Research ArticleDiarrheagenic Escherichia coli Phylogroups Are Associated withAntibiotic Resistance and Duration of Diarrheal Episode
Susan Mosquito,1 Maria J. Pons,2,3 Maribel Riveros,1
Joaquim Ruiz,2 and Theresa J. Ochoa1,4
1 Instituto de Medicina Tropical Alexander von Humboldt, Universidad Peruana Cayetano Heredia, Lima 031, Peru2ISGlobal, Barcelona Centre for International Health Research (CRESIB), Hospital Clnic, Universitat de Barcelona,08036 Barcelona, Spain3Universidad Peruana de Ciencias Aplicadas (UPC), Lima 09, Peru4School of Public Health, University of Texas, Houston, TX 77030, USA
Correspondence should be addressed toTheresa J. Ochoa; email@example.com
Received 28 July 2014; Revised 7 February 2015; Accepted 11 February 2015
Academic Editor: Boris Martinac
Copyright 2015 Susan Mosquito et al.This is an open access article distributed under theCreative CommonsAttribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Conventionally, in Escherichia coli, phylogenetic groups A and B1 are associated with commensal strains while B2 and D areassociated with extraintestinal strains.The aim of this study was to evaluate diarrheagenic (DEC) and commensal E. coli phylogenyand its association with antibiotic resistance and clinical characteristics of the diarrheal episode. Phylogenetic groups and antibioticresistance of 369 E. coli strains (commensal strains and DEC from children with or without diarrhea) isolated from Peruvianchildren 0.01). In summary, DEC-diarrhea strains were more associated with group D than strains from healthy controls.
Conventionally, Escherichia coli, a common isolate in clinicallaboratories, is classified into two major groups: commen-sal and pathogenic. Additionally pathogenic isolates mayproduce different diseases, being then subdivided in diar-rheogenic and extraintestinal E. coli. Human infectionscaused by extraintestinal E. coli include meningitis, urinarytract infections, sepsis, pneumonia, surgical site infections,and infections in other extraintestinal locations . However,when classified into subtypes, E. coli mainly fall into fourphylogenetic groups: A, B1, B2, and D . Previous studieshave shown that commensal E. coli strains tend to be asso-ciated within phylogenetic groups A and B1 [1, 2], whereasthe extraintestinal pathotypes fall within phylogenetic groupsB2 and D [3, 4]. Regarding uropathogenic E. coli (UPEC)strains determinants including phylogroups markers are well
established. However, for diarrheagenic E. coli (DEC), thescenario remains unclear. There is no information about theassociation between the phylogenetic group and the clinicaldata of the diarrheal episode .
Previous reports describe the emerging antibiotic resis-tance in commensal and diarrheagenic E. coli in Peru . However, there is no sufficient data in the correlationof phylogeny and antibiotic resistance . Therefore weconducted this study to determine the association betweenthe phylogenetic group and antibiotic resistance in a largenumber of E. coli (commensal and diarrheagenic) strainsfrom Peruvian infants.
2. Materials and Methods
2.1. Samples. Commensal and diarrheagenic E. coli strainswere isolated during a previous passive surveillance diarrhea
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study . In this study 1032 children were followed from2 to 12 months of life, obtaining a total of 1079 E. colistrains that were analyzed by a real time multiplex PCR todetermine DEC pathotypes . A total of 369 isolates fromthis study were randomly selected and analyzed, including74 commensal E. coli, 94 DEC from asymptomatic children(DEC-control), and 201 DEC isolated from children withdiarrhea (DEC-diarrhea). DEC pathotypes included in thisstudy were enteropathogenic E. coli (EPEC), enterotoxi-genic E. coli (ETEC), enteroaggregative E. coli (EAEC),and diffusely adherent E. coli pathotypes (DAEC); Shigatoxin-producing E. coli (STEC) and enteroinvasive E. coli(EIEC) strains were not included due to their low prevalence.
2.2. Phylogenetic Group Determination. The phylogeneticgroups were determined as previously described . In allcases the bacteria DNA was extracted boiling.
2.3. Clinical Data of Diarrheal Episodes. Variables such asepisode duration (days), maximum number of stools per day,total number of stools per episode, and a modified Vesikarimodified score  were analyzed and associated with phylo-genetic groups in those cases in which no coinfections werepreviously reported .
2.4. Antibiotic Resistance. Antibiotic susceptibility toampicillin (10 g), trimethoprim-sulfamethoxazole (23.75/1.25 g), chloramphenicol (30 g), nalidixic acid (30 g),and tetracycline (30 g) was determined by disk diffusionin accordance with the CLSI guidelines . Multiresistancewas defined as resistance to three ormore unrelated antibioticfamilies.
2.5. Statistical Analysis. Vesikari severity score was expressedby mean standard deviation and median (range) valueswere given for duration of the episode and number of stools.The comparisons between groups were made using chi-squared or Fishers exact test. Students -test was used for thecomparison of Vesikari severity scores between groups.
3.1. Phylogenetic Group Frequency. The E. coli strains (DECand commensals) were distributed in the four phylogeneticgroups: A (147 isolates, 40%), D (116 isolates, 31%), B1(76 isolates, 21%), and B2 (30 isolates, 8%). No significantdifference in the prevalence of phylogenetic groupswas foundwithin each pathotype when analyzed by control/diarrhea.In total were analyzed 87 EPEC (38 DEC-control, 49 DEC-diarrhea), 83 ETEC (26 DEC-control, 57 DEC-diarrhea),94 EAEC (24 DEC-control, 70 DEC-diarrhea), 31 DAEC(6 DEC-control; 25 DEC-diarrhea), and 74 commensalisolates.
The phylogroupAwas themost common, in both groups,control and diarrhea, in EPEC (45 isolates, 52%) and ETEC(44, 53%), while mostly DAEC isolates (27 isolates, 87%)belong to phylogroup D. Regarding EAEC, differences were
A B1 B2 D
DEC diarrheaDEC control
Figure 1: Percentage of phylogenetic groups in commensal E. colistrains ( = 74), diarrheagenic E. coli from healthy controls (DEC-control) ( = 94), and DEC from children with diarrhea (DEC-diarrhea) ( = 201).
found between those isolates causing diarrhea and thoserecovered from healthy children.Thus EAEC isolates causingdiarrhea were mostly classified as phylogroup D (29 isolates,41%), while those recovered from healthy children predomi-nantly belong to the phylogroupA (10 isolates, 42%) (Table 1).Analyzing together the DEC isolates, those classified as DEC-control strains were more associated with A group (50%)while the DEC-diarrhea strains were more associated withD group (34%) ( < 0.05). Meanwhile, commensal E. coli( = 74) were more associated with A (26 isolates, 35%) andD (28 isolates, 38%) phylogroups.The commensal group alsohad a high prevalence of B2 group (12 isolates, 16%) unlikeboth DEC groups ( < 0.05). Both DEC-control (24 isolates,26%) and DEC-diarrhea (54 isolates, 27%) groups were moreassociated with the phylogroup B1 than commensals strains(7 isolates, 14%) ( < 0.05) (Figure 1).
3.2. Clinical Data of Diarrheal Episodes. From the 201 DEC-diarrhea isolates analyzed in the study, 127 strains were iso-lated from diarrhea episodes in which no other pathogen wasdetected. In these 127 patients, no significant differences werefound for the studied variables among the four phylogeneticgroups. In general, the episode duration was 5 days (125),the maximum number of stools/day was 5 (311), the totalnumber of stools/episode was 20 (3128), and the Vesikariscore was 6 2.6. We found a higher proportion ( = 0.06)of persistent diarrhea (14 or more days) among B2-D groups(23.9%) compared to among A-B1 groups (9.88%). No differ-ences were found for either acute or prolonged diarrhea (714days) between severe (B2-D) and nonsevere (A-B1) groups.
3.3. Antibiotic Resistance. Resistance to trimethoprim-sul-famethoxazol, tetracycline, chloramphenicol, and nalidixicacid and multiresistance were significantly different amongthe four phylogenetic groups ( < 0.05) (Figure 2). Ingeneral, B2 and D groups presented higher percentage ofantibiotic resistance than A and B1 groups. In the caseof multiresistance D group presented significantly higherpercentages than the rest of the groups ( < 0.01)(Figure 2).