Outer Membrane Permeabilization Research Articles

Integrated multiplex analysis of cell death regulators in stage II colorectal cancer suggests patients with ‘persister’ cell profiles fail to benefit from adjuvant chemotherapy

ObjectiveInducing tumour cell apoptosis is a primary objective of chemotherapy but, to date, there are no validated biomarkers of apoptosis sensitivity or resistance. Our objective was to image multiple apoptosis...

Nuclear Chloride Ion-Selective Fluorescent Probe and Its Biological Applications.

Owing to the biological significance of Cl- in cells, several chemical fluorescent probes and biosensors have been constructed to monitor this anion in the cytosol and subcellular organelles. However, a fluorescent probe for the selective detection of nuclear Cl- has not been described thus far. In the current study, we developed the first nuclear Cl--selective biosensor, Cl-YFP-NLS, whose fluorescence was effectively quenched by this anion, and demonstrated that it is an efficient and powerful tool for determining the levels of nuclear Cl-. The results of cell studies using Cl-YFP-NLS as the probe suggested that the level of Cl- in the nucleus is lower than that in the cytosol. In addition, Cl-YFP-NLS along with lysosomal (Lyso-MQAE) and mitochondrial Cl--selective fluorescent probes (Mito-MQAE) were utilized to determine the effects of various substances on the levels of Cl- in subcellular organelles. The results showed that lysosomotropic agents decrease the lysosomal Cl- concentration and increase the levels of mitochondrial and nuclear Cl-. Also, observations suggested that substances capable of inducing mitochondrial outer membrane permeabilization without inducing lysosomal membrane permeabilization increase mitochondrial and nuclear Cl- concentrations but they do not affect the level of lysosomal Cl-. Moreover, a substance directly disrupting nuclear pore complexes increased the level of nuclear Cl- and did not change the levels of lysosomal and mitochondrial Cl-. Finally, nucleus-affecting substances that cause deoxyribonucleic acid damage and activate p53 and Bax increased the levels of mitochondrial and nuclear Cl- without influencing the level of lysosomal Cl-.

Anti-Biofilm Action of Cineole and Hypericum perforatum to Combat Pneumonia-Causing Drug-Resistant P. aeruginosa

Hospital-acquired antibiotic-resistant pneumonia is one of the major causes of mortality around the world that pose a catastrophic threat. Pseudomonas aeruginosa is one of the most significant opportunistic pathogens responsible for hospital-acquired pneumonia and gained resistance to the majority of conventional antibiotics. There is an urgent need for antibiotic alternatives to control drug-resistant pneumonia and other related respiratory infections. In the present study, we explored the antibacterial potential of cineole in combination with homeopathic medicines against biofilm-forming drug-resistant P. aeruginosa. Out of 26 selected and screened homeopathic medicines, Hypericum Perforatum (HyPer) was found to eradicate biofilm-forming drug-resistant P. aeruginosa most effectively when used in combination with cineole. Interestingly, the synergistic action of HyPer and cineole was also found to be similarly effective against planktonic cells of P. aeruginosa. Further, the potential synergistic killing mechanisms of cineole and HyPer were determined by analyzing zeta membrane potential, outer membrane permeability, and DNA release from P. aeruginosa cells upon treatment with cineole and HyPer. Additionally, molecular docking analysis revealed strong binding affinities of hypericin (an active ingredient of HyPer) with the PqsA (a quorum sensing protein) of P. aeruginosa. Overall, our findings revealed the potential synergistic action of cineole and HyPer against biofilm-forming drug-resistant P. aeruginosa. Cineole and HyPer could be used in combination with other bronchodilators as inhalers to control the biofilm-forming drug-resistant P. aeruginosa.

Electrophysiological Insights into Antibiotic Translocation and Resistance: The Impact of Outer Membrane Proteins.

The alarming rise of antibiotic resistance in Gram-negative bacteria has emerged as a major global health challenge. A key factor contributing to this crisis is the low permeability of the bacterial outer membrane, which acts as a barrier that prevents antibiotics from entering the cell. Protein channels embedded in this outer membrane selectively regulate the influx of hydrophilic compounds, including antibiotics. To combat antibiotic resistance, understanding the molecular mechanisms governing antibiotic permeability through bacterial membrane channels is crucial. This knowledge is key towards elucidating their roles in studing antibiotic resistance. By compiling and analysing the flux data from multiple electrophysiological reversal potential experimental studies, which involves measuring zero-current potentials and the corresponding single-channel conductance, we can calculate the flux of charged antibiotics/compounds across different Gram-negative bacterial outer membrane channels. Through this comprehensive synthesis, this review aims to advance our understanding and stimulate discussions about the physicochemical factors influencing the flux of antibiotics through bacterial membrane protein channels, ultimately enhancing our knowledge in this area.

The C-terminal sequences of Bcl-2 family proteins mediate interactions that regulate cell death.

Programmed cell death via the both intrinsic and extrinsic pathways is regulated by interactions of the Bcl-2 family protein members that determine whether the cell commits to apoptosis via mitochondrial outer membrane permeabilization (MOMP). Recently the conserved C-terminal sequences (CTSs) that mediate localization of Bcl-2 family proteins to intracellular membranes, have been shown to have additional protein-protein binding functions that contribute to the functions of these proteins in regulating MOMP. Here we review the pivotal role of CTSs in Bcl-2 family interactions including: (1) homotypic interactions between the pro-apoptotic executioner proteins that cause MOMP, (2) heterotypic interactions between pro-apoptotic and anti-apoptotic proteins that prevent MOMP, and (3) heterotypic interactions between the pro-apoptotic executioner proteins and the pro-apoptotic direct activator proteins that promote MOMP.

Screening and Evaluation of Potential Efflux Pump Inhibitors with a Seaweed Compound Diphenylmethane-Scaffold against Drug-Resistant Escherichia coli.

Drug-resistant efflux pumps play a crucial role in bacterial antibiotic resistance. In this study, potential efflux pump inhibitors (EPIs) with a diphenylmethane scaffold were screened and evaluated against drug-resistant Escherichia coli. Twenty-four compounds were docked against the drug-binding site of E. coli multidrug transporter AcrB, and 2,2-diphenylethanol (DPE), di-p-tolyl-methanol (DPT), and 4-(benzylphenyl) acetonitrile (BPA) were screened for their highest binding free energy. The modulation assay was further used for EPI evaluation, revealing that DPE, DPT, and BPA could reduce the drug IC50 value in E. coli strains overexpressing AcrB, indicating their modulation activity. Only DPE and BPA enhanced intracellular dye accumulation and inhibited the efflux of ethidium bromide and erythromycin. In addition, DPE and BPA showed an elevated post-antibiotic effect on drug-resistant E. coli, and they did not damage the permeability of the bacterial outer membrane. The cell toxicity test showed that DPE and BPA had limited human-cell toxicity. Therefore, DPE and BPA demonstrate efflux pump inhibitory activity, and they should be further explored as potential enhancers to improve the effectiveness of existing antibiotics against drug-resistant E. coli.

Mitochondrial Quality Control Processes at the Crossroads of Cell Death and Survival: Mechanisms and Signaling Pathways.

Biological aging results from an accumulation of damage in the face of reduced resilience. One major driver of aging is cell senescence, a state in which cells remain viable but lose their proliferative capacity, undergo metabolic alterations, and become resistant to apoptosis. This is accompanied by complex cellular changes that enable the development of a senescence-associated secretory phenotype (SASP). Mitochondria, organelles involved in energy provision and activities essential for regulating cell survival and death, are negatively impacted by aging. The age-associated decline in mitochondrial function is also accompanied by the development of chronic low-grade sterile inflammation. The latter shares some features and mediators with the SASP. Indeed, the unloading of damage-associated molecular patterns (DAMPs) at the extracellular level can trigger sterile inflammatory responses and mitochondria can contribute to the generation of DAMPs with pro-inflammatory properties. The extrusion of mitochondrial DNA (mtDNA) via mitochondrial outer membrane permeabilization under an apoptotic stress triggers senescence programs. Additional pathways can contribute to sterile inflammation. For instance, pyroptosis is a caspase-dependent inducer of systemic inflammation, which is also elicited by mtDNA release and contributes to aging. Herein, we overview the molecular mechanisms that may link mitochondrial dyshomeostasis, pyroptosis, sterile inflammation, and senescence and discuss how these contribute to aging and could be exploited as molecular targets for alleviating the cell damage burden and achieving healthy longevity.

Enhancing food preservation and safety: Synergistic effects of Allium-derived organosulfur compounds and outer membrane permeabilization peptide L-11

Ready-to-eat (RTE) foods, characterized by the lack of a terminal heating step prior to consumption, offer enhanced organoleptic qualities compared to their cooked counterparts, as they retain more of their natural flavors, textures, and nutritional content. However, these untreated products pose a risk for foodborne illnesses if not promptly consumed and/or adequately stored under refrigeration or in a frozen state. This investigation delves into the synergistic antimicrobial effects of a novel human-mimetic peptide, L-11, in conjunction with organosulfur compounds derived from Allium species—namely allicin, diallyl sulfide (DAS), dipropyl sulfide (DPS), propyl propane thiosulfinate (PTS), and propyl propane thiosulfonate (PTSO)—on the inhibition and control of Gram-negative foodborne pathogens across various food matrices. Notably, despite the minimal individual antimicrobial activity of peptide L-11 and the evaluated organosulfur compounds against target bacteria (Escherichia coli, Salmonella enterica, Yersinia enterocolitica, and Shigella sonnei), their combinations exhibited remarkable synergistic effects in vitro with until 32-fold reduction for some combinations. Specifically, combinations including PTS and PTSO demonstrated up to a 99% reduction in bacterial proliferation within RTE food models such as salmorejo, aioli, pumpkin cream, and spinach cream. These findings highlight the potential applications of these synergistic combinations in extending the shelf-life and enhancing the safety of RTE foods. To the best of our knowledge, this represents the inaugural report of such synergistic interactions between organosulfur compounds and peptides in extending the shelf-life of RTE foods.

Subminimum Inhibitory Concentrations Tetracycline Antibiotics Induce Biofilm Formation in Minocycline-Resistant Klebsiella pneumonia by Affecting Bacterial Physical and Chemical Properties and Associated Genes Expression.

Biofilm formation of Klebsiella pneumoniae can protect bacteria from antibiotics and is difficult to eradicate. Thus, the influence of subinhibitory concentrations of antibiotics on bacteria is becoming increasingly important. Our study showed that subminimum inhibitory concentrations (sub-MICs) of tetracycline antibiotics can increase biofilm formation in minocycline-resistant Klebsiella pneumoniae clinical strains. However, in the bacterial adhesion and invasion experiments, the adhesion and invasion ability decreased and the survival rate of Galleria mellonella increased. Under sub-MICs of tetracycline antibiotics treatment, abnormal stretching of bacteria was observed by scanning electron microscopy. Treatment with sub-MICs of tetracyclines leads to increased surface hydrophobicity and eDNA content and decreased outer membrane permeability. The expression levels of the fimA, luxS, qseB, and qseC genes decreased, the expression level of mrkA increased, and the expression level of acrA was inconsistent under different tetracycline antibiotics treatments. Together, our results suggested that the increase in Klebsiella pneumoniae biofilm formation caused by sub-MICs of tetracycline antibiotics may occur by affecting bacterial physical and chemical properties and associated genes expression.

Smart Galactosidase-Responsive Antimicrobial Dendron: Towards More Biocompatible Membrane-Disruptive Agents.

Antimicrobial resistance is a global healthcare challenge that urgently needs the development of new therapeutic agents. Antimicrobial peptides and mimics thereof are promising candidates but mostly suffer from inherent toxicity issues due to the non-selective binding of cationic groups with mammalian cells. To overcome this toxicity issue, this work herein reports the synthesis of a smart antimicrobial dendron with masked cationic groups (Gal-Dendron) that could be uncaged in the presence of β-galactosidase enzyme to form the activated Enz-Dendron and confer antimicrobial activity. Enz-Dendron show bacteriostatic activity toward Gram-negative (P. aeruginosa and E. coli) and Gram-positive (S. aureus) bacteria with minimum inhibitory concentration values of 96 µm and exerted its antimicrobial mechanism via a membrane disruption pathway, as indicated by inner and outer membrane permeabilization assays. Crucially, toxicity studies confirmed that the masked prodrug Gal-Dendron exhibited low hemolysis and is at least 2.4 times less toxic than the uncaged cationic Enz-Dendron, thus demonstrating the advantage of masking the cationic groups with responsive immolative linkers to overcome toxicity and selectivity issues. Overall, this study highlights the potential of designing new membrane-disruptive antimicrobial agents that are more biocompatible via the amine uncaging strategy.

A novel antimicrobial peptide S24 combats serious wound infections caused by Pseudomonas aeruginosa and Acinetobacter baumannii.

Pseudomonas aeruginosa and Acinetobacter baumannii are ranked as top-priority organisms by WHO. Antimicrobial peptides (AMPs) are promising antimicrobial agents that are highly effective against serious bacterial infections. In our previous study, a series of α-helical AMPs were screened using a novel multiple-descriptor strategy. The current research suggested that S24 exhibited strong antimicrobial activity against major pathogenic bacteria, and displayed minimal haemolysis, good serum stability and maintained salt resistance. We found that S24 exerted an antimicrobial effect by destroying outer membrane permeability and producing a strong binding effect on bacterial genomic DNA that inhibits genomic DNA migration. Furthermore, S24 exerted a strong ability to promote healing in wound infected by P. aeruginosa, A. baumannii and mixed strains in a mouse model. Overall, S24 showed good stability under physiological conditions and excellent antimicrobial activity, suggesting it may be a potential candidate for the development of serious bacterial infection treatment.

Abstract IA018: Vulnerabilities of TP53-mutated AML and therapeutic implications

Abstract Acute myeloid leukemia (AML) is a complex and genetically diverse with an overall survival rate of less than 32%. Despite the remarkable success of targeted therapy in mediating remission, the emergence of acquired resistance remains a major clinical challenge to overcome. The prevailing understanding of acquired resistance identifies successive genetic changes as the primary cause.TP53 mutations are found in 70-80% of acute myeloid leukemia (AML) patients with complex karyotypes and associated with resistance towards both conventional chemotherapy and newly approved venetoclax plus azacytidine (VEN/AZA) combination. By using CRISPR-Cas9-edited isogenic AML cells harboring, mutation (6 missense mutations) or deletion (KO) of TP53, we found that TP53 mutant/KO cells are less sensitive to etoposide or VEN-AZA induced apoptosis compared to WT without defect in G1 arrest. Surprisingly, we found that TP53-mutant and TP53-wild-type (WT) isogenic AML cells and primary tumors (n=40) had comparable mitochondrial outer membrane permeabilization (MOMP) at baseline, despite the key role of TP53 in transcriptionally activating proapoptotic regulators of MOMP (such as BAX, PUMA, and NOXA). Based on these findings, we hypothesize that the targets downstream of mitochondrial permeabilization drive resistance to HMA/VEN combinations in TP53 mutant disease. By leveraging unbiased bulk RNA-seq and proteomics, and whole genome CRISPR-cas9 screen we identified IAPs as functional vulnerability. Collectively we reveal novel chemoresistance mechanisms in TP53 mutant/KO downstream of MOMP and provide a targeting strategy to improve existing therapy by targeting non-transcriptional function of TP53 in overcoming therapy resistance. Citation Format: Shruti Bhatt. Vulnerabilities of TP53-mutated AML and therapeutic implications [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr IA018.

Repression of resistance mechanismsof Pseudomonas aeruginosa: implications of thecombination of antibiotics and phytoconstituents.

Antimicrobial resistance is a prevalent problem witnessed globally and creating an alarming situation for the treatment of infections caused by resistant pathogens. Available armaments such as antibiotics often fail to exhibit the intended action against resistant pathogens, leading to failure in the treatments that are causing mortality. New antibiotics or a new treatment approach is necessary to combat this situation. P. aeruginosais an opportunistic drug resistant pathogen and is the sixth most common cause of nosocomial infections. P. aeruginosadue to its genome organization and other factors are exhibiting resistance against drugs.Bacterial biofilm formation, low permeability of outer membrane, the production of the beta-lactamase, and the production of several efflux systems limits the antibacterial potential of several classes of antibiotics. Combination of phytoconstituents with antibiotics is a promising strategy to combat multidrug resistantP. aeruginosa.Phytoconstituents such as flavonoids, terpenoids, alkaloids, polypeptides, phenolics, and essential oils are well known antibacterial agents. In this review, the activity of combination of the phytoconstituents and antibiotics, and their corresponding mechanism of action was discussed elaborately. The combination of antibiotics and plant-derived compounds exhibited better efficacy compared to antibiotics alone against the antibiotic resistance P. aeruginosainfections.

A novel senolytic drug for pulmonary fibrosis: BTSA1 targets apoptosis of senescent myofibroblasts by activating BAX.

Idiopathic pulmonary fibrosis is a progressive and age-related disease that results from impaired lung repair following injury. Targeting senescent myofibroblasts with senolytic drugs attenuates pulmonary fibrosis, revealing a detrimental role of these cells in pulmonary fibrosis. The mechanisms underlying the occurrence and persistence of senescent myofibroblasts in fibrotic lung tissue require further clarification. In this study, we demonstrated that senescent myofibroblasts are resistant to apoptosis by upregulating the proapoptotic protein BAX and antiapoptotic protein BCL-2 and BCL-XL, leading to BAX inactivation. We further showed that high levels of inactive BAX-mediated minority mitochondrial outer membrane permeabilization (minority MOMP) promoted DNA damage and myofibroblasts senescence after insult by a sublethal stimulus. Intervention of minority MOMP via the inhibition of caspase activity by quinolyl-valyl-O-methylaspartyl-[2,6-difluorophenoxy]-methyl ketone (QVD-OPH) or BAX knockdown significantly reduced DNA damage and ultimately delayed the progression of senescence. Moreover, the BAX activator BTSA1 selectively promoted the apoptosis of senescent myofibroblasts, as BTSA1-activated BAX converted minority MOMP to complete MOMP while not injuring other cells with low levels of BAX. Furthermore, therapeutic activation of BAX with BTSA1 effectively reduced the number of senescent myofibroblasts in the lung tissue and alleviated both reversible and irreversible pulmonary fibrosis. These findings advance the understanding of apoptosis resistance and cellular senescence mechanisms in senescent myofibroblasts in pulmonary fibrosis and demonstrate a novel senolytic drug for pulmonary fibrosis treatment.

Comprehensive pan-cancer analysis of mitochondrial outer membrane permeabilisation activity reveals positive immunomodulation and assists in identifying potential therapeutic targets for immunotherapy resistance.

Mitochondrial outer membrane permeabilisation (MOMP) plays a pivotal role in cellular death and immune activation. A deeper understanding of the impact of tumour MOMP on immunity will aid in guiding more effective immunotherapeutic strategies. A comprehensive pan-cancer dataset comprising 30 cancer-type transcriptomic cohorts, 20 immunotherapy transcriptomic cohorts and three immunotherapy scRNA-seq datasets was collected and analysed to determine the influence of tumour MOMP activity on clinical prognosis, immune infiltration and immunotherapy effectiveness. Leveraging 65 scRNA-Seq datasets, the MOMP signature (MOMP.Sig) was developed to accurately reflect tumour MOMP activity. The clinical predictive value of MOMP.Sig was explored through machine learning models. Integration of the MOMP.Sig model and a pan-cancer immunotherapy CRISPR screen further investigated potential targets to overcome immunotherapy resistance, which subsequently underwent clinical validation. Our research revealed that elevated MOMP activity reduces mortality risk in cancer patients, drives the formation of an anti-tumour immune environment and enhances the response to immunotherapy. This finding emphasises the potential clinical application value of MOMP activity in immunotherapy. MOMP.Sig, offering a more precise indicator of tumour cell MOMP activity, demonstrated outstanding predictive efficacy in machine-learning models. Moreover, with the assistance of the MOMP.Sig model, FOXO1 was identified as a core modulator that promotes immune resistance. Finally, these findings were successfully validated in clinical immunotherapy cohorts of skin cutaneous melanoma and triple-negative breast cancer patients. This study enhances our understanding of MOMP activity in immune modulation, providing valuable insights for more effective immunotherapeutic strategies across diverse tumours.

Effects of pleiotropic ompR and envZ alleles of Escherichia coli on envelope stress and antibiotic sensitivity.

The EnvZ-OmpR two-component system of Escherichia coli regulates the expression of the ompF and ompC porin genes in response to medium osmolarity. However, certain mutations in envZ confer pleiotropy by affecting the expression of genes of the iron and maltose regulons not normally controlled by EnvZ-OmpR. In this study, we obtained two novel envZ and ompR pleiotropic alleles, envZT15P and ompRL19Q, among revertants of a mutant with heightened envelope stress and an outer membrane (OM) permeability defect. Unlike envZ, pleiotropic mutations in ompR have not been described previously. The mutant alleles reduced the expression of several outer membrane proteins (OMPs), overcame the temperature-sensitive growth defect of a protease-deficient (ΔdegP) strain, and lowered envelope stress and OM permeability defects in a background lacking the BamB protein of an essential β-barrel assembly machinery complex. Biochemical analysis showed OmpRL19Q, like wild-type OmpR, is readily phosphorylated by EnvZ, but the EnvZ-dependent dephosphorylation of OmpRL19Q~P was drastically impaired compared to wild-type OmpR. This defect would lead to a prolonged half-life for OmpRL19Q~P, an outcome remarkably similar to what we had previously described for EnvZR397L, resulting in pleiotropy. By employing null alleles of the OMP genes, it was determined that the three pleiotropic alleles lowered envelope stress by reducing OmpF and LamB levels. The absence of LamB was principally responsible for lowering the OM permeability defect, as assessed by the reduced sensitivity of a ΔbamB mutant to vancomycin and rifampin. Possible mechanisms by which novel EnvZ and OmpR mutants influence EnvZ-OmpR interactions and activities are discussed.IMPORTANCEMaintenance of the outer membrane (OM) integrity is critical for the survival of Gram-negative bacteria. Several envelope homeostasis systems are activated when OM integrity is perturbed. Through the isolation and characterization of novel pleiotropic ompR/envZ alleles, this study highlights the involvement of the EnvZ-OmpR two-component system in lowering envelope stress and the OM permeability defect caused by the loss of proteins that are involved in OM biogenesis, envelope homeostasis, and structural integrity.

Endolysins: a new antimicrobial agent against antimicrobial resistance. Strategies and opportunities in overcoming the challenges of endolysins against Gram-negative bacteria.

Antibiotic-resistant bacteria are rapidly emerging, and the increasing prevalence of multidrug-resistant (MDR) Acinetobacter baumannii poses a severe threat to humans and healthcare organizations, due to the lack of innovative antibacterial drugs. Endolysins, which are peptidoglycan hydrolases encoded by a bacteriophage, are a promising new family of antimicrobials. Endolysins have been demonstrated as an effective therapeutic agent against bacterial infections of A. baumannii and many other Gram-positive and Gram-negative bacteria. Endolysin research has progressed from basic in vitro characterization to sophisticated protein engineering methodologies, including advanced preclinical and clinical testing. Endolysin are therapeutic agent that shows antimicrobial properties against bacterial infections caused by drug-resistant Gram-negative bacteria, there are still barriers to their implementation in clinical settings, such as safety concerns with outer membrane permeabilizers (OMP) use, low efficiency against stationary phase bacteria, and stability issues. The application of protein engineering and formulation techniques to improve enzyme stability, as well as combination therapy with other types of antibacterial drugs to optimize their medicinal value, have been reviewed as well. In this review, we summarize the clinical development of endolysin and its challenges and approaches for bringing endolysin therapies to the clinic. This review also discusses the different applications of endolysins.

Combined effect of SAR-endolysin LysKpV475 with polymyxin B and Salmonella bacteriophage phSE-5.

Endolysins are bacteriophage (or phage)-encoded enzymes that catalyse the peptidoglycan breakdown in the bacterial cell wall. The exogenous action of recombinant phage endolysins against Gram-positive organisms has been extensively studied. However, the outer membrane acts as a physical barrier when considering the use of recombinant endolysins to combat Gram-negative bacteria. This study aimed to evaluate the antimicrobial activity of the SAR-endolysin LysKpV475 against Gram-negative bacteria as single or combined therapies, using an outer membrane permeabilizer (polymyxin B) and a phage, free or immobilized in a pullulan matrix. In the first step, the endolysin LysKpV475 in solution, alone and combined with polymyxin B, was tested in vitro and in vivo against ten Gram-negative bacteria, including highly virulent strains and multidrug-resistant isolates. In the second step, the lyophilized LysKpV475 endolysin was combined with the phage phSE-5 and investigated, free or immobilized in a pullulan matrix, against Salmonella enterica subsp. enterica serovar Typhimurium ATCC 13311. The bacteriostatic action of purified LysKpV475 varied between 8.125 μg ml-1 against Pseudomonas aeruginosa ATCC 27853, 16.25 μg ml-1 against S. enterica Typhimurium ATCC 13311, and 32.50 μg ml-1 against Klebsiella pneumoniae ATCC BAA-2146 and Enterobacter cloacae P2224. LysKpV475 showed bactericidal activity only for P. aeruginosa ATCC 27853 (32.50 μg ml-1) and P. aeruginosa P2307 (65.00 μg ml-1) at the tested concentrations. The effect of the LysKpV475 combined with polymyxin B increased against K. pneumoniae ATCC BAA-2146 [fractional inhibitory concentration index (FICI) 0.34; a value lower than 1.0 indicates an additive/combined effect] and S. enterica Typhimurium ATCC 13311 (FICI 0.93). A synergistic effect against S. enterica Typhimurium was also observed when the lyophilized LysKpV475 at ⅔ MIC was combined with the phage phSE-5 (m.o.i. of 100). The lyophilized LysKpV475 immobilized in a pullulan matrix maintained a significant Salmonella reduction of 2 logs after 6 h of treatment. These results demonstrate the potential of SAR-endolysins, alone or in combination with other treatments, in the free form or immobilized in solid matrices, which paves the way for their application in different areas, such as in biocontrol at the food processing stage, biosanitation of food contact surfaces and biopreservation of processed food in active food packing.

Treatment Strategies of Colistin Resistance Acinetobacter baumannii Infections.

Acinetobacter baumannii has emerged as a pressing challenge in clinical practice, mainly due to the development of resistance to multiple antibiotics, including colistin, one of the last-resort treatments. This review highlights all the possible mechanisms of colistin resistance and the genetic basis contributing to this resistance, such as modifications to lipopolysaccharide or lipid A structures, alterations in outer membrane permeability via porins and heteroresistance. In light of this escalating threat, the review also evaluates available treatment options. The development of new antibiotics (cefiderocol, sulbactam/durlobactam) although not available everywhere, and the use of various combinations and synergistic drug combinations (including two or more of the following: a polymyxin, ampicillin/sulbactam, carbapenems, fosfomycin, tigecycline/minocycline, a rifamycin, and aminoglycosides) are discussed in the context of overcoming colistin resistance of A. baumannii infections. Although most studied combinations are polymyxin-based combinations, non-polymyxin-based combinations have been emerging as promising options. However, clinical data remain limited and continued investigation is essential to determine optimal therapeutic strategies against colistin-resistant A. baumannii.

Loss and gain of ceftazidime-avibactam susceptibility in a non-carbapenemase-producing K1-ST23 hypervirulent Klebsiella pneumoniae

ABSTRACT Objectives This study aimed at revealing the underlying mechanisms of the loss and gain of ceftazidime-avibactam susceptibility in a non-carbapenemase-producing hypervirulent Klebsiella pneumoniae (hvKp). Methods Here we longitudinally recovered 3 non-carbapenemase-producing K1-ST23 hvKp strains at a one-month interval (KP29105, KP29499 and KP30086) from an elderly male. Antimicrobial susceptibility testing, whole genome sequencing, transcriptomic sequencing, gene cloning, plasmid conjugation, quantitative real-time PCR (qRT-PCR), and SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) were conducted. Results Among the 3 hvKp strains, KP29105 was resistant to the third- and fourth-generation cephalosporins, KP29499 acquired resistance to both ceftazidime-avibactam and carbapenems, while KP30086 restored its susceptibility to ceftazidime-avibactam, imipenem and meropenem but retained low-level resistance to ertapenem. KP29105 and KP29499 carried plasmid-encoded genes bla CTX-M-15 and bla CTX-M-71, respectively, but KP30086 lost both. Cloning of gene bla CTX-M-71 and conjugation experiment of bla CTX-M-71-carrying plasmid showed that the transformant and transconjugant were susceptible to ceftazidime-avibactam but had a more than 8-fold increase in MICs. Supplementation with an outer membrane permeabilizer could reduce the MIC of ceftazidime-avibactam by 32 folds, indicating that porins play a key role in ceftazidime-avibactam resistance. The OmpK35 of the 3 isolates was not expressed, and the OmpK36 of KP29499 and KP30086 had a novel amino acid substitution (L359R). SDS-PAGE and qRT-PCR showed that the expression of porin OmpK36 of KP29499 and KP30086 was significantly down-regulated compared with KP29105. Conclusions In summary, we reported the rare ceftazidime-avibactam resistance in a non-carbapenemase-producing hvKp strain. Resistance plasmid carrying bla CTX-M-71 and mutated OmpK36 had a synergetic effect on the resistance.