Survival and Virulence Potential of Drug-Resistant E. coli in Simulated Gut Conditions and Antibiotic Challenge
Abstract
:1. Introduction
2. Materials and Methods
2.1. Bacterial Strains and Growth Conditions
2.2. Screening of Virulence Genes
2.3. Extraction and Profiling of Plasmid DNA
2.4. Conjugation Experiment
2.5. Growth Kinetics of MDR and Sensitive E. coli under In-Vitro Gut Conditions
2.6. Determination of Minimum Inhibitory Concentration (MIC) on Exposure to In-Vitro Gut Conditions
2.7. Effect of Simulated Gut Conditions and Ciprofloxacin Shock on the Expression of Different Virulence Genes
2.8. Bacterial Adhesion and Invasion Assay
2.9. Transcriptome Analysis of MDR Strain Grown in 3% Bile
2.9.1. Bile Exposure
2.9.2. Total RNA Isolation, cDNA Library Construction and Analysis
2.9.3. Gene Ontology and Cluster Analysis
2.9.4. Transcriptome Validation by qPCR
2.9.5. Validation of Transcriptome Data by Motility Test
2.9.6. Statistical Analysis
3. Results
3.1. Screening of Virulence Genes–Occurrence of traT Gene in Majority of the Isolates
3.2. Presence of Multiple Plasmids in MDR Strains
3.3. Trans-Conjugation Ablity of Quinolone Resitant Isolates
3.4. Reduction in Growth Kinetics of MDR and Sensitive E. coli upon Bile Challenge
3.5. Variation in Minimum Inhibitory Concentration (MIC) on Exposure to In-Vitro Gut Conditions
3.6. Effect of In-Vitro Gut Conditions and Ciprofloxacin Shock on the Expression of Virulence Genes
3.7. Adhesion and Invasion Potential of MDR and Sensitive Isolate
3.8. Functional Classification of Differentially Expressed Genes (DEGs) in MDR Isolate on Exposure to Bile and Combination of Bile and Ciprofloaxcin
3.8.1. Identification of Significantly Expressed Genes Using Hierarchical Clustering
3.8.2. Downregulation of Various Metabolic Pathways on Exposure to Bile and Combination of Bile and Ciprofloxacin
3.9. Transcriptome Validation of Differentially Expressed Genes by qPCR
Decreased Motility on Exposure to Bile and Combination of Bile and Ciprofloxacin
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Functional Annotation Cluster | Enrichment Score | Gene Count |
---|---|---|
C vs. B | ||
Annotation Cluster 1 | 3.27 | |
Transposition | 10 | |
DNA Recombination | 16 | |
Annotation Cluster 2 | 2.71 | |
Bacterial type flagellum-dependent cell motility | 11 | |
Bacterial type flagellum dependent swarming facility | 8 | |
Annotation Cluster 3 | 2.31 | |
Flagellar assembly | 14 | |
C vs. BAB | ||
Annotation Cluster 1 | 3.9 | |
Biosynthesis of secondary metabolites | 62 | |
Carbon metabolism | 31 | |
Pyruvate metabolism | 19 | |
Citric acid cycle | 12 | |
Annotation Cluster 2 | 3.55 | |
DNA recombination | 19 | |
Transposition | 15 | |
Annotation Cluster 3 | 2.82 | |
Glycolysis/Gluconeogenesis | 15 | |
Annotation Cluster 4 | 2.6 | |
NADP binding | 20 | |
B vs. BAB | ||
Annotation Cluster 1 | 2.8 | |
Metabolic pathways | 12 | |
Annotation Cluster 2 | 2.17 | |
ATP binding | 10 | |
Response to heat | 6 |
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Aditya, V.; Kotian, A.; Sanil, A.; Thaseena, P.M.-a.; Karunasagar, I.; Deekshit, V.K. Survival and Virulence Potential of Drug-Resistant E. coli in Simulated Gut Conditions and Antibiotic Challenge. Int. J. Environ. Res. Public Health 2022, 19, 12805. https://doi.org/10.3390/ijerph191912805
Aditya V, Kotian A, Sanil A, Thaseena PM-a, Karunasagar I, Deekshit VK. Survival and Virulence Potential of Drug-Resistant E. coli in Simulated Gut Conditions and Antibiotic Challenge. International Journal of Environmental Research and Public Health. 2022; 19(19):12805. https://doi.org/10.3390/ijerph191912805
Chicago/Turabian StyleAditya, Vankadari, Akshatha Kotian, Anisha Sanil, Poidal Mohammed-ali Thaseena, Indrani Karunasagar, and Vijaya Kumar Deekshit. 2022. "Survival and Virulence Potential of Drug-Resistant E. coli in Simulated Gut Conditions and Antibiotic Challenge" International Journal of Environmental Research and Public Health 19, no. 19: 12805. https://doi.org/10.3390/ijerph191912805