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Introduction: Diarrheagenic Escherichia coli (DEC) are responsible for about 30–40% of acute diarrhoea episodes in developing countries. Up to 10% of isolates of E. coli from stool specimens have been reported to be non-lactose fermenting (NLF E. coli). There is scarcity of literature available regarding the pathogenicity of NLF E. coli causing infectious diarrhoea. Aim: We aimed to elucidate the importance of NLF E. coli in causing diarrhoea in both adults as well as children by detecting various DEC pathotypes among NLF E. coli in stool samples from gastroenteritis cases. Material and Methods: A total of 376 non-lactose fermenting (NLF) E. coli isolates isolated from 3110 stool samples of diarrhoea/ gastroenteritis patients were included in the study. Upto 3 non-lactose fermenting colonies which did not confirm as V.cholerae, Aeromonas spp., Salmonella spp., Shigella spp., but were identified as E.coli on MALDI-TOF were selected from each MacConkey agar plate and streaked onto fresh, sterilized nutrient agar and biochemical reactions were put up. Multiplex PCR was put up for EAEC, EPEC, ETEC, EHEC pathotypes and PCR for ipaH gene was put up for EIEC. Antibiotic sensitivity testing was performed with the disc diffusion method. Results: Using multiplex PCR and ipaH PCR, a total of 63 pathotypes of DEC were obtained, EAEC being the most predominant (n= 31) followed by EIEC (n=22), EPEC (n=8) and ETEC (n=2). To further differentiate EIEC from Shigella, additional biochemicals were performed which include acetate utilization, mucate and salicin fermentation, and esculin hydrolysis. Antimicrobial susceptibility testing (AST) showed that maximum resistance was seen against ciprofloxacin (82.5%) followed by ampicillin (77.8%) and cotrimoxazole (68.2%) and minimum resistance was seen against ertapenem (4.8%). Conclusion: In our study two pathotypes (EAEC, EIEC) were predominant among NLF E. coli and these were important etiological agents not only in children but also in adults. Our study also sheds light on epidemiology of EIEC which is one of the most neglected DEC pathotype as hardly any microbiological laboratory process NLF E. coli for EIEC.

For the first time in the world, we report Enteroaggregative E.coli harbouring blaNDM−4 and blaNDM−7 that were isolated from healthy children. NDM-4 and NDM-7 are associated with increased carbapenemase activity. Clonal spread, inter-strain and interspecies horizontal transfer of such resistant determinants in a healthy population will be a cause of concern.

Urinary tract infections (UTIs) are a serious health concern worldwide. Treatment of UTIs is becoming a challenge as uropathogenic Escherichia coli (UPEC), which is the most common etiological agent, has developed resistance to the main classes of antibiotics. Small molecules that interfere with metabolic processes rather than growth are attractive alternatives to conventional antibiotics. Repurposing of already known drugs for treating infectious diseases could be an attractive avenue for finding novel therapeutics against infections caused by UPEC. Virtual screenings enable the rapid and economical identification of target ligands from large libraries of compounds, reducing the cost and time of traditional drug discovery. Moreover, the drugs that have been approved by the FDA have low cytotoxicity and good pharmacological characteristics. In this work, we targeted the HisC enzyme of the histidine biosynthetic pathway as enzymes of this pathway are absent in humans. We screened the library of FDA-approved drugs against HisC via molecular docking, and four hits (Docetaxel, Suramin, Digitoxin, and Nystatin) showing the highest binding energy were selected. These were further tested for antibacterial activity, which was observed only for Docetaxel (MIC value of 640 μg/ml); therefore, Docetaxel was further tested for its efficacy in vivo in murine catheter UTI model and antibiofilm activity using crystal violet staining and scanning electron microscopy. Docetaxel inhibited biofilm formation and reduced the bacterial load in urine, kidney, and bladder. Docking studies revealed that Docetaxel acts by blocking the binding site of HisC to the native substrate by competitive inhibition. Docetaxel may be a potential new inhibitor for UPEC with antibacterial and antibiofilm capability.

Purpose Emergence and spread of resistance among Vibrio cholerae have become a global public health problem. In India, no consolidated data is available on antimicrobial susceptibility patterns and antibiotic resistance genes. Materials & methods A total of 110 representative isolates obtained over a period of 14 years were included. Antimicrobial susceptibility was tested by disc diffusion and micro broth dilution. Presence of 13 antimicrobial resistance genes was ascertained by using PCR. Results Antimicrobial resistance fluctuated for most of the antibiotics. Resistance to cotrimoxazole in our study was 92.72% and the SXT element was present in all isolates. Resistance to nalidixic acid, tetracycline, and cefotaxime was found to be 98.18%, 7.27%, and 10.9% respectively. Resistance to ampicillin saw a fluctuating trend with a recent fall. Resistance to ciprofloxacin and azithromycin was 12.72% and 29% by MIC. blaTEM was the most common ESBL gene (94.5%). Other were blaCMY (26.36%) and blaNDM (2.7%). We report blaCTX-M-15 and blaOXA-48 and ermB for the first time in the world. Newer antimicrobials like prulifloxacin and rifaximin were tested for the first time from India. Conclusion Our study has shown very high levels of resistance to older antibiotics and the emergence of resistance to some of the newer classes of antibiotics. There is an urgent need for increased surveillance studies, rational use of the antimicrobials and preventive measures to control the disease.

To study the significance of enteroaggregative Escherichia coli (EAEC) as a pathogen causing acute diarrhea and a commensal in healthy nourished and malnourished children younger than five years of age in the Chandigarh region and to address possible traits of EAEC virulence genes, biofilm formation, phylogroups, and antibiotic resistance that would be correlated with diarrhea or carriage.Stool samples were obtained from children with acute diarrhea (n = 548), as well as nourished (n = 550), and malnourished controls without diarrhea (n = 110). E coli isolates were confirmed as EAEC by pCVD432 polymerase chain reaction. Multiplex polymerase chain reactions were used to identify 22 virulence-related genes and phylogeny. Antibiotic susceptibility, adherence, and biofilm-forming potential also were studied.Overall, 16.6% of children were malnourished. EAEC detection was greater among children with acute diarrhea (16%) than nourished (6%) and malnourished nondiarrheal controls (2.7%). We found an association of EAEC infections with age2 years (P = .0001) in the diarrheal group. Adhesive variants adhesion fimbriae IV and adhesion fimbriae II were significantly associated with diarrhea. The aggR and aar genes showed a positive and negative association with the severity of disease (P = .0004 and P = .0003). A high degree of multidrug resistance was found (73.8%) in the diarrheal group. Most EAEC strains from the diarrheal group belonged to B2 and D phylogroups, whereas strains from non-diarrheal groups, which belonged to phylogroup B1.EAEC is a significant contributor to childhood diarrhea, its presence as a commensal, and the significance of the association of various virulence factors among the EAEC isolated from diarrheal and non-diarrheal stools. These data reinforce the importance of aggR and aar as positive and negative regulators and the contribution of AAF/II and AAF/IV fimbria for the pathobiology of EAEC.

Horizontal gene transfer of virulence genes (VGs) from different Escherichia coli pathotypes results in the evolution of hybrid strains. Hybrid genotypes of enteroaggregative E. coli and uropathogenic E. coli (EAEC/UPEC) have been reported in sporadic infections and outbreaks of extraintestinal origin. Yet, their association with routine infections is still underrated.In this study, we analysed 163 isolates of E. coli from cases of urinary tract infection seeking hybrid (EAEC/UPEC) strains. Using multiplex polymerase chain reaction, we investigated VGs (adhesive and toxin genes) of UPEC along with EAEC marker genes (aap and agg R), ast A (toxin genes) and serine protease autotransporters of Enterobacteriaceae, pet (plasmid-encoded toxin) and pic (mucinase gene). Those UPEC strains which had characteristic defining genes of EAEC (agg R/aap or their combination) were considered UPEC/EAEC hybrids.Molecular predictors of EAEC (aap and aggR) were detected in 20.2% (33/163) of the strains. The pap C was also detected in 36% of the EAEC/UPEC hybrid strains. Phylogenetic analysis revealed that hybrid strains belonged to Group D (60.6%). Nearly 73.8% of UPEC and 75.7% of UPEC/EAEC hybrid strains were multidrug-resistant. Among UPEC isolates, 72.3% and in hybrid UPEC/EAEC, 78.7% isolates were able to produce biofilm.Our results indicated a closer relationship among EAEC and UPEC, which suggested that some EAEC strains can be potential uropathogens. Ours is a first study documenting the existence of EAEC pathotypes VGs in UPEC strains of nosocomial origin; further studies are required to understand the diarrhoeagenic potential of these hybrids.

Urinary tract infections (UTIs) are one of the commonest bacterial infections worldwide. The most common etiological agent is uropathogenic Escherichia coli. The occurrence of high rates of antimicrobial resistance among UPEC has complicated therapeutic management and emphasized the need for new drug targets or newer approaches to tackle the increasing trend of antimicrobial resistance. The use of OMICS technologies provides an attractive alternative to experimental methods to provide momentum in the discovery of drugs and decrease the chances of drug failure in later stages of drug development. The availability of the full genome sequence of many disease-causing organisms and humans in public databases has significantly increased the search for promising drug targets. The present study was performed to identify and characterize promising drug targets against a highly virulent UPEC strain CFT073 by utilizing a novel hierarchical in silico approach. We conducted the study in four phases. In phase 1, three sets of proteins were mined through a chokepoint, virulence, and resistance genes analysis. The proteins selected from phase 1 were further subjected to the nonhomology analysis in phase 2. In phase 3, co-evolution analysis was done to find non-homologous proteins which interact with virulence and resistance genes at high correlation values (≥0.9). We found eight putative drug targets (MurB, MurC, HisC, HyaB, HybO, FliN, FliH, and MotB) that interacted with five virulence (chuU, chuW, iucA, fepA, and hylD) and two resistance genes (arnA and acrB). Most of the identified drug targets are part of distinct metabolic pathways (viz., peptidoglycan biosynthesis, histidine biosynthesis etc). In phase 4, qualitative characterization of the above drug targets including protein location in the bacterial cell, broad-spectrum analysis, interactome analysis, evaluation of druggability by similarity search against drug-target database, and essentiality analysis was performed. PCR-based confirmation of identified drug targets in clinical isolates of UPEC was also carried out. The findings of this study may aid in the discovery of new antibacterial agents for better treatment of E. coli UTIs.

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.

Background: Horizontal gene transfer of virulence genes (VGs) from different Escherichia coli pathotypes results in the evolution of hybrid strains. Hybrid genotypes of enteroaggregative E. coli and uropathogenic E. coli (EAEC/UPEC) have been reported in sporadic infections and outbreaks of extraintestinal origin. Yet, their association with routine infections is still underrated. Materials and Methods: In this study, we analysed 163 isolates of E. coli from cases of urinary tract infection seeking hybrid (EAEC/UPEC) strains. Using multiplex polymerase chain reaction, we investigated VGs (adhesive and toxin genes) of UPEC along with EAEC marker genes (aap and agg R), ast A (toxin genes) and serine protease autotransporters of Enterobacteriaceae, pet (plasmid-encoded toxin) and pic (mucinase gene). Those UPEC strains which had characteristic defining genes of EAEC (agg R/aap or their combination) were considered UPEC/EAEC hybrids. Results: Molecular predictors of EAEC (aap and aggR) were detected in 20.2% (33/163) of the strains. The pap C was also detected in 36% of the EAEC/UPEC hybrid strains. Phylogenetic analysis revealed that hybrid strains belonged to Group D (60.6%). Nearly 73.8% of UPEC and 75.7% of UPEC/EAEC hybrid strains were multidrug-resistant. Among UPEC isolates, 72.3% and in hybrid UPEC/EAEC, 78.7% isolates were able to produce biofilm. Conclusions: Our results indicated a closer relationship among EAEC and UPEC, which suggested that some EAEC strains can be potential uropathogens. Ours is a first study documenting the existence of EAEC pathotypes VGs in UPEC strains of nosocomial origin; further studies are required to understand the diarrhoeagenic potential of these hybrids.