BACKGROUND: Escherichia coli (E. coli) is a multidrug resistant opportunistic pathogen that can cause secondary bacterial infections in patients with COVID-19. This study aimed to determine the antimicrobial resistance profile of E. coli as a secondary bacterial infection in patients with COVID-19 and to assess the prevalence and characterization of genes related to efflux pumps and porin.
METHODS: A total of 50 nonduplicate E. coli isolates were collected as secondary bacterial infections in COVID-19 patients. The isolates were cultured from sputum samples. Confirmation and antibiotic susceptibility testing were conducted by Vitek 2. PCR was used to assess the prevalence of the efflux pump and porin-related genes in the isolates. The phenotypic and genotypic evolution of antibiotic resistance genes related to the efflux pump was evaluated.
RESULTS: The E. coli isolates demonstrated high resistance to ampicillin (100%), cefixime (62%), cefepime (62%), amoxicillin-clavulanic acid (60%), cefuroxime (60%), and ceftriaxone (58%). The susceptibility of E. coli to ertapenem was greatest (92%), followed by imipenem (88%), meropenem (86%), tigecycline (80%), and levofloxacin (76%). Regarding efflux pump gene combinations, there was a significant association between the acrA gene and increased resistance to levofloxacin, between the acrB gene and decreased resistance to meropenem and increased resistance to levofloxacin, and between the ompF and ompC genes and increased resistance to gentamicin.
CONCLUSIONS: The antibiotics ertapenem, imipenem, meropenem, tigecycline, and levofloxacin were effective against E. coli in patients with COVID-19. Genes encoding efflux pumps and porins, such as acrA, acrB, and outer membrane porins, were highly distributed among all the isolates. Efflux pump inhibitors could be alternative antibiotics for restoring tetracycline activity in E. coli isolates.

The PTEN-induced kinase 1 (PINK1)-Parkin pathway plays a vital role in maintaining a healthy pool of mitochondria in higher eukaryotic cells. While the downstream components of this pathway are well understood, the upstream triggers remain less explored. In this study, we conducted an extensive analysis of inhibitors targeting various mitochondrial electron transport chain (ETC) complexes to investigate their potential as activators of the PINK1-Parkin pathway. We identified cloflucarban, an antibacterial compound, as a novel pathway activator that simultaneously inhibits mitochondrial complexes III and V, and V. RNA interference (RNAi) confirmed that the dual inhibition of these complexes activates the PINK1-Parkin pathway. Intriguingly, we discovered that albumin, specifically bovine serum albumin (BSA) and human serum albumin (HSA) commonly present in culture media, can hinder carbonyl cyanide m-chlorophenyl hydrazone (CCCP)-induced pathway activation. However, cloflucarban\'s efficacy remains unaffected by albumin, highlighting its reliability for studying the PINK1-Parkin pathway. This study provides insights into the activation of the upstream PINK1-Parkin pathway and underscores the influence of culture conditions on research outcomes. Cloflucarban emerges as a promising tool for investigating mitochondrial quality control and neurodegenerative diseases.

Duck plague virus (DPV) is extremely infectious and lethal, so antiviral drugs are urgently needed. Our previous study shows that DPV infection with duck embryo fibroblast (DEF) induces reactive oxygen species (ROS) changes and promotes apoptosis. In this study, we tested the antiviral effect of the carbonyl cyanide m-chlorophenyl hydrazone (CCCP), a common mitochondrial autophagy inducer. Our results demonstrated a dose-dependent anti-DPV effect of CCCP, CCCP-treatment blocked the intercellular transmission of DPV after infection, and we also proved that CCCP could have an antiviral effect up to 48 hpi. The addition of CCCP reversed the DPV-induced ROS changes, CCCP can inhibit virus-induced apoptosis; meanwhile, CCCP can affect mitochondrial fusion and activate mitophagy to inhibit DPV. In conclusion, CCCP can be an effective antiviral candidate against DPV.

For Raman hyperspectral detection and imaging in live cells, it is very desirable to create novel probes with strong and unique Raman vibrations in the biological silent region (1800-2800 cm-1). The use of molecular probes in Raman imaging is a relatively new technique in subcellular research; however, it is developing very rapidly. Compared with the label-free method, it allows for a more sensitive and selective visualization of organelles within a single cell. Biological systems are incredibly complex and heterogeneous. Directly visualizing biological structures and activities at the cellular and subcellular levels remains by far one of the most intuitive and powerful ways to study biological problems. Each organelle plays a specific and essential role in cellular processes, but importantly for cells to survive, mitochondrial function must be reliable. Motivated by earlier attempts and successes of biorthogonal chemical imaging, we develop a tool supporting Raman imaging of cells to track biochemical changes associated with mitochondrial function at the cellular level in an in vitro model. In this work, we present a newly synthesized highly sensitive RAR-BR Raman probe for the selective imaging of mitochondria in live endothelial cells.

Multidrug efflux pumps are the frontline defense mechanisms of Gram-negative bacteria, yet little is known of their relative fitness trade-offs under gut conditions such as low pH and the presence of antimicrobial food molecules. Low pH contributes to the proton-motive force (PMF) that drives most efflux pumps. We show how the PMF-dependent pumps AcrAB-TolC, MdtEF-TolC, and EmrAB-TolC undergo selection at low pH and in the presence of membrane-permeant phytochemicals. Competition assays were performed by flow cytometry of co-cultured Escherichia coli K-12 strains possessing or lacking a given pump complex. All three pumps showed negative selection under conditions that deplete PMF (pH 5.5 with carbonyl cyanide 3-chlorophenylhydrazone or at pH 8.0). At pH 5.5, selection against AcrAB-TolC was increased by aromatic acids, alcohols, and related phytochemicals such as methyl salicylate. The degree of fitness cost for AcrA was correlated with the phytochemical\'s lipophilicity (logP). Methyl salicylate and salicylamide selected strongly against AcrA, without genetic induction of drug resistance regulons. MdtEF-TolC and EmrAB-TolC each had a fitness cost at pH 5.5, but salicylate or benzoate made the fitness contribution positive. Pump fitness effects were not explained by gene expression (measured by digital PCR). Between pH 5.5 and 8.0, acrA and emrA were upregulated in the log phase, whereas mdtE expression was upregulated in the transition-to-stationary phase and at pH 5.5 in the log phase. Methyl salicylate did not affect pump gene expression. Our results suggest that lipophilic non-acidic molecules select against a major efflux pump without inducing antibiotic resistance regulons.IMPORTANCEFor drugs that are administered orally, we need to understand how ingested phytochemicals modulate drug resistance in our gut microbiome. Bacteria maintain low-level resistance by proton-motive force (PMF)-driven pumps that efflux many different antibiotics and cell waste products. These pumps play a key role in bacterial defense by conferring resistance to antimicrobial agents at first exposure while providing time for a pathogen to evolve resistance to higher levels of the antibiotic exposed. Nevertheless, efflux pumps confer energetic costs due to gene expression and pump energy expense. The bacterial PMF includes the transmembrane pH difference (ΔpH), which may be depleted by permeant acids and membrane disruptors. Understanding the fitness costs of efflux pumps may enable us to develop resistance breakers, that is, molecules that work together with antibiotics to potentiate their effect. Non-acidic aromatic molecules have the advantage that they avoid the Mar-dependent induction of regulons conferring other forms of drug resistance. We show that different pumps have distinct selection criteria, and we identified non-acidic aromatic molecules as promising candidates for drug resistance breakers.

Presenilin 1 (PS1) is a transmembrane proteolytic subunit of γ-secretase that cleaves amyloid precursor proteins. Mutations in PS1 (mPS1) are associated with early-onset familial Alzheimer\'s disease (AD). The link between mutated PS1, mitochondrial calcium regulation, and AD has been studied extensively in different test systems. Despite the wide-ranging role of mPS1 in AD, there is a paucity of information on the link between PS1 and neuronal cell death, a hallmark of AD. In the present study, we employed the selective mitochondrial uncoupler carbonyl cyanide chlorophenylhydrazone (CCCP) and compared the reactivity of mPS1-transfected cultured rat hippocampal neurons with PS1 and control neurons in a situation of impaired mitochondrial functions. CCCP causes a slow rise in cytosolic and mitochondrial calcium in all three groups of neurons, with the mPS1 neurons demonstrating a faster rise. Consequently, mPS1 neurons were depolarized by CCCP and measured with TMRM, a mitochondrial voltage indicator, more than the other two groups. Morphologically, CCCP produced more filopodia in mPS1 neurons than in the other two groups, which were similarly affected by the drug. Finally, mPS1 transfected neurons tended to die from prolonged exposure to CCCP sooner than the other groups, indicating an increase in vulnerability associated with a lower ability to regulate excess cytosolic calcium.

The purpose of this study was to evaluate the antimicrobial activity of indole-3-carbinol (I3C) with membrane-active agents, namely carbonyl cyanide 3-chlorophenylhydrazone (CCCP) and ethylenediaminetetraacetic acid (EDTA) against multidrug-resistant (MDR) Gram-negative bacteria and bacterial persisters. The determination of minimal inhibitory concentration (MIC) showed that I3C was effective against Acinetobacter baumannii (3.13‒6.25 × 10-3 mol l-1), Klebsiella pneumoniae (8 × 10-3 mol l-1), Pseudomonas aeruginosa (6.25‒12.5 × 10-3 mol l-1), and Escherichia coli (6.25‒12.5 × 10-3 mol l-1). Our study demonstrated that EDTA synergistically enhanced the bactericidal activity of I3C against most MDR Gram-negative bacteria isolates and contributed to an 8- to 64-fold MIC reduction compared with that of I3C alone, yet CCCP only displayed synergy with I3C against P. aeruginosa and A. baumannii. The EDTA-I3C combination also significantly reduced the viable number of testing bacteria (P = 7.2E-05), effectively reduced bacterial persisters, and repressed bacterial growth compared with that the use of I3C alone. Our data demonstrate that use of EDTA as adjuvant molecules can effectively improve the antibacterial activity of I3C and may help to reduce the development of antimicrobial resistance.

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BACKGROUND: Over the last several decades, the AcrAB and OqxAB efflux pumps have been found to cause multidrug resistance (MDR) in various bacteria, most notably Klebsiella pneumoniae. Antibiotic resistance surges with increased expression of the acrAB and oqxAB efflux pumps.
METHODS: In accordance with CLSI guidelines, a disk diffusion test was carried out using 50 K. pneumoniae isolates obtained from various clinical samples. CT was computed in treated samples and compared to a susceptible ciprofloxacin strain (A111). The final finding is presented as the fold change in the target gene\'s expression in treated samples relative to a control sample (A111), normalized to a reference gene. As ΔΔCT = 0 and 2 to the power of 0 = 1, relative gene expression for reference samples is often set to 1 Results: The highest rates of resistance were recognized with cefotaxime (100%), cefuroxime (100%), cefepime (100%), levofloxacin (98%), trimethoprimsulfamethoxazole (80%), and gentamicin (72%), whereas imipenem (34%) had the lowest rates. Overexpression of acrA and acrB, oqxA and oqxB, regulators marA, soxS, and rarA were greater in ciprofloxacin-resistant isolates compared to the reference strain (strain A111). There was also a moderate connection between ciprofloxacin MIC and acrAB gene expression and a moderate connection between ciprofloxacin MIC and oqxAB gene expression.
CONCLUSIONS: This work provides a deeper knowledge of the role of efflux pump genes, particularly acrAB and oqxAB, as well as transcriptional regulators marA, soxS, and rarA, in bacterial resistance to ciprofloxacin.

Improving the efficacy of existing antibiotics is significant for combatting antibiotic resistance that poses a major threat to human health. Carbonyl cyanide m-chlorophenylhydrazine (CCCP), a well-known protonophore for dissipating proton motive force (PMF), has been widely used to block the PMF-dependent uptake of aminoglycoside antibiotics and thus suppress aminoglycoside lethality. Here, we report that CCCP and its functional analog FCCP, but not other types of protonophores, unprecedently potentiate aminoglycosides (e.g., tobramycin and gentamicin) by 3-4 orders of magnitude killing of Escherichia coli, Staphylococcus aureus, Shigella flexneri, and Vibrio alginolyticus cells in stationary phase but not these cells in exponential phase nor other 12 bacterial species we examined. Overall, the effect of CCCP on aminoglycoside lethality undergoes a gradual transition from suppression against E. coli exponential-phase cells to potentiation against late stationary-phase cells, with the cell growth status and culture medium being crucial. Consistently, disturbance of the PMF by changing transmembrane proton gradient (ΔpH) or electric potential (ΔΨ) also potentiates tobramycin. Nevertheless, CCCP neither increases the intracellular concentration of tobramycin nor decreases the MIC of the antibiotic, thus excluding that CCCP acts as an efflux pump inhibitor to potentiate aminoglycosides. Rather, we show that the combined treatment dramatically enhances the cellular level of hydroxyl radical under both aerobic and anaerobic culturing conditions, under which the antioxidant N-acetyl cysteine fully suppresses both hydroxyl radical accumulation and cell death. Together, these findings open a new avenue to develop certain protonophores as aminoglycoside adjuvants against pathogens in stationary phase and also illustrate an essential role of hydroxyl radical in aminoglycoside lethality regardless of aerobic respiration.