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.

Exploring the Prevalence, Predictors, and Impact of Bacterial Infections to Guide Empiric Antimicrobial Decisions in Cirrhosis (EPIC-AD).

Background/Aims: This study delved into cirrhosis-related infections to unveil their epidemiology, risk factors, and implications for antimicrobial decisions. Methods: We analyzed acutely decompensated cirrhosis patients (n = 971) from North India between 2013-2023 at a tertiary center. Microbiological and clinical features based on infection sites (EASL criteria) and patient outcomes were assessed. Results: Median age was 45 years; 87% were males with 47% having alcoholic hepatitis. Of these, 675 (69.5%) had infections; 305 (45%) were culture-confirmed. Notably, 71% of confirmed cases were multi-drug resistant organisms (MDRO)-related, chiefly carbapenem-resistant (48%). MDRO prevalence was highest in pulmonary (80.5%) and skin-soft-tissue infections (76.5%). Site-specific distribution and antimicrobials were suggested. Predictive models identified prior hospitalization [OR:2.23 (CI:1.58-3.14)], norfloxacin prophylaxis [OR:2.26 (CI:1.44-3.55)], prior broad-spectrum antibiotic exposure [OR:1.61 (CI:1.12-2.30)], presence of systemic inflammatory response-SIRS [OR:1.75 (CI: 1.23-2.47)], procalcitonin [OR:4.64 (CI:3.36-6.40)], and HE grade [OR:1.41 (CI:1.04-1.90)], with an area under curve; AUC of 0.891 for infection prediction. For MDRO infection prediction, second infection [OR: 7.19 (CI: 4.11-12.56)], norfloxacin prophylaxis [OR: 2.76 (CI: 1.84-4.13)], CLIF-C OF [OR: 1.10 (CI: 1.01-1.20)], prior broad-spectrum antibiotic exposure [OR: 1.66 (CI: 1.07-2.55)], rifaximin [OR: 040 (0.22-0.74)] multisite [OR: 3.67 (CI: 1.07-12.56)], and polymicrobial infection [OR: 4.55 (CI: 1.45-14.17)] yielded an AUC of 0.779 and 93% specificity. Norfloxacin prophylaxis, multisite infection, mechanical ventilation, prior broad-spectrum antibiotic exposure, and infection as acute precipitant predicted carbapenem-resistant infection (AUC: 0.821). Infections (culture-proven or probable), MDROs, carbapenem/pan-drug resistance, and second infections independently linked with mortality (P < 0.001), adjusted for age, leucocytosis, and organ failures. A model incorporating age [HR:1.02 (CI: 1.01-1.03), infection [HR:1.52 (CI: 1.05-2.20)], prior hospitalization [HR:5.33 (CI: 3.75-7.57)], norfloxacin [HR:1.29 (CI: 1.01-1.65)], multisite infection [HR:1.47 (CI:1.06-2.04)], and chronic liver failure consortium-organ failure score; CLIF-C OF [HR:1.17 (CI: 1.11-1.23)] predicted mortality with C-statistics of 0.782 (P < 0.05). Conclusion: High MDRO burden, especially carbapenem-resistant, necessitates urgent control measures in cirrhosis. Site-specific epidemiology and risk models can guide empirical antimicrobial choices in cirrhosis management.

Extensively Drug-Resistant Klebsiella pneumoniae Associated with Complicated Urinary Tract Infection in Northern India.

Klebsiella pneumoniae (Kp), which is associated with hospital-acquired infections, is extensively drug-resistant (XDR), making treatment difficult. Understanding the genetic epidemiology of XDR-Kp can help determine its potential to be hypervirulent (hv) through the presence of siderophores. We characterized the genomes of 18 colistin-resistant XDR-Kp isolated from 14 patients with complicated tract infection at an Indian healthcare facility. The 18 organisms comprised the following sequence types (STs): ST14 (n = 9), ST147 (n = 5), ST231 (n = 2), ST2096 (n = 1), and ST25 (n = 1). Many patients in each ward were infected with the same ST, suggesting a common source of infection. Some patients had recurrent infections with multiple STs circulating in the ward, providing evidence of hospital transmission. ß-lactamase genes (blaCTX-M-1, blaSHV, and blaampH) were present in all isolates. blaNDM-1 was present in 15 isolates, blaOXA-1 in 16 isolates, blaTEM-1D in 13 isolates, and blaOXA-48 in 3 isolates. Disruption of mgrB by various insertion sequences was responsible for colistin resistance in 6 isolates. The most common K-type among isolates was K2 (n = 10). One XDR convergent hvKp ST2096 mutation (iuc+ybt+blaOXA-1+blaOXA-48) was associated with prolonged hospitalization. Convergent XDR-hvKp has outbreak potential, warranting effective antimicrobial stewardship and infection control.

Diptheroids can cause nosocomial UTI and be multidrug resistant: A case report of Corynebacterium striatum, first from India.

Gram positive bacilli in the urine are usually dismissed as contaminants in urine specimens as these are commensal flora of skin and mucous membranes. Corynebacterium species were misidentified in the past due to complex biochemicals but the advent of modern diagnostics has made their identification quicker and accurate. Corynebacterium species have recently emerged as pathogens of nosocomial outbreak potential. C. striatum has been identified as opportunistic nosocomial pathogen causing various infections. We report first case of C. striatum as nosocomial urinary tract infection (UTI) pathogen in a child with bilateral renal disease. C. striatum causing UTI is very rarely reported.

Neisseria mucosa: A not so Benign Culprit of urinary tract infection: A case report.

Neisseria mucosa is saprophytic human commensal but reported as a causative agent in a couple of urinary tract infections [UTI] in susceptible individuals. In the present case, a young girl with long standing neurological problems presented with bladder outlet obstruction and fever. Her urine culture yielded Neisseria mucosa which was susceptible to broad spectrum penicillins, aminoglycosides, cephalosporins, ciprofloxacin, and azithromycin. She recovered with suitable dosage of amoxicillin clavulanic acid and was discharged. Isolation of N. mucosa here becomes clinically significant as this girl had various ureteric and lower limb weaknesses in past and was symptomatic for UTI with this infection.

Diarrheal woes in transplantation from real world settings with special focus on clostridium difficile infection.

Background: Diarrhea is the major cause of discomfort and morbidity of patients undergoing hematopoietic stem cell transplant (HSCT). The cause of diarrhea may be infective or non-infective. Methods: This is a prospective single center observational study from North India conducted over a period of approximately 4 years among 105 patients who underwent HSCT (autologous-72, allogeneic-33). The objective of the study was to identify the overall incidence and characteristics of diarrhea in HSCT in the real world, to evaluate any differences among allogeneic or autologous transplants, incidence of C Difficile among diarrheal patients, and antimicrobial usage among these patients. Results: Diarrhea was present in 89 of 105 patients (84.7%). The mean diarrheal duration was of 8.39±4.57 days (range: 1-24 days). There was non statistical difference between the incidence of diarrhea amongst allogeneic and autologous transplants (78.9% Vs 87.5%). Out of 89 patients with diarrhea, 13 were CDTA positive. We could isolate Clostridium difficile in culture in only 7.6% of patients with CDTA positivity. Metronidazole was the antibiotic of choice for diarrhea in our post-transplant settings. Metronidazole was prescribed for a median duration of 8 days (Range: 3-18 days). Seventeen patients received oral vancomycin with a median duration of 8 days (Range: 5-14 days). Conclusion: We conclude by saying that diarrhea was a common post-transplant morbidity. Clostridium difficile is not common in patients with the diarrhea post hematopoietic stem cell transplant. All cases of diarrhea need not be infective particularly in allogeneic settings.

Differential invasiveness & expression of antimicrobial peptides in Shigella serotypes.

Background & objectives: The study of Shigella pathogenesis at present is severely hampered by the lack of a relevant animal model that replicates human bacillary dysentery. Different Shigella serogroups cause varying severity of clinical illness. Ex vivo colonization of Shigella flexneri, S. dysenteriae and S. sonnei were characterized in human paediatric colonic pinch biopsies in the in vitro organ culture (IVOC) model to study the invasiveness of Shigella by gentamicin protection assay (GPA). Furthermore, the expression of antimicrobial peptides (AMPs) in response to different serotypes of Shigella was also studied in IVOC model. Methods: IVOC explants were inoculated with 109 colony forming units of different serotypes of Shigella and recovery of bacteria studied. Histopathological analysis was carried out to study inflammatory immune responses. GPA was done to elucidate the invasiveness of different serotypes of Shigella. Secretions of AMPs were measured by enzyme-linked immunosorbent assay (ELISA). Western blotting was performed to check the expression of AMPs and nuclear factor kappa B in IVOC explants. Results: After 24 h post-infection, the colon biopsies showed intense inflammatory reaction. In both IVOC and GPA, S. dysenteriae 1 was the most invasive as compared to S. flexneri and S. sonnei. S. sonnei was the least invasive. ELISA demonstrated that S. sonnei dampened the HBD (human ß-defensin)-2 responses whereas there was augmentation by S. dysenteriae and there was a modest but non-significant increase by S. flexneri. A modest increase in HBD-3 by S. sonnei and S. flexneri was observed but was not found to be significant. However, western blotting data showed upregulation of all AMPs by all serotypes. Western blotting is more sensitive than ELISA. Interpretation & conclusions: In the present study, differences in invasiveness and AMP production induced by different serotypes of Shigella were found. Human intestinal IVOC represents a model system to investigate early interaction between pathogenic bacteria and the human gut.

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.

Exploring Synergistic Combinations in Extended and Pan-Drug Resistant (XDR and PDR) Whole Genome Sequenced Acinetobacter baumannii.

Background: The diminishing antimicrobial options for the treatment of XDR and PDR Acinetobacter baumannii is an increasing concern. In this study, we assessed the in vitro synergy of the fosfomycin (FOS) with meropenem (MEM), amikacin (AK), tigecycline (TGC), and colistin (CL) in whole genome sequenced isolates. Methods: Non-replicate whole genome sequenced (illumina next-generation sequencing platform, Clevergene, India), A. baumanii (7 XDR, 1PDR) were subjected to in vitro synergy testing by checkerboard (CB) and time kill assay (TKA) after MIC determination, with glucose-6-phosphate being incorporated in all runs. FOS was used as a cornerstone drug in four combinations and colistin in one. ResFinder, MLST, PlasmidFinder, and CSIPhylogeny tools were used. Results: Mortality occurred in three patients. Diverse MLST were observed, ST-1962 (3 isolates) and one each of ST2062, ST2063, ST1816, ST1806, ST234. FOS MICs ranged from 32 to 128 mg/L, MEM MIC: 16-64 mg/L, TGC MIC: ≤2-≤4 mg/L and AK MIC: >512 mg/L. CL: MIC range, 0.25-≤2 mg/L, PDR MIC > 16 mg/L. Synergy results by CB: FOS-MEM: synergy in ⅞ (90%) isolates. Synergy lowered MEM MICs to susceptibility breakpoints in 6/8 cases. CL-MEM: Excellent synergy (3/3) isolates. FOS-AK: Indifference in ⅞, antagonism ⅛ (AK-susceptible isolate). FOS-TGC: Partial synergy (PS) in 8/8 (TGC MIC dropped to ≤0.25 mg/L in 3/8). In the PDR isolate, synergy was seen in FOS-MEM, CL-MEM, PS in FOS-CL, FOS-TGC, indifference in FOS-AK. TKA: Excellent synergy was observed with FOS-MEM from 4 h, while FOS-AK and FOS-TGC demonstrated synergy at 24 h. Synergy was achieved despite presence of widespread resistance markers against aminoglycosides (AacAad, AadA, AadB, Aph3″Ia, ArmA, Arr, StrA, StrB), beta-lactams (ADC, BlaA1, BlaA2, Zn-dependent_hydrolase, OXA-23, OXA-51, PER-1,TEM-1D, CARB-5, Mbl), sulphonamides (SulII, SulI), phenicols (CatBx, CmlA), macrolides (MphE, MsrE) and tetracycline (TetB) were widespread. Carbapenemase, CARB-5 was present in one isolate. Beta-lactamase genes OXA-23, OXA-51, BlaA2, Zn-dependent_hydrolase, ADC, Mbl and macrolide resistance genes MphE, MsrE were present in all 8 isolates. Conclusions: FOS-MEM and CL-MEM are promising combinations against A. baumannii. Synergy of FOS-MEM in intrinsically resistant A. baumannii shows that this antibiotic combination might be useful in treating such XDR and PDR pathogens.

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.

Evaluation of an automated rapid urine culture method for urinary tract infection: Comparison with gold standard conventional culture method.

PURPOSE: Urinary tract infection is one of the most prevalent disease affecting people from all age groups. For its diagnosis, conventional culture and antibiotic susceptibility is the gold standard. However, its major limitation is that the results take minimum of 24 â€‹h and antibiotic susceptibility is available after 48 â€‹h. Automated culture methods having comparable sensitivity and specificity as compared to conventional culture should be evaluated for routine diagnostics. With this aim we evaluated the diagnostic accuracy of automated urine culture method HB&L uroquattro by comparing with the gold standard conventional culture method. MATERIALS AND METHODS: A total of 1220 urine samples were included in the study. Semi-quantitative urine culture was performed using standard methods on cysteine lactose electrolyte deficient medium. For the automated culture, HB&L Uroquattro (Alifax, Polverara, PD, Italy), standard guidelines given in the manual of the instrument were followed. Diagnostic performance in terms of sensitivity, specificity, positive and negative likelihood ratios, positive and negative predictive values were calculated. RESULTS: Based on the final interpretation of conventional culture for the total 1220 samples, 26 samples (2.1%) showed major non-concordance as they were identified as sterile by HB&L but had significant growth by conventional culture and 19.9% showed minor non-concordance. At 100-999 colony forming unit/ml, HB&L has high negative predictive value i.e. 96.6% with 95% CI (95.2%-97.6%) and sensitivity i.e. 92.66% with 95% CI of (89.42%-95.15%). CONCLUSION: The HB&L Uroquattro seems to be a reliable instrument to obtain urine microbiological results in a timely manner. This technique can give presumptive report to the clinician within 5 â€‹h only for initiation of empirical antibiotics in cases of positive results.

The genomic characterization of Salmonella Paratyphi A from an outbreak of enteric fever in Vadodara, India.

Salmonella enterica Typhi (S. Typhi) and Paratyphi A (S. Paratyphi A) are the causative agents of enteric fever, a systemic human disease with a burden of 300 000 cases per year in India. The majority of enteric fever cases are associated with S. Typhi, resulting in a paucity of data regarding S. Paratyphi A, specifically with respect to genomic surveillance and antimicrobial resistance (AMR). Here, we exploited whole-genome sequencing (WGS) to identify S. Paratyphi A genotypes and AMR determinants associated with an outbreak of S. Paratyphi A in Vadodara, India, from December 2018 to December 2019. In total 117 S. Paratyphi A were isolated and genome sequenced, most were genotype 2.4.2 (72.6 % of all cases), which is the globally dominant genotype. The remainder were genotype 2.3 (25.6 %), while only two isolates belonged to genotype 2.4.1. A single base-pair mutation in gyrA, associated with reduced susceptibility to fluoroquinolones, was present in all of the outbreak isolates; with 74.35 % of isolates having a S83F substitution and the remainder having an S83Y substitution. Our surveillance study suggests that S. Paratyphi A is an emergent pathogen in South Asia, which may become increasingly relevant with the introduction of Vi conjugate vaccines.

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.

Identification of novel inhibitors against Escherichia coli utilizing HisC as a target from histidine biosynthesis pathway.

Escherichia coli is a gram-negative bacterial pathogen that poses a significant challenge both clinically and epidemiologically. Large numbers of multi-drug resistant E. coli have emerged in the last decade, because of the selection pressure generated by the inadequate use of antibiotics. Although research to combat antibiotic resistance has been going on extensively but still lags in the rate of development of newer antibiotics. Therefore, newer approaches are required to speed up the rate of discovery of antibiotics. Computational methods for screening of inhibitors have made a significant contribution to the discovery of novel antimicrobials. The present study utilized histidinol-phospho aminotransferase (HisC) as a target. HisC is an enzyme that plays a crucial role in the biosynthesis of histidine and its absence in mammals makes it an attractive drug target. A ZINC library of 5000 natural compounds was screened against HisC (PDB ID: 1FG7) using PyRx and the first 500 hits were selected for secondary screening after sorting the result on the basis of binding score. Fifteen compounds passed the secondary filter ADME and out of these five passed toxicity filters; the best among five hits was selected on the basis of its binding score and inhibition constants. Further, molecular dynamics simulations and free binding were computed of selected five compounds and two natural compounds ZINC402598829 and ZINC31157928 complexed with HisC were found as highly stable. Overall, our results indicated that these natural sources could be used as potential HisC inhibitors.Communicated by Ramaswamy H. Sarma.

Genome Analysis and Antibiofilm Activity of Phage 590B against Multidrug-Resistant and Extensively Drug-Resistant Uropathogenic Escherichia coli Isolates, India.

Urinary tract infections (UTIs) are among the most common bacterial infections in humans. Uropathogenic Escherichia coli (UPEC), which are the most frequent agents causing community as well as hospital-acquired UTIs, have become highly drug-resistant, thus making the treatment of these infections challenging. Recently, the use of bacteriophages (or 'phages') against multidrug-resistant (MDR) and extensively drug-resistant (XDR) microorganisms has garnered significant global attention. Bacterial biofilms play a vital role in the pathogenesis of UTIs caused by UPEC. Phages have the potential to disrupt bacterial biofilms using lytic enzymes such as EPS depolymerases and endolysins. We isolated a lytic phage (590B) from community sewage in Chandigarh, which was active against multiple MDR and XDR biofilm-forming UPEC strains. During whole-genome sequencing, the 44.3 kb long genome of phage 590B encoded 75 ORFs, of which 40 were functionally annotated based on homology with similar phage proteins in the database. Comparative analysis of associated phage genomes indicated that phage 590B evolved independently and had a distinct taxonomic position within the genus Kagunavirus in the subfamily Guernseyvirinae of Siphoviridae. The phage disrupted biofilm mass effectively when applied to 24 h old biofilms formed on the Foley silicon catheter and coverslip biofilm models. To study the effect of intact biofilm architecture on phage predation, the biofilms were disrupted. The phage reduced the viable cells by 0.6-1.0 order of magnitude after 24 h of incubation. Regrowth and intact bacterial cells were observed in the phage-treated planktonic culture and biofilms, respectively, which indicated the emergence of phage-resistant bacterial variants. The phage genome encoded an endolysin which might have a role in the disruption and inhibition of bacterial biofilms. Moreover, the genome lacked genes encoding toxins, virulence factors, antibiotic resistance, or lysogeny. Therefore, lytic phage 590B may be a good alternative to antibiotics and can be included in phage cocktails for the treatment of UTIs caused by biofilm-forming MDR and XDR UPEC strains.

Health-care-associated bloodstream and urinary tract infections in a network of hospitals in India: a multicentre, hospital-based, prospective surveillance study.

BACKGROUND: Health-care-associated infections (HAIs) cause significant morbidity and mortality globally, including in low-income and middle-income countries (LMICs). Networks of hospitals implementing standardised HAI surveillance can provide valuable data on HAI burden, and identify and monitor HAI prevention gaps. Hospitals in many LMICs use HAI case definitions developed for higher-resourced settings, which require human resources and laboratory and imaging tests that are often not available. METHODS: A network of 26 tertiary-level hospitals in India was created to implement HAI surveillance and prevention activities. Existing HAI case definitions were modified to facilitate standardised, resource-appropriate surveillance across hospitals. Hospitals identified health-care-associated bloodstream infections and urinary tract infections (UTIs) and reported clinical and microbiological data to the network for analysis. FINDINGS: 26 network hospitals reported 2622 health-care-associated bloodstream infections and 737 health-care-associated UTIs from 89 intensive care units (ICUs) between May 1, 2017, and Oct 31, 2018. Central line-associated bloodstream infection rates were highest in neonatal ICUs (>20 per 1000 central line days). Catheter-associated UTI rates were highest in paediatric medical ICUs (4·5 per 1000 urinary catheter days). Klebsiella spp (24·8%) were the most frequent organism in bloodstream infections and Candida spp (29·4%) in UTIs. Carbapenem resistance was common in Gram-negative infections, occurring in 72% of bloodstream infections and 76% of UTIs caused by Klebsiella spp, 77% of bloodstream infections and 76% of UTIs caused by Acinetobacter spp, and 64% of bloodstream infections and 72% of UTIs caused by Pseudomonas spp. INTERPRETATION: The first standardised HAI surveillance network in India has succeeded in implementing locally adapted and context-appropriate protocols consistently across hospitals and has been able to identify a large number of HAIs. Network data show high HAI and antimicrobial resistance rates in tertiary hospitals, showing the importance of implementing multimodal HAI prevention and antimicrobial resistance containment strategies. FUNDING: US Centers for Disease Control and Prevention cooperative agreement with All India Institute of Medical Sciences, New Delhi. TRANSLATION: For the Hindi translation of the abstract see Supplementary Materials section.

Whole genome sequencing and in vitro activity data of Escherichia phage NTEC3 against multidrug-resistant Uropathogenic and extensively drug-resistant Uropathogenic E. coli isolates.

This data article describes the whole-genome sequencing and in vitro activity data of Escherichia phage NTEC3 isolated from a community sewage sample in Chandigarh, India. The phage NTEC3 was active against multi-drug-resistant (MDR) and extensively drug-resistant (XDR) biofilm-forming Uropathogenic Escherichia coli (UPEC) strains. The genome of this phage was linear, double-stranded, and 44.2 kb long in size. A total of 72 ORFs (open reading frames) were predicted and 30 ORFs were encoded for functional proteins. The phage belonged to the Kagunavirus genus of the Siphoviridae family. Phylogenetic analysis using DNA polymerase was performed to understand the phage evolutionary relationships. Genes encoding for lysogeny, virulence, toxins, antibiotic resistance, and the CRISPR/CRISPR-like system were not found during screening. The annotated genome was deposited in Genbank under the accession number OK539620.