Phenotypic and genotypic characterization of enteroaggregative Escherichia coli isolates from pediatric population in Pakistan

Abstract

Enteroaggregative Escherichia coli (EAEC) are a leading cause of diarrhea among children. The objective of this study was to define the frequency of EAEC among diarrheal children from flood-affected areas as well as sporadic cases, determine multidrug resistance, and evaluation of virulence using an in vivo model of pathogenesis. Stool samples were collected from 225 diarrheal children from 2010 to 2011 from flood-affected areas as well as from sporadic cases in Pakistan. Identified EAEC isolates were characterized by phylogrouping, antibiotic resistance patterns including the extended-spectrum beta lactamase spectrum, single nucleotide polymorphism detection in gyrA and parC, and virulence potential using wax worm, G. mellonella. A total of 35 (12.5%) confirmed EAEC isolates were identified among 225 E. coli isolates. EAEC isolates displayed high resistance to tetracycline, ampicillin, and cefaclor. A total of 34.28% were ESBL positive. Single nucleotide polymorphism detection revealed 37.14% and 68.57% isolates were positive for SNPs in gyrA (A660 -T660 ) and parC (C330 -T330 ), respectively. Phylogrouping revealed that B2 phylogroup was more prevalent among all EAEC isolates tested followed by D, A, B1, and non-typeable (NT). Infection of G. mellonella with EAEC showed that killing infective dose was 100% higher than E. coli DH5 alpha control. EAEC are prevalent among Pakistani children with diarrhea, they are highly resistant to antibiotics, and predominantly fall into B2 phylogroup. Epidemiologic surveillance of EAEC and other E. coli pathotypes is critical to assess not only the role of these pathogens in diarrheal disease but also to determine the extent of multidrug resistance among the population.

Drivers of antimicrobial resistance amongst intestinalEscherichia coliisolated from children in South Asia and sub-Saharan Africa

“…The high AMR frequencies for first-line drugs, in particular penicillins, detected in this study are similar to reports of other intestinal, extra-intestinal and environmental isolates of E. coli 22,37 . Of note, the high levels of ciprofloxacin-resistance found in this study at the Asian sites corresponded to high levels reported in clinical cases in India, Bangladesh and Pakistan 17,18,21,23,24,38 . Further, while ESBL-producing aEPEC were detected in this study in Asia, the frequency of these was lower than those reported from hospitals in both Asia and Africa 18,21,31,38 .…”

“…While limited AMR data for E. coli are available from the seven countries where the GEMS sites were located, there are no national surveillance data from any of these countries. Furthermore, AMR data on E. coli in these countries predominately pertain to E. coli causing extra-intestinal infections and to a limited number of drugs, mainly third-generation cephalosporins and fluoroquinolones 21,[23][24][25]31 . Here we present the first comprehensive AMR phenotypic data and molecular characterization for multiple antimicrobials, detecting several common mobile elements mediating the AMR including small plasmids, class I integrons with different cassettes and transposons including Tn6029, Tn10 and Tn1721 ( Fig S3).…”

“…Of greatest concern are multi-drug resistant (MDR) E. coli (non-susceptible to one or more agents in at least three antimicrobial categories) 16 , particularly those that are fluoroquinolone resistant and/or produce extended-spectrum beta-lactamases (ESBL). While several recent studies have reported increases in ESBLs 17,18 , gentamicin 19,20 , ciprofloxacin [20][21][22][23][24] and revealed diverse AMR profiles, these data have largely come from E. coli responsible for disease in hospital settings. Thus, there remain significant gaps in knowledge of the prevalence of AMR in human intestinal E. coli populations globally, particularly in the developing nations of sub-Saharan Africa and South Asia 25 .…”

Typical and Atypical Enteroaggregative Escherichia coli Are Both Virulent in the Galleria mellonella Model

“…For ethical reasons, studies with human volunteers that would best reproduce bacterial virulence is not currently feasible (Nataro et al, 1995), therefore, studies make use of animal models, mainly rodents, as a way to investigate aspects of the EAEC pathogenesis and, in recent years, for ethical and costs reasons, the use of invertebrate models has been implemented for studies of several microorganisms, even for EAEC (Morin et al, 2010; Roche et al, 2010; França et al, 2013; Jønsson et al, 2016; Khalil et al, 2016). In this context, the larvae of Galleria mellonella (Lepidoptera: Pyralidae) shows several morphological, physiological and immunological advantages over other invertebrate models (Tsai et al, 2016).…”

“…In addition to the hemocyte-mediated cellular immune response, G. mellonella also has a humoral immune response consisting of effector molecules, including opsonins (Park et al, 2005; Shaik and Sehnal, 2009), antimicrobial peptides (Lee et al, 2004; Brown et al, 2009), and melanin (Soderhall and Cerenius, 1998). Previously, Jønsson et al (2016) and Khalil et al (2016) analyzed only typical EAEC strains in this same model, comparing it with non-pathogenic E. coli and validating the model for EAEC pathogenicity studies.…”