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. Author summary Enteroaggregative E. coli (EAEC) are an increasingly important cause of diarrhea. E. coli belonging to this category cause watery diarrhea, which is often persistent and can be inflammatory. It is also associated with traveler’s diarrhea in children and adults in middle and high-income countries. EAEC are defined by their ability to adhere to epithelial cells in a characteristic stacked brick-like pattern. However, the identification of these pathogenic strains remains elusive because of its heterogeneous nature. Genes that could contribute to the pathogenicity of EAEC encode adhesions, toxins, and other factors. Due to the heterogeneity of EAEC strains and differing host immune responses, not all EAEC infections are symptomatic. A critical factor in both recognizing EAEC pathogenesis and defining typical EAEC (tEAEC) strains is AggR, a transcriptional control for many EAEC virulence genes. The central role of aggR in virulence confers a strong priority to understand its pathogenicity. To identify EAEC, the CVD432 probe has been used. The CVD432 is a DNA probe from pAA plasmid of EAEC, has been reported to be specific for the detection of EAEC. The lack of sensitivity comes from the genetic heterogeneity of the EAEC strains and the wide geographic dispersal of strains. In our study, we performed a large surveillance of EAEC from North India among the pediatric population. Samples were collected by the microbiology staff at the Postgraduate Institute of Medical Education and Research (PGIMER) and referral system labs in Chandigarh (Manimajra), Punjab (Ludhiana), Haryana (Panchkula and Ambala Cantt), Himachal Pradesh (Hamirpur, Shimla, and Tanda), and Uttarakhand (Rishikesh, Rudrapur, and Haridwar)]. PGIMER is the largest tertiary care hospital in North India and serves patients from across Punjab, Jammu and Kashmir, Himachal Pradesh and Haryana. EAEC infections were detected using molecular methods. In our finding, astA, aap, and aggR genes were most frequently found in the EAEC from the diarrheal population. Within tEAEC, this gene combination is present in more than 50% of strains and helps to differentiate tEAEC from aEAEC. Our collection of EAEC strains helps in finding an appropriate marker for the early detection of EAEC. Our signature sequence (astA, aap, and aggR) will be ideal as focus genes for EAEC identification, as well as tEAEC and aEAEC. The multidrug resistance (MDR) was observed in 75.8% of the EAEC strains. tEAEC exhibits resistance to a greater number of antibiotics with respect to aEAEC. The phylogenetic analysis revealed that EAEC phylogeny is diverse and dispersed in all the phylogroups.