David Durand

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.

Fecal Leukocytes in Children Infected with Diarrheagenic Escherichia coli

ABSTRACT The purpose of this study was to determine the presence and quantity of fecal leukocytes in children infected with diarrheagenic Escherichia coli and to compare these levels between diarrhea and control cases. We analyzed 1,474 stool samples from 935 diarrhea episodes and 539 from healthy controls of a cohort study of children younger than 2 years of age in Lima, Peru. Stools were analyzed for common enteric pathogens, and diarrheagenic E. coli isolates were studied by a multiplex real-time PCR. Stool smears were stained with methylene blue and read by a blinded observer to determine the number of polymorphonuclear leukocytes per high-power field (L/hpf). Fecal leukocytes at >10 L/hpf were present in 11.8% (110/935) of all diarrheal episodes versus 1.1% (6/539) in controls (P < 0.001). Among stool samples with diarrheagenic E. coli as the only pathogen isolated (excluding coinfection), fecal leukocytes at >10 L/hpf were present in 8.5% (18/212) of diarrhea versus 1.3% (2/157) of control samples (P < 0.01). Ninety-five percent of 99 diarrheagenic E. coli diarrhea samples were positive for fecal lactoferrin. Adjusting for the presence of blood in stools, age, sex, undernutrition, and breastfeeding, enterotoxigenic E. coli (ETEC) isolation as a single pathogen, excluding coinfections, was highly associated with the presence of fecal leukocytes (>10 L/hpf) with an odds ratio (OR) of 4.1 (95% confidence interval [CI], 1.08 to 15.51; P < 0.05). Although diarrheagenic E. coli was isolated with similar frequencies in diarrhea and control samples, clearly it was associated with a more inflammatory response during symptomatic infection; however, in general, these pathogens elicited a mild inflammatory response.

Virulence factors and mechanisms of antimicrobial resistance in Shigella strains from periurban areas of Lima (Peru)

The study was aimed to describe the serotype, mechanisms of antimicrobial resistance, and virulence determinants in Shigella spp. isolated from Peruvian children. Eighty three Shigella spp. were serogrouped and serotyped being established the antibiotic susceptibility. The presence of 12 virulence factors (VF) and integrase 1 and 2, along with commonly found antibiotic resistance genes was established by PCR. S. flexneri was the most relevant serogroup (55 isolates, 66%), with serotype 2a most frequently detected (27 of 55, 49%), followed by S. boydii and S. sonnei at 12 isolates each (14%) and S. dysenteriae (four isolates, 5%). Fifty isolates (60%) were multi-drug resistant (MDR) including 100% of S. sonnei and 64% of S. flexneri. Resistance levels were high to trimethoprim-sulfamethoxazole (86%), tetracycline (74%), ampicillin (67%), and chloramphenicol (65%). Six isolates showed decreased azithromycin susceptibility. No isolate was resistant to nalidixic acid, ciprofloxacin, nitrofurantoin, or ceftriaxone. The most frequent resistance genes were sul2 (95%), tet(B) (92%), cat (80%), dfrA1 (47%), blaOXA-1like (40%), with intl1 and intl2 detected in 51 and 52% of the isolates, respectively. Thirty-one different VF profiles were observed, being the ipaH (100%), sen (77%), virA and icsA (75%) genes the most frequently found. Differences in the prevalence of VF were observed between species with S. flexneri isolates, particularly serotype 2a, possessing high numbers of VF. In conclusion, this study highlights the high heterogeneity of Shigella VF and resistance genes, and prevalence of MDR organisms within this geographic region.

In Vitro Development and Analysis of Escherichia coli and Shigella boydii Azithromycin–Resistant Mutants

The aim of this study was to develop and analyze in vitro azithromycin (AZM)-resistant mutants of Escherichia coli and Shigella boydii. Three clinical isolates of E. coli and one S. boydii isolated from feces samples collected from children under 5 years of age with diarrhea in Lima, Peru were inoculated onto Mueller-Hinton plates containing increasing serial dilutions of AZM ranging from their specific minimal inhibitory concentration (2 or 4 mg/l) to 64 mg/l. From these plates, 16 AZM-resistant mutants were selected to determine the stability of the resistance and the presence of cross resistance with other antibiotics. The role of Phe-Arg-β-Naphthylamide (PAβN)-inhibitible efflux pumps as well as the presence of mutations in the rplV, rplD, and rrlH (23S rRNA) genes and alterations in the outer membrane profiles were determined in these 16 mutants. The rate of mutation ranged from < 2.70×10(-10) to 2.17×10(-7) for E. coli and from < 9.58×10(-10) to 1.05×10(-8) for S. boydii. E. coli mutants showed an increase in the AZM-MIC up to sixfold with one strain achieving a MIC >256 mg/l. In contrast, S. boydii only presented increases of up to twofold in MIC levels. All the strains obtained, but one showed stable AZM resistance. In the presence of PAβN, the AZM MICs decreased to parental levels in Shigella mutants, while no MIC returned to parental levels among the E. coli mutants. No cross resistance to other classes of antibiotics was found. These results show the relevance of PAβN-inhibitible efflux pumps in the basal levels and development of AZM resistance. Further studies to characterize the remaining unidentified mechanisms of AZM resistance are needed.

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

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 sub-alleles, 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.

Detection of secretory immunoglobulin A in human colostrum as mucosal immune response against proteins of the type III secretion system of Salmonella, Shigella and enteropathogenic Escherichia coli.

Background: Some enteropathogens use the type III secretion system to secrete proteins that allows them to interact with enterocytes and promote bacterial attachment or intracellular survival. These proteins are Salmonella invasion proteins (Sip), invasion plasmid antigens (Ipa) of Shigella and Escherichia coli secreted proteins (Esp) of enteropathogenic E. coli. There are no previous studies defining the presence of colostral sIgA against all these 3 major enteric pathogens. Objective: To evaluate the presence of sIgA in colostrum against proteins of the type III secretion system of Salmonella, Shigella and enteropathogenic E. coli. Methods: We collected 76 colostrum samples from puerperal women in Lima, Peru. These samples were reacted with type III secretion system proteins extracted from bacterial culture supernatants and evaluated by Western Blot. Results: Antibodies were detected against Salmonella antigens SipA in 75 samples (99%), SipC in 62 (82%) and SipB in 31 (41%); against Shigella antigens IpaC in 70 (92%), IpaB in 68 (89%), IpaA in 66 (87%) and IpaD in 41 (54%); and against enteropathogenic E. coli EspC in 70 (92%), EspB-D in 65 (86%) and EspA in 41 (54%). Ten percent of samples had antibodies against all proteins evaluated and 42% against all except 1 protein. There was no sample negative to all these proteins. Conclusions: The extraordinarily high frequency of antibodies in colostrum of puerperal women detected in this study against these multiple enteric pathogens shows evidence of immunological memory and prior exposure to these pathogens, in addition to its possible protective role against infection.

pic gene of enteroaggregative Escherichia coli and its association with diarrhea in Peruvian children

Enteroaggregative Escherichia coli (EAEC) causes acute and persistent diarrhea among children, HIV-infected patients, and travelers to developing countries. We have searched for 18 genes-encoding virulence factors associated with aggregative adherence, dispersion, biofilm, toxins, serine protease autotransporters of Enterobacteriaceae (SPATEs) and siderophores, analyzed in 172 well-characterized EAEC strains (aggR(+)) isolated from stool samples of 97 children with diarrhea and 75 healthy controls from a passive surveillance diarrhea cohort study in Peru. Eighty-one different genetic profiles were identified, 37 were found only associated with diarrhea and 25 with control samples. The most frequent genetic profile was aggC(+)aatA(+)aap(+)shf(+)fyuA(+), present in 19 strains, including diarrhea and controls. The profile set1A(+)set1B(+)pic(+) was associated with diarrhea (P < 0.05). Of all genes evaluated, the most frequent were aatA (CVD 342) present in 159 strains (92.4%) and fyuA in 157 (91.3%). When EAEC strains were analyzed as a single pathogen (excluding co-infections), only pic was associated with diarrhea (P < 0.05) and with prolonged diarrhea (diarrhea ≥ 7 days) (P < 0.05). In summary, this is the first report on the prevalence of a large set of EAEC virulence genes and its association with diarrhea in Peruvian children. More studies are needed to elucidate the exact role of each virulence factor.

Diarrheagenic Escherichia coli: Prevalence and Pathotype Distribution in Children from Peruvian Rural Communities

Diarrheagenic Escherichia coli (DEC) are common pathogens of childhood gastrointestinal infections worldwide. To date, research tracking DEC has mainly been completed in urban areas. This study aims to determine the prevalence and pathotype distribution of DEC strains in children from rural Peruvian communities and to establish their association with malnutrition. In this prospective cohort, 93 children aged 6-13 months from rural communities of Urubamba (Andes) and Moyobamba (jungle) were followed for 6 months. Diarrheal and control stool samples were analyzed using multiplex real-time polymerase chain reaction to identify the presence of virulence genes of DEC strains. The overall isolation rate of DEC was 43.0% (352/820). Enteroaggregative E. coli (EAEC, 20.4%), enteropathogenic E. coli (EPEC, 14.2%), and diffusely aggregative E. coli (DAEC, 11.0%) were the most prevalent pathotypes. EAEC was more frequently found in Moyobamba samples (P < 0.01). EPEC was the only strain significantly more frequent in diarrheal than asymptomatic control samples (P < 0.01). DEC strains were more prevalent among younger children (aged 6-12 months, P < 0.05). A decline in height-for-age Z-score (HAZ) was observed in 75.7% of children overall. EAEC was more frequently isolated among children who had a greater HAZ decline (P < 0.05). In conclusion, DEC strains were frequently found in stool samples from children in rural communities of the highlands and jungle of Peru. In addition, children with a greater decline in their growth rate had higher EAEC isolation rates, highlighting the importance of this pathogen in child malnutrition.

Which mechanisms of azithromycin resistance are selected when efflux pumps are inhibited

The aim of this study was to develop in vitro azithromycin (AZM)-resistant mutants of Escherichia coli and Shigella spp. in the presence of Phe-Arg β-naphthylamide (PAβN) and to observe which AZM resistance mechanisms other than efflux pumps were inhibited by PAβN emerge. The frequency of mutation ranged between <6.32 × 10(-10) and 5.22 × 10(-7) for E. coli and between <5.32 × 10(-10) and 1.69 × 10(-7) for Shigella spp. The E. coli mutants showed an increase in the AZM minimum inhibitory concentration (MIC) up to 128-fold, whilst the Shigella spp. mutants presented increases in MIC levels of up to 8-fold. In one mutant, the insertion of nucleotides encoding the amino acid sequence IMPRAS was found in the rplV gene. Increases in OmpW expression were observed in all E. coli mutants compared with their respective parental isolates. The combination of antibiotics and efflux pump inhibitors appears to be a good option to reduce the frequency of mutation in clinical isolates.

PATRONES DE ADHERENCIA DE CEPAS DE Escherichia coli DIFUSAMENTE ADHERENTE (DAEC) PROVENIENTES DE NIÑOS CON Y SIN DIARREA

RESUMEN Introduccion. Las E. coli de adherencia difusa (DAEC) son el sexto grupo de E. coli diarrogenicas reconocidas. Su asociacion con diarrea es controversial. No se conoce la variabilidad en los patrones de adherencia de cepas clinicas. Objetivos. Comparar los patrones de adherencia entre cepas aisladas de ninos con y sin diarrea. Materiales y metodos. Se analizo 31 cepas DAEC, 25 de diarrea y 6 de ninos asintomaticos (control) aislados de un estudio de cohorte de ninos menores de 12 meses en el cono sur de Lima. Las DAEC fueron identificadas por PCR (gen daaD). Se evaluo el patron y grado de adherencia en cultivos de celulas HEp-2; la polimerizacion de actina se evaluo por la prueba de coloracion de fluorescencia de actina (f AS); y la motilidad se evaluo por metodos convencionales microbiologicos. Resultados. El patron de adherencia difusa se encontro en el 88% de muestras de diarrea y en el 100% de muestras control. La cantidad de bacterias adheridas por celula fue significativamente menor en las muestras de diarrea (p<0,05). Sin embargo, la polimerizacion de actina fue mayor en las muestras de diarrea (60% frente a 17%). La prueba de motilidad fue positiva en el 60% de las cepas de diarrea y en el total de muestras control. Conclusiones. Nuestros hallazgos sugieren la existencia de diferencia en los patrones de adherencia, polimerizacion de actina y motilidad entre cepas de DAEC correspondientes a los grupos de diarrea y control. La significancia de estos resultados debe confirmarse con mayor numero de cepas, asi como la determinacion de los genes de virulencia en las cepas. Palabras clave: Escherichia coli de Adherencia Difusa; Diarrea; Nino; Adhesion Bacteriana; (fuente: DeCS BIREME).