Maribel Riveros

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.

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.

Diarrheagenic Escherichia coli phylogroups are associated with antibiotic resistance and duration of diarrheal episode.

Conventionally, in Escherichia coli, phylogenetic groups A and B1 are associated with commensal strains while B2 and D are associated with extraintestinal strains. The aim of this study was to evaluate diarrheagenic (DEC) and commensal E. coli phylogeny and its association with antibiotic resistance and clinical characteristics of the diarrheal episode. Phylogenetic groups and antibiotic resistance of 369 E. coli strains (commensal strains and DEC from children with or without diarrhea) isolated from Peruvian children <1 year of age were determined by a Clermont triplex PCR and Kirby-Bauer method, respectively. The distribution of the 369 E. coli strains among the 4 phylogenetic groups was A (40%), D (31%), B1 (21%), and B2 (8%). DEC-control strains were more associated with group A while DEC-diarrhea strains were more associated with group D (P < 0.05). There was a tendency (P = 0.06) for higher proportion of persistent diarrhea (≥14 days) among severe groups (B2 and D) in comparison with nonsevere groups (A and B1). Strains belonging to group D presented significantly higher percentages of multidrug resistance than the rest of the groups (P > 0.01). In summary, DEC-diarrhea strains were more associated with group D than strains from healthy controls.

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.

Resistance to quinolones, cephalosporins and macrolides in Escherichia coli causing bacteraemia in Peruvian children

OBJECTIVES To characterise the β-lactam, quinolone and macrolide resistance levels and mechanisms in 62 Escherichia coli isolates causing bacteraemia in Peruvian children. METHODS Minimum inhibitory concentrations (MICs) of ciprofloxacin, nalidixic acid (NAL) and azithromycin were determined in the presence and absence of Phe-Arg-β-naphthylamide. Susceptibility to other 14 antimicrobial agents was also established. Extended-spectrum β-lactamases (ESBLs) were identified, and mutations in gyrA and parC as well as the presence of transferable mechanisms of quinolone resistance (TMQR) and macrolide resistance (TMMR) were determined. RESULTS Fifty isolates (80.6%) were multidrug-resistant. High proportions of resistance to ampicillin (93.5%), NAL (66.1%) and trimethoprim/sulfamethoxazole (66.1%) were observed. No isolate showed resistance to carbapenems and only two isolates were resistant to nitrofurantoin. Twenty-seven isolates carried ESBL-encoding genes: 2 blaSHV-12; 13 blaCTX-M-15; 4 blaCTX-M-2; 6 blaCTX-M-65; and 2 non-identified ESBLs. Additionally, 27 blaTEM-1 and 9 blaOXA-1-like genes were detected. All quinolone-resistant isolates showed target mutations, whilst TMQR were present in four isolates. Efflux pumps played a role in constitutive NAL resistance. The association between quinolone resistance and ESBL production was significant (P=0.0011). The mph(A) gene was the most frequent TMMR (16 isolates); msr(A) and erm(B) genes were also detected. Only one TMMR-carrying isolate [presenting mph(A) and erm(B) concomitantly] remained resistant to azithromycin when efflux pumps were inhibited. CONCLUSIONS A variety of ESBL-encoding genes and widespread of blaCTX-M-15 in Lima has been shown. The role of efflux pumps in azithromycin resistance needs to be further evaluated, as well as effective control of the use of antimicrobial agents.

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.

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.

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).

Virulence and Antimicrobial Resistance in Campylobacter spp. from a Peruvian Pediatric Cohort

The presence of virulence factors (VFs) and mechanisms of quinolones and macrolide resistance was analyzed in Campylobacter spp. from a pediatric cohort study in Lima. In 149 isolates (39 Campylobacter jejuni and 24 Campylobacter coli from diarrheic cases; 57 C. jejuni and 29 C. coli from controls), the presence of the cdtABC and cadF genes and iam marker was established. Nalidixic acid, ciprofloxacin, erythromycin, and azithromycin susceptibilities were established in 115 isolates and tetracycline-susceptibility was established in 100 isolates. The presence of mutations in the gyrA, parC, and 23S rRNA genes was determined. The cadF gene and all genes from the cdtABC operon were significantly more frequent among C. jejuni (P < 0.0001); the iam marker was more frequent in C. coli (P < 0.0001). No differences were observed in VFs between cases and controls. Almost all isolates were tetracycline-resistant; nalidixic acid and ciprofloxacin resistance reached levels of 90.4% and 88.7%, respectively. Resistance to macrolides was 13% (C. jejuni 4.3%; C. coli 26.1%). Resistance to ciprofloxacin was related to GyrA Thr86 substitutions, while 13 of 15 macrolide-resistant isolates possessed a 23S rRNA mutation (A2075G). Differences in the presence of VFs and alarming levels of resistance to tested antimicrobial agents were observed among C. jejuni and C. coli.

Pathogenicity Island O-122 in enteropathogenic Escherichia coli strains is associated with diarrhea severity in children from Lima Peru

EPEC is an attaching and effacing diarrheal pathogen that carries a large pathogenicity island, locus for enterocyte effacement (LEE). Recently, the pathogenicity island PAI O-122 was described among non-LEE effectors and found to be associated with diarrhea among atypical EPEC strains. It is unknown if incomplete PAI O-122 could be associated with diarrhea duration and severity. To identify these virulence determinants we analyzed 379 EPEC strains isolated from Peruvian children. EPEC was diagnosed by PCR(eae+, stx-) and classified as typical(t-EPEC) or atypical(a-EPEC). To characterize PAI O-122 we amplified three modules by PCR: Module 1(pagC), Module 2(senA, nleB and nleE) and Module 3(lifA/efa-1). To characterize the large ORF lifA/efa-1 we amplified the regions known as efa-N, efa-M and efa-C. Clinical information was obtained from the cohort study. A total of 379 EPEC strains were able to analyze PAI O-122 genes, 128 (10.4%) EPEC strains were isolated from 1235 diarrhea episodes and 251(9.2%) from 2734 healthy controls. t-EPEC strains were isolated from 14.8% (19/128) of children with diarrhea and 25/251(10.0%) from healthy controls. The most frequent PAI O-122 genes were nleE(37.7%), senA(34.6%) and nleB(37.5%), with similar prevalence among diarrhea and control samples. However, lifA/efa-1 was more common among diarrhea cases than healthy control cases (30.5% vs. 21.1%, p<0.05). The presence of complete PAI O-122 was associated with diarrhea episodes of higher severity among single pathogen infection (33.3% vs. 1.8%, p<0.05) mainly due to the presence of a complete lifA/efa-1 gene. In summary, the gene lifA/efa-1 is significantly associated with diarrheal episodes of higher severity, suggesting to be an important virulent factor.