determine the antimicrobial susceptibility and mechanisms of resistance in 205 enterotoxigenic Escherichia coli (ETEC) isolates from two cohort studies in children <24 months in Lima, Peru. METHODS ETEC were identified by an in-house multiplex real-time PCR. Susceptibility to 13 antimicrobial agents was tested by disk diffusion; mechanisms of resistance were evaluated by PCR. RESULTS ETEC isolates were resistant to ampicillin (64%), cotrimoxazole (52%), tetracycline (37%); 39% of the isolates were multidrug-resistant. Heat-stable toxin producing (ETEC-st) (48%) and heat-labile toxin producing ETEC (ETEC-lt) (40%) had higher rates of multidrug resistance than isolates producing both toxins (ETEC-lt-st) (21%), p<0.05. Only 10% of isolates were resistant to nalidixic acid and none to ciprofloxacin or cefotaxime. Ampicillin and sulfamethoxazole resistance were most often associated with blaTEM (69%) and sul2 genes (68%), respectively. Tetracycline resistance was associated with tet(A) (49%) and tet(B) (39%) genes. Azithromycin inhibitory diameters were ≤15 mm in 36% of isolates, with 5% of those presenting the mph(A) gene. CONCLUSIONS ETEC from Peruvian children are often resistant to older, inexpensive antibiotics, while remaining susceptible to ciprofloxacin, cephalosporins and furazolidone. Fluoroquinolones and azithromycin remain the drugs of choice for ETEC infections in Peru. However, further development of resistance should be closely monitored.