Genome sequence of pan drug-resistant enteroaggregative Escherichia coli belonging to ST38 clone from India, an emerging EAEC/UPEC hybrid pathotype.

Here, we report the genomic characterization of a pan drug-resistant (PDR) enteroaggregative Escherichia coli (EAEC) isolated from an immunocompromised infant who had diarrhea. The isolate belonged to the sequence type (ST) 38, which is a known enteroaggregative Escherichia coli (EAEC)/uropathogenic Escherichia coli (UPEC) hybrid strain having multi-drug resistance (MDR). The strain carried genes encoding multiple resistances to carbapenems, third-generation cephalosporins, polymyxin, fluoroquinolones, aminoglycosides, fosfomycin, nitrofurantoin, sulphonamides, and multiple efflux pump genes. Interspecies horizontal transfer, inter-strain, and clonal spread of these resistances to commensals and pathogens will be worrisome. We are concerned about the spread of such PDR strains. The genomic characterization of such strains will be useful in understanding the genetic makeup of EAEC/UPEC hybrid strains and developing new vaccines/diagnostics and therapeutics.

Association of Putative Virulence Genes with HEp-2 Cell Adherence and Biofilm Production in Enteroaggregative E. coli Strains Isolated from Acute Diarrheal and Healthy Children, India.

Enteroaggregative Escherichia coli (EAEC) is an emerging enteric pathogen that causes acute and chronic diarrhea in developed and industrialized countries in children. EAEC colonizes the human intestine and this ability to form colonies and biofilm is an important step in pathogenesis. Here, we investigated the relationship between known or putative 22 EAEC virulence genes and biofilm formation in isolates derived from acute diarrhea and healthy children and their aggregative adherence (AA) pattern with Hep-2 cell lines. A total of 138 EAEC isolates were recovered from 1210 stool samples from children (age < 10 years) suffering from acute diarrhea and 33 EAEC strains isolated from 550 healthy children (control group) of different Anganwadi centers in Chandigarh region were included. Polymerase chain reaction using the primer pair pCVD432 identified E. coli isolates as EAEC. A total of 22 virulence-related genes have been identified using M-PCR chain reactions. The crystal violet method was used for the quantitative biofilm assay. Aggregative adherence assay was also studied using HEp-2 cell lines. Of 138 EAEC isolates from the acute diarrheal group, 121 (87.6%) EAEC isolates produced biofilm. In our findings, typical EAEC (62%) isolates were strong biofilm producers (37.5%) in the diarrheal group. Among adhesive variants, agg4A (39.6%) and aggA (21.6%) were the most common and were statistically significant (p = 0.01 and p = 0.03 respectively). We reported that the aggR gene along with the typical AA pattern was present in 71.4% of the EAEC strains in the diarrheal group, whereas it was present in 44% of the control group. Other aggR non-dependent genes like ORF3 and eilA may also lead to biofilm formation. In conclusion, there is significant heterogeneity in putative virulence genes of EAEC isolates from children and biofilm formation is associated with the combination of many genes.

Are non-lactose-fermenting Escherichia coli important diarrhoeal pathogens in children and adults?

Introduction: Diarrhoeagenic Escherichia coli (DEC) remains one of the major causes of acute diarrhoea episodes in developing countries. The percentage of acute diarrhoea cases caused by DEC is 30-40 % in these countries. Approximately 10% of E. coli isolates obtained from stool specimens have been reported to be non-lactose-fermenting (NLF). The available literature is sparse 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 and children by detecting various DEC pathotypes among NLF E. coli in stool samples taken from gastroenteritis cases. Material and Methods: A total of 376 NLF E. coli isolates from 3110 stool samples from diarrhoea/gastroenteritis patients were included in the study. Up to three NLF colonies that were not confirmed as Vibrio cholerae , Aeromonas spp., Salmonella spp. or Shigella spp., but were identified as E. coli using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF), were carefully picked up from each MacConkey agar plate and then meticulously streaked onto freshly prepared, sterilized nutrient agar plates, and biochemical reactions were conducted. Multiplex PCR was conducted for the EAEC, EPEC, ETEC and EHEC pathotypes and PCR for the ipaH gene was conducted for EIEC. The disc diffusion method was used for antibiotic sensitivity testing. Results: Using multiplex PCR and ipaH PCR, a total of 63 pathotypes of DEC were obtained, with 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 biochemical tests were performed, including acetate utilization, mucate and salicin fermentation, and aesculin 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 not only important aetiological agents in children, but also in adults. Our study also sheds light on the epidemiology of EIEC, which is one of the most neglected DEC pathotypes, as hardly any microbiological laboratories process NLF E. coli for EIEC.

Computational Guided Drug Targets Identification against Extended-Spectrum Beta-Lactamase-Producing Multi-Drug Resistant Uropathogenic Escherichia coli.

Urinary tract infections (UTIs) are one of the most frequent bacterial infections in the world, both in the hospital and community settings. Uropathogenic Escherichia coli (UPEC) are the predominant etiological agents causing UTIs. Extended-spectrum beta-lactamase (ESBL) production is a prominent mechanism of resistance that hinders the antimicrobial treatment of UTIs caused by UPEC and poses a substantial danger to the arsenal of antibiotics now in use. As bacteria have several methods to counteract the effects of antibiotics, identifying new potential drug targets may help in the design of new antimicrobial agents, and in the control of the rising trend of antimicrobial resistance (AMR). The public availability of the entire genome sequences of humans and many disease-causing organisms has accelerated the hunt for viable therapeutic targets. Using a unique, hierarchical, in silico technique using computational tools, we discovered and described potential therapeutic drug targets against the ESBL-producing UPEC strain NA114. Three different sets of proteins (chokepoint, virulence, and resistance genes) were explored in phase 1. In phase 2, proteins shortlisted from phase 1 were analyzed for their essentiality, non-homology to the human genome, and gut flora. In phase 3, the further shortlisted putative drug targets were qualitatively characterized, including their subcellular location, broad-spectrum potential, and druggability evaluations. We found seven distinct targets for the pathogen that showed no similarity to the human proteome. Thus, possibilities for cross-reactivity between a target-specific antibacterial and human proteins were minimized. The subcellular locations of two targets, ECNA114_0085 and ECNA114_1060, were predicted as cytoplasmic and periplasmic, respectively. These proteins play an important role in bacterial peptidoglycan biosynthesis and inositol phosphate metabolism, and can be used in the design of drugs against these bacteria. Inhibition of these proteins will be helpful to combat infections caused by MDR UPEC.

Vibrio Phage VMJ710 Can Prevent and Treat Disease Caused by Pathogenic MDR V. cholerae O1 in an Infant Mouse Model.

Cholera, a disease of antiquity, is still festering in developing countries that lack safe drinking water and sewage disposal. Vibrio cholerae, the causative agent of cholera, has developed multi-drug resistance to many antimicrobial agents. In aquatic habitats, phages are known to influence the occurrence and dispersion of pathogenic V. cholerae. We isolated Vibrio phage VMJ710 from a community sewage water sample of Manimajra, Chandigarh, in 2015 during an outbreak of cholera. It lysed 46% of multidrug-resistant V. cholerae O1 strains. It had significantly reduced the bacterial density within the first 4-6 h of treatment at the three multiplicity of infection (MOI 0.01, 0.1, and 1.0) values used. No bacterial resistance was observed against phage VMJ710 for 20 h in the time-kill assay. It is nearest to an ICP1 phage, i.e., Vibrio phage ICP1_2012 (MH310936.1), belonging to the class Caudoviricetes. ICP1 phages have been the dominant bacteriophages found in cholera patients' stools since 2001. Comparative genome analysis of phage VMJ710 and related phages indicated a high level of genetic conservation. The phage was stable over a wide range of temperatures and pH, which will be an advantage for applications in different environmental settings. The phage VMJ710 showed a reduction in biofilm mass growth, bacterial dispersal, and a clear disruption of bacterial biofilm structure. We further tested the phage VMJ710 for its potential therapeutic and prophylactic properties using infant BALB/c mice. Bacterial counts were reduced significantly when phages were administered before and after the challenge of orogastric inoculation with V. cholerae serotype O1. A comprehensive whole genome study revealed no indication of lysogenic genes, genes associated with possible virulence factors, or antibiotic resistance. Based on all these properties, phage VMJ710 can be a suitable candidate for oral phage administration and could be a viable method of combatting cholera infection caused by MDR V. cholerae pathogenic strains.

In Vitro and In Vivo Studies of Heraclenol as a Novel Bacterial Histidine Biosynthesis Inhibitor against Invasive and Biofilm-Forming Uropathogenic Escherichia coli.

Globally, urinary tract infections (UTIs) are one of the most frequent bacterial infections. Uropathogenic Escherichia coli (UPEC) are the predominant etiological agents causing community and healthcare-associated UTIs. Biofilm formation is an important pathogenetic mechanism of UPEC responsible for chronic and recurrent infections. The development of high levels of antimicrobial resistance (AMR) among UPEC has complicated therapeutic management. Newer antimicrobial agents are needed to tackle the increasing trend of AMR and inhibit biofilms. Heraclenol is a natural furocoumarin compound that inhibits histidine biosynthesis selectively. In this study, for the first time, we have demonstrated the antimicrobial and antibiofilm activity of heraclenol against UPEC. The drug reduced the bacterial load in the murine catheter UTI model by ≥4 logs. The drug effectively reduced bacterial loads in kidney, bladder, and urine samples. On histopathological examination, heraclenol treatment showed a reversal of inflammatory changes in the bladder and kidney tissues. It reduced the biofilm formation by 70%. The MIC value of heraclenol was observed to be high (1024 µg/mL), though the drug at MIC concentration did not have significant cytotoxicity on the Vero cell line. Further molecular docking revealed that heraclenol binds to the active site of the HisC, thereby preventing its activation by native substrate, which might be responsible for its antibacterial and antibiofilm activity. Since the high MIC of heraclenol is not achievable clinically in human tissues, further chemical modifications will be required to lower the drug's MIC value and increase its potency. Alternatively, its synergistic action with other antimicrobials may also be studied.

Repurposing of FDA approved drugs against uropathogenic Escherichia coli: In silico, in vitro, and in vivo analysis.

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.

Phenotypic and genotypic characterization of antimicrobial resistance in clinical isolates of Vibrio cholerae over a decade (2002-2016).

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. 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. CONCLUSIONS: 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.

Prevalence, Virulence Gene Profiling, and Characterization of Enteroaggregative Escherichia coli from Children with Acute Diarrhea, Asymptomatic Nourished, and Malnourished Children Younger Than 5 Years of Age in India.

OBJECTIVE: 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. STUDY DESIGN: 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. RESULTS: 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 age <2 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. CONCLUSIONS: 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.

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

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.

Molecular, phylogenetic and antibiotic resistance analysis of enteroaggregative escherichia coli/uropathogenic Escherichia coli hybrid genotypes causing urinary tract infections.

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.