Comparative analysis of virulence determinants, phylogroups, and antibiotic susceptibility patterns of typical versus atypical Enteroaggregative E. coli in India

Abstract

Enteroaggregative Escherichia coli (EAEC) is an evolving enteric pathogen that causes acute and chronic diarrhea in developed and industrialized nations in children. EAEC epidemiology and the importance of atypical EAEC (aEAEC) isolation in childhood diarrhea are not well documented in the Indian setting. A comparative analysis was undertaken to evaluate virulence, phylogeny, and antibiotic sensitivity among typical tEAEC versus aEAEC. A total of 171 EAEC isolates were extracted from a broad surveillance sample of diarrheal (N = 1210) and healthy children (N = 550) across North India. Polymerase chain reaction (PCR) for the aggR gene (master regulator gene) was conducted to differentiate tEAEC and aEAEC. For 21 virulence genes, we used multiplex PCR to classify possible virulence factors among these strains. Phylogenetic classes were identified by a multiplex PCR for chuA, yjaA, and a cryptic DNA fragment, TspE4C2. Antibiotic susceptibility was conducted by the disc diffusion method as per CLSI guidelines. EAEC was associated with moderate to severe diarrhea in children. The prevalence of EAEC infection (11.4%) was higher than any other DEC group (p = 0.002). tEAEC occurrence in the diarrheal group was higher than in the control group (p = 0.0001). tEAEC strain harbored more virulence genes than aEAEC. astA, aap, and aggR genes were most frequently found in the EAEC from the diarrheal population. Within tEAEC, this gene combination was present in more than 50% of strains. Also, 75.8% of EAEC strains were multidrug-resistant (MDR). Phylogroup D (43.9%) and B1 (39.4%) were most prevalent in the diarrheal and control group, respectively. Genetic analysis revealed EAEC variability; the comparison of tEAEC and aEAEC allowed us to better understand the EAEC virulence repertoire. Further microbiological and epidemiological research is required to examine the pathogenicity of not only typical but also atypical EAEC.

Fig 1. Representation of sampling sites of the regional health centers of different cities across the North India region [Chandigarh (PGIMER), Civil hospital Manimajra; Haryana (Civil hospital Panchkula and Civil hospital Ambala Cantt); Himachal Pradesh (Government Medical College (GMC) Hamirpur, Indira Gandhi Medical College (IGMC) Shimla, and Rajendra Prasad Government Medical College (RPGMC) Tanda); Punjab (Civil hospital and private laboratory Mohali and Private laboratory Ludhiana); and Uttarakhand (All India Institute of Medical Science (AIIMS) Rishikesh, District hospital Rudrapur and District hospital Haridwar)] from where stool samples of diarrheal and healthy children were collected during 2015–17.

The map was made with Natural Earth using QGIS software version 3.14.6.

Fig 2. Distribution of diarrheagenic Escherichia coli (DEC) groups in the diarrheal group.

EPEC: enteropathogenic E. coli, EAEC: enteroaggregative E. coli, ETEC: enterotoxigenic E. coli and hybrid strains.

Fig 3. A representative gel electrophoresis profile of different DEC using M-PCR.

lane 1, negative sample; lane 2, negative control (NC); lane 3, positive control (PC) EAEC (pCVD432); lane 4, EPEC (Eae); lane 5, EAEC (pCVD432); lane 6, EAEC (pCVD432); lane 7 EAEC (pCVD432); lane 8, EAEC (pCVD432); lane 9, ladder (100 bp); and lane 10, ETEC (LT and Sth).