Biofilm and Gene Expression Characteristics of the Carbapenem-Resistant Enterobacterales, Escherichia coli IMP, and Klebsiella pneumoniae NDM-1 Associated with Common Bacterial Infections.

open access articleIn light of the limited therapeutic options with Carbapenem-Resistant Enterobacterales (CRE) infections, understanding the bacterial risk factors, such as biofilm formation and related gene expression of CRE, is vital. This study investigates the biofilm formation and biofilm-related gene expression of two enteric Enterobacterales with major CR determinants Escherichia coli IMP and Klebsiella pneumoniae NDM-1, which were seen in high prevalence in most common bacterial infections over the past few years. To our knowledge, this is the first study that demonstrated the relationship between biofilm formation and the related gene expression, to understand the potential molecular mechanisms during the biofilm formation in CRE. Biofilms were quantified by tissue culture plate assay at the stages of the biofilm development: initial attachment (6 h), microcolony formation (12 h), maturation (24 h), and dispersion (48 h). In a dispersion, event bacteria detach without any mechanical means and colonise another area. To investigate the influence of different growth conditions on biofilm formation, biofilms were quantified under different growth conditions. In parallel, quantitative real-time PCR (qPCR) assessed the biofilm-related gene expression of a cluster of genes, including biofilm maturation, quorum sensing, stress survival, and antibiotic resistance. Structural changes during biofilm development were assessed via confocal laser scanning microscopy (CLSM). We observed that the biofilm formation of CRE is correlated with the biofilm development stages, with maximum biofilm observed at 24 h at the maturation stage. Our data also showed that biofilm growth, under the condition tested, is the major factor influencing the variability of biofilm gene expression quantification assays. qPCR analyses have demonstrated that the expression of biofilm-related genes is highly correlated with phenotypic biofilm development, and these findings can be further expanded to understand the variation in regulation of such genes in these significant CRE pathogens. Our study demonstrated that both CRE strains, E. coli IMP and K. pneumoniae NDM-1, are high biofilm formers, and genes involved in biofilm development are upregulated during biofilm growth. The characteristic of the increased biofilm formation with the upregulation of antibiotic-resistant and biofilm-related genes indicates the successful pathogenic role of biofilms of these selected CRE and is attributed to their multi-drug resistance ability and successful dissemination of CRE in common bacterial infections

The Anti-Virulence Effect of Cranberry Active Compound Proanthocyanins (PACs) on Expression of Genes in the Third-Generation Cephalosporin-Resistant Escherichia coli CTXM-15 Associated with Urinary Tract Infection

open access book chapterBackground: Urinary tract infections (UTIs) are one of the most common infections found in humans, with uropathogenic Escherichia coli (UPEC) being the most common cause. Prevention of UTI is a major global concern due to its recurrent nature, medical cost, and most importantly, the increased antimicrobial resistance among UPEC. The resistance in UPEC is mainly due to the Extended-Spectrum β-lactamases (ESBL), particularly the E. coli CTXM-15 type which is known for its rapid dissemination worldwide. Treatment options for E.coli CTXM-15 have become limited over recent years because of their multi-drug resistance, hence anti-virulent strategies based on herbal remedies, have considered as a viable option. The cranberry product, Cysticlean® capsules, contain 240 mg of proanthocyanins (PACs), which have been shown to significantly inhibit E. coli adherence, both in vitro and ex vivo, to uroepithelial cells. Method: In this study, the cephalosporin-resistant E. coli isolate NCTC 1553 (E. coli CTXM-15) was analysed by qRT-PCR (quantitative Reverse Transcriptase -Polymerase Chain Reaction) for the expression of virulence factors after treatment with Cysticlean®. qRT-PCR was carried out to detect virulence determinants encoding for toxins SAT, and USP, the iron acquisition system ChuA, the protectins SoxS, KPSM, TraT and RecA, the antibiotic resistance gene CTX-M (encode β-lactamases), and the transporters IdfB and HcaT. Results: Cysticlean® significantly reduced the expression of all ten selected genes encoding for virulence factors and β-lactamases. Conclusion: Cranberry product Cysticlean® could represent a practicable alternative option for the prevention of recurrent UTI caused by multi-drug resistant E. coli CTXM-15, as the product acts on multiple bacterial target

Bacterial Filaments Induced by Antibiotic Minimal Inhibitory Concentrations in Persister Cells

Background: The ability of minor subpopulations among clonal populations to survive antibiotics is referred to as bacterial persistence. It is believed that persisters come from latent cells, where antibiotic target areas are less active and incapable of being affected. Objective: 112 clinical Escherichia coli isolates were acquired out of diverse medical samples and genetically identified using the uspA gene, which is part of the housekeeping genes. Methods: The examination of persister cells was carried out by subjecting isolates of E. coli in the exponential phase with high dose of ciprofloxacin (20 fold MIC) and calculating the surviving persister cells using CFU (colony forming units) counts. The detection and measurement of bacterial filament production was done using scanning electron and light microscopy. Results: Results showed that the bacterial filament cells kept on lengthen but cease to divide (no septa formation) at sub-minimal inhibitory doses of ciprofloxacin. Persistent isolates were shown to exhibit a wide range of form and size variations, with cells up to 4.5 times longer than usual. Conclusions: The results showed the importance of antibiotic stress on persisted cells that result in the production of filaments as a means of survival and the need to examine these rare phenotypic variations. These occurrences may be the beginning of the spread of bacterial resistance

Phytochemical Composition, In Silico Molecular Docking Analysis and Antibacterial Activity of Lawsonia inermis Linn Leaves Extracts against Extended Spectrum Beta-Lactamases-Producing Strains of Klebsiella pneumoniae

Klebsiella pneumoniae is an opportunistic Gram-negative bacterium in the Enterobacteriaceae family associated with a wide range of diseases, such as pneumonia, bloodstream infections, meningitis and urinary tract infections. Infections caused by drug-resistant strains of Klebsiella pneumoniae pose a significant threat to the effectiveness of conventional antibiotics. Hence, this has led to the need to explore alternative antimicrobial therapies, especially natural products derived from plant sources. This study assessed the phytochemical composition and antibacterial properties and performed a molecular docking analysis of Henna leaves (Lawsonia inermis L.) extracts on strains of Klebsiella pneumoniae. Crude ethanol and methanol extracts of L. inermis L. were prepared at different concentrations (25, 50, 75 and 100 mg/mL) and tested on extended spectrum beta-lactamases (ESBLs)-producing strains of Klebsiella pneumoniae. Phytocompounds were identified using gas chromatography–mass spectrometry (GC-MS) and further subjected to virtual ligands screening with DataWarrior (v05.02.01) and a molecular docking analysis using AutoDock4.2 (v4.2.6). The active compounds of L. inermis L. were determined by the docking analysis, including phytochemical, physicochemical, pharmacokinetics and docking score. The GC-MS analysis identified 27 phytoconstituents, including ethyl acetate, sclareol, 2-[1,2-dihydroxyethyl]-9-[β-d-ribofuranosyl] hypoxanthine, α-bisabolol and 2-Isopropyl-5-methylcyclohexyl 3-(1-(4-chlorophenyl)-3-oxobutyl)-coumarin-4-yl carbonate. The 27 compounds were then screened for their physicochemical and pharmacokinetic properties. The results revealed that the methanol extracts at 100 mg/mL showed significantly higher (p < 0.05) zones of inhibition (13.7 ± 1.2 mm), while the ethanol extracts at 50 mg/mL were significantly lower (6.3 ± 0.6 mm) compared to all the other treatments. The docking analysis revealed that out of the 27 compounds identified, only twelve (12) compounds have a drug-likeness activity. The 12 compounds were further subjected to docking analysis to determine the binding energies with the CTX-M protein of Klebsiella pneumoniae. Only one compound [CID_440869; (2-[1,2-dihydroxyethyl]-9-[β-d-ribofuranosyl] hypoxanthine)] had the best binding energy of −9.76 kcal/mol; hence, it can be considered a potentially suitable treatment for infections caused by ESBLs-producing strains of Klebsiella pneumoniae. This study has demonstrated that L. inermis L. extracts have antibacterial effects. Further research could explore the potential antimicrobial applications of L. inermis L. extracts to many bacterial strains.Unfunde

Autoregulation of the Escherichia coli melR promoter: repression involves four molecules of MelR

The Escherichia coli MelR protein is a transcription activator that autoregulates its own promoter by repressing transcription initiation. Optimal repression requires MelR binding to a site that overlaps the melR transcription start point and to upstream sites. In this work, we have investigated the different determinants needed for optimal repression and their spatial requirements. We show that repression requires a complex involving four DNA-bound MelR molecules, and that the global CRP regulator plays little or no role

Autoregulation of the Escherichia coli melR

promoter: repression involves four molecule

Transient Structure Associated with the Spindle Pole Body Directs Meiotic Microtubule Reorganization in S. pombe

SummaryBackgroundVigorous chromosome movements driven by cytoskeletal assemblies are a widely conserved feature of sexual differentiation to facilitate meiotic recombination. In fission yeast, this process involves the dramatic conversion of arrays of cytoplasmic microtubules (MTs), generated from multiple MT organizing centers (MTOCs), into a single radial MT (rMT) array associated with the spindle pole body (SPB), the major MTOC during meiotic prophase. The rMT is then dissolved upon the onset of meiosis I when a bipolar spindle emerges to conduct chromosome segregation. Structural features and molecular mechanisms that govern these dynamic MT rearrangements are poorly understood.ResultsElectron tomography of the SPBs showed that the rMT emanates from a newly recognized amorphous structure, which we term the rMTOC. The rMTOC, which resides at the cytoplasmic side of the SPB, is highly enriched in γ-tubulin reminiscent of the pericentriolar material of higher eukaryotic centrosomes. Formation of the rMTOC depends on Hrs1/Mcp6, a meiosis-specific SPB component that is located at the rMTOC. At the onset of meiosis I, Hrs1/Mcp6 is subject to strict downregulation by both proteasome-dependent degradation and phosphorylation leading to complete inactivation of the rMTOC. This ensures rMT dissolution and bipolar spindle formation.ConclusionsOur study reveals the molecular basis for the transient generation of a novel MTOC, which triggers a program of MT rearrangement that is required for meiotic differentiation