Search Results (49)

This study presents a quantitative electrophysiological method to directly measure the passive transport of ammonium ions through bacterial outer membrane porins. Using a zero-current reversal potential assay in planar lipid bilayers under defined bi-ionic gradients, this study evaluates the permeability of ammonium salts through two general diffusion porins: Omp-Pst2 from Providencia stuartii and OmpF from Escherichia coli. Under matched ionic conditions, Omp-Pst2 exhibited significantly higher ammonium flux—approximately 6000 ions per second per monomer at a 1 µM gradient—compared to ~4000 ions per second for OmpF. Importantly, the identity of the accompanying anion (chloride vs. sulfate) modulated both the ion selectivity and flux rate, highlighting the influence of counterion interactions on porin-mediated transport. These findings underscore how structural differences between porins—such as pore geometry and charge distribution—govern ion permeability. The method applied here provides a robust framework for quantifying nutrient flux at the single-channel level and offers novel insights into how Gram-negative bacteria may adapt their membrane transport mechanisms under nitrogen-limited conditions. This work not only enhances our understanding of outer membrane permeability to small ions like ammonium, but also has implications for antimicrobial strategy development and biotechnological applications in nitrogen assimilation. Full article

Antibiotic resistance to Klebsiella pneumoniae poses a major public health threat, particularly in intensive care unit (ICU) settings. The emergence of extensively drug-resistant (XDR) strains complicates treatment options, requiring a deeper understanding of their genetic makeup and potential therapeutic targets. This research delineated an extensively drug-resistant (XDR) Klebsiella pneumoniae strain obtained from an ICU patient and telomeric temperate phage derived from hospital effluent. The bacteria showed strong resistance to multiple antibiotics, including penicillin (≥16 μg/mL), ceftriaxone (≥32 μg/mL), and meropenem (≥8 μg/mL), which was caused by SHV-11 beta-lactamase, NDM-1 carbapenemase, and porin mutations (OmpK37, MdtQ). The strain was categorized as K46 and O2a types and carried virulence genes involved in iron acquisition, adhesion, and immune evasion, as well as plasmids (IncHI1B_1_pNDM-MAR, IncFIB) and eleven prophage regions, reflecting its genetic adaptability and resistance dissemination. The 172,025 bp linear genome and 46.3% GC content of the N-15-like phage showed strong genomic similarities to phages of the Sugarlandvirus genus, especially those that infect K. pneumoniae. There were structural proteins (11.8%), DNA replication and repair enzymes (9.3%), and a toxin–antitoxin system (0.4%) encoded by the phage genome. A protelomerase and ParA/B partitioning proteins indicate that the phage is replicating and maintaining itself in a manner similar to the N15 phage, which is renowned for maintaining a linear plasmid prophage throughout lysogeny. Understanding the dynamics of antibiotic resistance and pathogen development requires knowledge of phages like this one, which are known for their temperate nature and their function in altering bacterial virulence and resistance profiles. The regulatory and structural proteins of the phage also provide a model for research into the biology of temperate phages and their effects on microbial communities. The importance of temperate phages in bacterial genomes and their function in the larger framework of microbial ecology and evolution is emphasized in this research. Full article

Developing effective antibiotics against Gram-negative bacteria remains challenging due to their protective outer membrane. With this study, we investigated the relationship between antibiotic permeation through the OmpF porin of Escherichia coli and antimicrobial efficacy. We measured the relative permeability coefficients (RPCs) through the bacterial porin by liposome swelling assays, including non-antibacterial molecules, and the minimum inhibitory concentrations (MICs) against E. coli. We developed a machine learning (ML) approach by combining classification and regression models to correlate these data sets. Our strategy allowed us to quantify the negative correlation between RPC and MIC values, clearly indicating that increased permeability through OmpF generally leads to improved antimicrobial activity. Moreover, the correlation was remarkable only for compounds with significant permeability coefficients. Conversely, when permeation ability is low, other factors play the most significant role in antimicrobial potency. Importantly, the proposed ML-based approach was set by exploiting the available seminal information from previous investigations in order to keep the number of molecular descriptors to the minimum for greater interpretability. This provided valuable insights into the complex interplay between different molecular properties in defining the overall outer membrane permeation and, consequently, the antimicrobial efficacy. From a practical perspective, the presented approach does not aim at identifying the “golden rule” for boosting antibiotic potency. The automated protocol presented here could be used to inspect, in silico, many alternatives of a given molecular structure, with the output being the list of the best candidates to be then synthesized and tested. This could be a valuable in silico tool for researchers in both academia and industry to rapidly evaluate novel potential compounds and reduce costs and time during the early drug discovery stage. Full article

Clinical emergent bacterial pathogens are a great threat to the global health system, chiefly Gram-negative carbapenem-resistant Enterobacterales and the Klebsiella pneumoniae species complex. Here, we present the molecular and phenotypic characterization of Klebsiella quasipneumoniae subs. similipneumoniae IEC57090 strain, belonging to ST138 and showing a multidrug resistance phenotype. The bla_NDM-7 present in one of the two resistance plasmids carried by the isolate, the antibiotic resistance genes fosA, oqxAB, and acrR, and gene mutations on porins ompK36 and ompK37, both associated with cephalosporin and carbapenem resistance, were detected. Virulence factors such as the clusters of type I and III fimbria, type IV pili genes, and genes associated with the K1 capsule, siderophore production, and multiple mobile genetic elements (MGE) were predicted. The emergence of silent pathogens in clinical environments highlights the importance of active research on new threads that may compromise the last resources of antimicrobials, such as carbapenems, specifically on mobile genetic elements containing carbapenemases in emergent pathogens, which can spread these antimicrobial resistance elements. This study reinforces that molecular biology vigilance can prevent outbreaks and help to better understand antimicrobial resistance and pathogens in clinical environment dynamics. Full article

Phenolic compounds are industrially versatile chemicals that have been successfully produced in microbial cell factories. Unfortunately, most phenolic compounds are highly toxic to cells in specific cellular environments or above a particular concentration because they form a complex with iron and promote hydroxyl radical production in Fenton reactions, resulting in the ferroptosis of cells. Here, we demonstrated that overexpression of efflux pumps and porins, including porins LamB and OmpN, and efflux pumps EmrAB, MdtABC, and SrpB, can enhance Escherichia coli phloroglucinol (PG) tolerance by inhibiting the generation of hydroxyl radicals. In addition, LamB and OmpN overexpression improved the bioproduction of PG. Furthermore, efflux pumps and porins can enhance bacterial tolerance to various phenolic compounds, including phenol, catechol, resorcinol, pyrogallol, and 2-naphthol. LamB and MdtABC confer a generalized tolerance to phenols. However, EmrAB, OmpN, and SrpB showed inconsistent effects of bacterial tolerance to different phenolic compounds. Our results will theoretically support the construction of phenolic compound-tolerant bacteria strains, which should be more efficient in the biosynthesis of phenols. Full article

Background/Objectives: Previously, we reported that 3-O-alkyl difluoroquercetins (di-F-Q) potentiates the antimicrobial activity of aztreonam (ATM) against metallo-β-lactamase (MBL)-producing P. aeruginosa through simultaneous inhibition of MBLs and efflux pumps. However, the ATM-potentiating activity of the 3-O-alkyl di-F-Q was observed only at high and potentially toxic concentrations (32 mg/L). Methods: As both MBLs and efflux pumps reside in the periplasm of Gram-negative bacteria, their inhibitors should accumulate in the periplasmic space. However, the outer membrane porins, the major entry pathway in Gram-negative bacteria, allow the passive diffusion of hydrophilic polar molecules across the outer membrane. Thus, we reasoned that the introduction of a polar substituent at 7-OH position of 3-O-alkyl di-F-Q would enhance its periplasmic concentration to result in potentiation of ATM at lower concentrations. Results: The title compound 5 exhibited inhibitory activity against NDM-1 as well as the efflux pump of P. aeruginosa, which resulted in synergistical potentiation of ATM. A combination of ATM (8 mg/L) and 5 (8 mg/L) inhibited 80% of the ATM-resistant CPPA, while ATM alone did not show any inhibition. In addition, only 4 mg/L of 5 was needed to reduce the MIC₉₀ of ATM four-fold in ATM-resistant CPPA (n = 15). The time–kill data further supported the effectiveness of the combined treatment of ATM with 5, and the combination of ATM (1xMIC) with 8 mg/L of 5 showed bactericidal effects in every bacterial strain tested (PA-002, bla_IMP, PA-003, bla_VIM, PA-014, bla_GES, and PA-017, bla_NDM) by reducing the bacterial loads by 5.1 log₁₀~8.9 log₁₀. Conclusions: The title compound 5 exhibited inhibitory activity against NDM-1 as well as the efflux pump of P. aeruginosa, and the combined inhibitory activity resulted in synergistical potentiation of ATM. It should be noted that most CPPA isolates tested were sensitized to 8 mg/L of ATM upon combination with 4~8 mg/L of 5. Full article

Background/Objectives: A. baumannii is a prominent nosocomial pathogen due to its drug-resistant phenotype, representing a public health problem. In this study, the aim was to determine the effect of different antimicrobial combinations against selected multidrug-resistant (MDR) or extensive drug-resistant (XDR) isolates of A. baumannii. Methods: MDR or XDR A. baumannii isolates were characterized by assessing genes associated with drug resistance, efflux pumps, porin expression, and biofilm formation. The activities of antimicrobial combinations including tigecycline, ampicillin/sulbactam, meropenem, levofloxacin, and colistin were evaluated using checkerboard and time-to-kill assays on isolates with different susceptibility profiles and genetic characteristics. Results: Genetic characterization of MDR/XDR strains (n = 100) included analysis of OXA-24/40 gene carbapenemase (98%), genes encoding aminoglycoside-modifying enzymes (44%), and parC gene mutations (10%). AdeIJK, AdeABC, and AdeFGH efflux pumps were overexpressed in 17–34% of isolates. Omp33-36, OmpA, and CarO membrane porins were under-expressed in 50–76% of isolates; CarO was overexpressed in 22% of isolates. Isolates showed low biofilm production (11%). Synergistic activity was observed with levofloxacin-ampicillin/sulbactam and meropenem-colistin, which were able to inhibit bacterial growth. Conclusions: Genetic characteristics of A. baumannii were highly variable among the strains. Synergistic activity was observed with meropenem-colistin and levofloxacin-ampicillin/sulbactam combinations in the checkerboard method, but not in the time-to-kill assays. These discrepancies among both methods indicate that further studies are needed to determine the best therapeutic combination for treating infections by A. baumannii. Full article

Acinetobacter baumannii (A. baumannii) has emerged as a prominent multidrug-resistant (MDR) pathogen, significantly complicating treatment strategies due to its formidable resistance mechanisms, particularly against carbapenems. Reduced membrane permeability, active antibiotic efflux, and enzymatic hydrolysis via different β-lactamases are the main resistance mechanisms displayed by A. baumannii, and they are all effective against successful treatment approaches. This means that alternate treatment approaches, such as combination therapy that incorporates beta-lactams, β-lactamase inhibitors, and novel antibiotics like cefiderocol, must be investigated immediately. Cefiderocol, a new catechol-substituted siderophore cephalosporin, improves antibacterial activity by allowing for better bacterial membrane penetration. Due to its unique structure, cefiderocol can more efficiently target and destroy resistant bacteria by using iron transport systems. Through its inhibition of peptidoglycan formation through binding to penicillin-binding proteins (PBPs), cefiderocol avoids conventional resistance pathways and induces bacterial cell lysis. The possibility of resistance development due to β-lactamase synthesis and mutations in PBPs, however, emphasizes the need for continued investigation into cefiderocol’s efficacy in combination treatment regimes. Cefiderocol’s siderophore mimic mechanism is especially important in iron-limited conditions because it can use ferric-siderophore transporters to enter cells. Additionally, its passive diffusion through bacterial porins increases its intracellular concentrations, making it a good option for treating carbapenem-resistant A. baumannii, especially in cases of severe infections and ventilator-associated diseases (IVACs). Cefiderocol may reduce MDR infection morbidity and mortality when combined with customized antimicrobial treatments, but further investigation is needed to improve patient outcomes and address A. baumannii resistance issues. Full article

The association between sepsis and thrombotic complications is still not well known. Different mechanisms have been shown to be involved in the sepsis-induced prothrombotic state, but clinical scenarios may differ. In this review, we have summarized the role that bacterial products such as porins and toxins can have in the induction of the prothrombotic state during sepsis and the interaction that they can have with each other. Furthermore, the above-mentioned mechanisms might be involved in the pattern of the clinical presentation of thrombotic events during bacterial sepsis, which would secondarily explain the association between sepsis and venous thromboembolism, the association between sepsis and disseminated intravascular coagulation, and the association between sepsis and microangiopathic venous thromboembolism. Full article

Bacterial resistance to antibiotics can lead to long-lasting, hard-to-cure infections that pose significant threats to human health. One key mechanism of antimicrobial resistance (AMR) is to reduce the antibiotic permeation of cellular membranes. For instance, the lack of outer membrane porins (OMPs) can lead to elevated AMR levels. However, knowledge on whether mutations of OMPs can also influence antibiotic susceptibility is limited. This work aims to address this question and identified an A226D mutation in OmpC, a trimeric OMP, in Escherichia coli. Surveillance studies found that this mutation is present in 50 E. coli strains for which whole genomic sequences are available. Measurement of minimum inhibition concentrations (MICs) found that this mutation leads to a 2-fold decrease in MICs for β-lactams ampicillin and piperacillin. Further survival assays confirmed the role this mutation plays in β-lactam susceptibility. With molecular dynamics, we found that the A226D mutation led to increased overall flexibility of the protein, thus facilitating antibiotic uptake, and that binding with piperacillin was weakened, leading to easier antibiotic penetration. This work reports a novel mutation that plays a role in antibiotic susceptibility, along with mechanistic studies, and further confirms the role of OMPs in bacterial tolerance to antibiotics. Full article

Combining microbial products with different mechanisms of action can produce synergistic insecticidal effects and slow down the development of resistance. This study evaluated the insecticidal activity of mixtures containing the commercial Photorhabdus luminescens (Pl) strain ATCC 29,999, the local isolate Pl 2103-UV, and the commercial Bacillus thuringiensis subsp. kurstaki (Bt) ABTS-351 against Spodoptera frugiperda. As the proportion of Bt increased in the mixtures, insecticidal activity increased, with the synergistic ratios reaching 1.98 for ATCC 29,999 and 5.29 for 2103-UV at a1:5 Pl:Bt ratio, representing approximately twofold and fivefold reductions, respectively, in the Bt dosage required for mortality. Hemolymph analysis revealed the highest Pl bacterial loads in the 1:5 treatments within the S. frugiperda hemocoel. Histopathology also showed exacerbated midgut vacuolation in the 1:5 ATCC 29,999:Bt treatment. Whole-genome analysis showed that 2103-UV produced more porins, potentially contributing to its higher insecticidal activity. This study demonstrated that Bt assists the invasion of Pl into the hemocoel and enhances synergistic insecticidal efficiency. The findings provide a reference for integrating Pl with other microbial products to sustainably manage significant and severe lepidopteran pests. Full article

Although biofilms contribute to bacterial tolerance to desiccation and survival in low-moisture foods, the molecular mechanisms underlying biofilm formation have not been fully understood. This study created a mutant library from Salmonella Enteritidis using mini-Tn10 transposon mutagenesis. The biofilm-forming potential of acquired mutants was assessed before the genomic DNA of the mutants that formed significantly (p ≤ 0.05) less biofilm mass than their wildtype parent strain was extracted for deep DNA sequencing. The gene of each mutant interrupted by mini-Tn10 insertion was identified by aligning obtained sequencing data with the reference Genbank sequences using a BLAST search. Sixty-four mutant colonies were selected, and five mutants that formed the least amount of biofilm mass compared to the wildtype parent strain were selected for sequencing analysis. The results of the BLAST search revealed that the gene interrupted by mini-Tn10 in each mutant is responsible for the biosynthesis of aldehyde dehydrogenase (EutE), cysteine desulfurase (SufS or SufE), a transporter protein, porin OmpL, and a ribbon–helix–helix protein from the CopG family, respectively. Knock-off mutant construction is a possible approach to verify the potential of the identified genes to serve as targets of antimicrobial intervention to control Salmonella colonization on low-moisture foods and in their production environment. Full article

Mitochondria are most likely descendants of strictly aerobic prokaryotes from the class Alphaproteobacteria. The mitochondrial matrix is surrounded by two membranes according to its relationship with Gram-negative bacteria. Similar to the bacterial outer membrane, the mitochondrial outer membrane acts as a molecular sieve because it also contains diffusion pores. However, it is more actively involved in mitochondrial metabolism because it plays a functional role, whereas the bacterial outer membrane has only passive sieving properties. Mitochondrial porins, also known as eukaryotic porins or voltage-dependent anion-selective channels (VDACs) control the permeability properties of the mitochondrial outer membrane. They contrast with most bacterial porins because they are voltage-dependent. They switch at relatively small transmembrane potentials of 20 to 30 mV in closed states that exhibit different permeability properties than the open state. Whereas the open state is preferentially permeable to anionic metabolites of mitochondrial metabolism, the closed states prefer cationic solutes, in particular, calcium ions. Mitochondrial porins are encoded in the nucleus, synthesized at cytoplasmatic ribosomes, and post-translationally imported through special transport systems into mitochondria. Nineteen beta strands form the beta-barrel cylinders of mitochondrial and related porins. The pores contain in addition an α-helical structure at the N-terminal end of the protein that serves as a gate for the voltage-dependence. Similarly, they bind peripheral proteins that are involved in mitochondrial function and compartment formation. This means that mitochondrial porins are localized in a strategic position to control mitochondrial metabolism. The special features of the role of mitochondrial porins in apoptosis and cancer will also be discussed in this article. Full article

Klebsiella pneumoniae (K. pneumoniae) exhibits the ability to form biofilms as a means of adapting to its adverse surroundings. K. pneumoniae in this biofilm state demonstrates remarkable resistance, evades immune system attacks, and poses challenges for complete eradication, thereby complicating clinical anti-infection efforts. Moreover, the precise mechanisms governing biofilm formation and disruption remain elusive. Recent studies have discovered that fingolimod (FLD) exhibits biofilm properties against Gram-positive bacteria. Therefore, the antibiofilm properties of FLD were evaluated against multidrug-resistant (MDR) K. pneumoniae in this study. The antibiofilm activity of FLD against K. pneumoniae was assessed utilizing the Alamar Blue assay along with confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM), and crystal violet (CV) staining. The results showed that FLD effectively reduced biofilm formation, exopolysaccharide (EPS), motility, and bacterial abundance within K. pneumoniae biofilms without impeding its growth and metabolic activity. Furthermore, the inhibitory impact of FLD on the production of autoinducer-2 (AI-2) signaling molecules was identified, thereby demonstrating its notable anti-quorum sensing (QS) properties. The results of qRT-PCR analysis demonstrated that FLD significantly decreased the expression of genes associated with the efflux pump gene (AcrB, kexD, ketM, kdeA, and kpnE), outer membrane (OM) porin proteins (OmpK35, OmpK36), the quorum-sensing (QS) system (luxS), lipopolysaccharide (LPS) production (wzm), and EPS production (pgaA). Simultaneously, FLD exhibited evident antibacterial synergism, leading to an increased survival rate of G. mellonella infected with MDR K. pneumoniae. These findings suggested that FLD has substantial antibiofilm properties and synergistic antibacterial potential for colistin in treating K. pneumoniae infections. Full article

Due to their two-cell membranes, Gram-negative bacteria are particularly resistant to antibiotics. Recent investigations aimed at exploring new target proteins involved in Gram-negative bacteria adaptation helped to identify environmental changes encountered during infection. One of the most promising approaches in finding novel targets for antibacterial drugs consists of blocking noncoding RNA-based regulation using the protein cofactor, Hfq. Although Hfq is important in many bacterial pathogens, its involvement in antibiotics response is still unclear. Indeed, Hfq may mediate drug resistance by regulating the major efflux system in Escherichia coli, but it could also play a role in the influx of antibiotics. Here, using an imaging approach, we addressed this problem quantitatively at the single-cell level. More precisely, we analyzed how Hfq affects the dynamic influx and efflux of ciprofloxacin, an antibiotic from the group of fluoroquinolones that is used to treat bacterial infections. Our results indicated that the absence of either whole Hfq or its C-terminal domain resulted in a more effective accumulation of ciprofloxacin, irrespective of the presence of the functional AcrAB-TolC efflux pump. However, overproduction of the MicF small regulatory RNA, which reduces the efficiency of expression of the ompF gene (coding for a porin involved in antibiotics influx) in a Hfq-dependent manner, resulted in impaired accumulation of ciprofloxacin. These results led us to propose potential mechanisms of action of Hfq in the regulation of fluoroquinolone fluxes across the E. coli envelope. Full article