Multidrug-resistant Bacterial Pathogens Research Articles

Rifaximin prophylaxis causes resistance to the last-resort antibiotic daptomycin.

Multidrug-resistant bacterial pathogens like vancomycin-resistant Enterococcus faecium (VREfm) are a critical threat to human health1. Daptomycin is a last-resort antibiotic for VREfm infections with a novel mode of action2, but for which resistance has been widely reported but is unexplained. Here we show that rifaximin, an unrelated antibiotic used prophylactically to prevent hepatic encephalopathy in patients with liver disease3, causes cross-resistance to daptomycin in VREfm. Amino acid changes arising within the bacterial RNA polymerase in response to rifaximin exposure cause upregulation of a previously uncharacterized operon (prdRAB) that leads to cell membrane remodelling and cross-resistance to daptomycin through reduced binding of the antibiotic. VREfm with these mutations are spread globally, making this a major mechanism of resistance. Rifaximin has been considered 'low risk' for the development of antibiotic resistance. Our study shows that this assumption is flawed and that widespread rifaximin use, particularly in patients with liver cirrhosis, may be compromising the clinical use of daptomycin, a major last-resort intervention for multidrug-resistant pathogens. These findings demonstrate how unanticipated antibiotic cross-resistance can undermine global strategies designed to preserve the clinical use of critical antibiotics.

Isolation and preliminary characterization of a novel bacteriophage vB_KquU_φKuK6 that infects the multidrug-resistant pathogen Klebsiella quasipneumoniae.

Klebsiella quasipneumoniae (previously known as K. pneumoniae K6) strains are among the multidrug-resistant hypervirulent bacterial pathogens. Phage therapy can help treat infections caused by such pathogens. Here we report some aspects of virology and therapeutic potentials of vB_KquU_φKuK6, a bacteriophage that infects Klebsiella quasipneumoniae. K. quasipneumoniae (ATCC 700603) was used to screen wastewater lytic phages. The isolate vB_KquU_φKuK6 that consistently created large clear plaques was characterized using standard virological and molecular methods. vB_KquU_φKuK6 has a complex capsid with an icosahedral head (~60 nm) and a slender tail (~140 nm × 10 nm). The phage has a 51% AT-rich linear dsDNA genome (51,251 bp) containing 121 open reading frames. The genome contains genes encoding spanin, endolysin, and holin proteins necessary for lytic infection and a recombinase gene possibly involved in lysogenic infection. vB_KquU_φKuK6 is stable at -80 to +67°C, pH 4-9, and brief exposure to one volume percent of chloroform. vB_KquU_φKuK6 has a narrow host range. Its lytic infection cycle involves a latency of 20 min and a burst size of 435 plaque-forming units. The phage can cause lysogenic infection, and the resulting lysogens are resistant to lytic infection by vB_KquU_φKuK6. vB_KquU_φKuK6 reduces the host cells' ability to form biofilm but fails to eliminate that ability. vB_KquU_φKuK6 demonstrates phage-antibiotic synergy and reduces the minimum inhibitory concentration of chloramphenicol and neomycin sulfate by about 8 folds. vB_KquU_φKuK6 cannot be directly used for phage therapy because it is a temperate bacteriophage. However, genetically modified strains of vB_KquU_φKuK6 alone or combined with antibiotics or other lytic Klebsiella phages can have therapeutic utilities in treating K. quasipneumoniae infections.

Imidazoles and Quaternary Ammonium Compounds as Effective Therapies against (Multidrug-Resistant) Bacterial Wound Infections.

The rise and spread of antimicrobial resistance complicates the treatment of bacterial wound pathogens, further increasing the need for newer, effective therapies. Azoles such as miconazole have shown promise as antibacterial compounds; however, they are currently only used as antifungals. Previous research has shown that combining azoles with quaternary ammonium compounds yields synergistic activity against fungal pathogens, but the effect on bacterial pathogens has not been studied yet. In this study, the focus was on finding active synergistic combinations of imidazoles and quaternary ammonium compounds against (multidrug-resistant) bacterial pathogens through checkerboard assays. Experimental evolution in liquid culture was used to evaluate the possible emergence of resistance against the most active synergistic combination. Several promising synergistic combinations were identified against an array of Gram-positive pathogens: miconazole/domiphen bromide, ketoconazole/domiphen bromide, clotrimazole/domiphen bromide, fluconazole/domiphen bromide and miconazole/benzalkonium chloride. Especially, miconazole with domiphen bromide exhibits potential, as it has activity at a low concentration against a broad range of pathogens and shows an absence of strong resistance development over 11 cycles of evolution. This study provides valuable insight into the possible combinations of imidazoles and quaternary ammonium compounds that could be repurposed for (topical) wound treatment. Miconazole with domiphen bromide shows the highest application potential as a possible future wound therapy. However, further research is needed into the mode of action of these compounds and their efficacy and toxicity in vivo.

Synergistic invitro antimicrobial activity of polyherbal combination of Morinda lucida fruit and Pterocarpus santalinoides seed against multi-drug resistant clinical bacterial isolates

Background: Synergistic drug combination has been shown to be a way of bypassing drug resistance and reducing the amounts of antimicrobials consumed. As infections caused by multi-drug resistant organisms (MDROs) continue to pose global threat, it is important to search for new antimicrobial combinations of plant origin that are safe and readily available. The objective of this study is to evaluate the synergistic antimicrobial activity of extracts of Morinda lucida fruit and Pterocarpus santalinoides seed against multi-drug resistant (MDR) bacterial isolates Methodology: Morinda lucida and P. santalinoides fruits were plucked and washed clean, and the fruits of P. santalinoides were deseeded to remove the seeds. They were cut into smaller pieces and dried under shade before being milled into smooth powder and extracted with methanol. The clinical bacterial isolates used were MDR Escherichia coli, Proteus mirabilis, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Staphylococcus aureus. The phytochemical constituents of the extracts were determined using the standard method. The invitro antimicrobial assay of the extracts was done at a concentration of 50 to 400 mg/ml using the agar well diffusion technique. The minimum inhibitory concentration (MIC) and fractional inhibitory concentration (FIC) of each extract were determined using the Checker-board micro-titration method, and the FIC result obtained for each isolate was used to calculate the fractional inhibitory concentration index (FICI) of the combined extracts. The time kill assay was performed to confirm the synergistic and bactericidal activities from the MIC values obtained. Results: The phytochemical analysis revealed that the extracts of both herbal plants contained alkaloids, phenol, tannin, saponin, glycosides, and terpenoids. The antimicrobial assay results showed that the polyherbal combination of the extracts was active against all the MDR bacterial isolates tested with varying zones of inhibition. The mean inhibition zone diameters of individual M. lucida and P. santalinoides ranged from 18.0±0.9mm to 26.0±1.4mm and 17.0±0.1mm to 24.0±0.2mm respectively, while the MIC ranged from 6.25mg/ml to 12.5mg/ml for M. lucida and 12.5 mg/ml for P. santalinoides. The mean inhibition zone diameter of the polyherbal combination of the two plant extracts ranged from 25.00±00mm to 34.0±0.4mm, which compared favorably with that of levofloxacin control (28.0±0.0mm to 32.00±0.0mm), while their MIC ranged from 0.39mg/ml to 1.56mg/ml. The FIC of M. lucida extract ranged from 0.06mg/ml to 0.25mg/ml while that of P. santalinoides ranged from 0.03mg/ml to 0.13mg/ml. The FICI of combined extracts ranged from 0.09mg/ml to 0.38 mg/ml for all the MDR isolates, which is less than 0.5mg/ml, indicating synergism against all the isolates. The time of kill assay confirmed the synergistic and bactericidal activities with a 3log10 CFU/ml decrease in the number of viable cells within 6 hours of incubation.Conclusion: Our findings showed synergistic antibacterial actions of extracts of M. lucida fruit and P. santalinoides seed against MDR clinical bacterial isolates, comparable to levofloxacin. Combination of these herbal plants may serve as alternative sources of antimicrobial agents for the treatment of infections caused by MDR bacterial pathogens.

In vitro evaluation of Morinda lucida root extracts against multi-drug resistant bacterial pathogens isolated from diabetic foot ulcers

Background: The continuous rise in microbial resistance to orthodox antimicrobial drugs has led to the search for alternative sources with proven efficacy to solve the challenges of antimicrobial resistance (AMR). The preferred alternatives are plant sources, and this has led to the evaluation of constituents and potency of medicinal plants to provide scientific justification for their use. Methodology: The root of Morinda lucida plant was dug up from the ground, washed clean, and cut into smaller pieces and dried. The root was then ground into fine powder and extracted with water (aqueous), methanol, ethyl acetate and n-hexane. Multi-drug resistant (MDR) Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis and Pseudomonas aeruginosa were isolated from wound swab samples collected from patients with diabetic foot ulcers using conventional cultures, biochemical identification, and antimicrobial susceptibility tests. The phytochemical and proximate contents of the extracts were assessed by standard technique. The in vitro antimicrobial activities of the extracts were determined at a concentration of 200mg/ml of the extract using the agar well diffusion technique. The minimum inhibitory and bactericidal concentrations were determined by serial doubling dilution. The methanol extract time-kill assay was performed to determine the time of kill of the bactericidal concentration. Results: The phytochemical analysis showed that M. lucida root contains essential secondary metabolites such as flavonoids, alkaloids, tannin, saponin, glucosides, anthraquinone and quinine. The methanol and aqueous extracts showed higher in vitro antibacterial activity, producing the highest zone of inhibition of 27mm against S. aureus but a lower activity of 18mm with n-hexane extract against all isolates except S. aureus. The MIC ranges from 3.125mg/ ml and 25mg/ml. The time-kill assay of methanol extract at 2x and 3x MIC showed that bactericidal activity occurred within 0-8 hours of incubation, indicating high activity. Conclusion: The antibacterial potency of M. lucida root extract and the phytochemical components from this study shows that it can serve as a source of alternative antimicrobial agent that may be effective in the treatment MDR bacterial infections.

Detection of the CTX-M Gene Associated with Extended-Spectrum β-Lactamase (ESBL) in Broiler Chickens in Surabaya Traditional Markets

A common indicator used to examine the frequency and distribution of antibiotic resistance against other enteric bacteria in humans and animals is the commensal enteric bacterium, Escherichia coli. The transmission of plasmids harboring ESBL enzymes, primarily generated by E. coli, is the cause of this resistance. The purpose of this study was to identify the CTX-M gene in ESBL-producing E. coli from broiler chicken cloacal swabs in traditional Surabaya markets. The samples used were 96 cloacal swabs from broiler chickens in the traditional markets of Dukuh Kupang, Keputran, Pacar Keling, and Pucang. The antibiotic disks used in this study belonged to five different antibiotic classes; they are aztreonam (monobactam), chloramphenicol (phenicol), kanamycin (aminoglycoside), ciprofloxacin (fluoroquinolone), and tetracycline (tetracycline). Presumptive ESBL strains were then molecularly screened for the presence of CTX-M gene. Results revealed that out of the 96 chicken cloacal swab samples collected, 58 (60.42%) were positive for E. coli based on morphological culture, Gram staining, and biochemical tests. Additionally, 15 out of the 58 E. coli isolates recovered from broiler chicken cloacal swabs were multidrug-resistant (MDR) while 7 of E. coli isolates harbored CTX-M gene. Conclusively, this study has shown that broiler chickens sold in traditional Surabaya markets harbor MDR E. coli which possess CTX-M gene. Conditions in traditional markets with low levels of cleanliness and chickens placed close together can spread resistance genes with serious public health consequences. Therefore, it is imperative to observe good hygienic practices in Surabaya traditional markets in order to curtail the spread of MDR bacterial pathogens in the food chain.

Recent Trends in Therapeutic Drug Monitoring of Peptide Antibiotics.

Antimicrobial peptides take a specific position in the field of antibiotics (ATBs), however, from a large number of available molecules only a few of them were approved and are used in clinics. These therapeutic modalities play a crucial role in the management of diseases caused by multidrug-resistant bacterial pathogens and represent the last-line therapy for bacterial infections. Therefore, there is a demand for a rationale use of such ATBs based on optimization of the dosing strategy to minimize the risk of resistance and ensure the sustainable efficacy of the drug in real clinical practice. Therapeutic drug monitoring, as a measurement of drug concentration in the body fluids or tissues, results in the optimization of the patient´s medication and therapy outcome. This strategy is beneficial and could result in tailored therapy for different types of infection and the prolongation of the use and efficacy of ATBs in hospitals. This review paper provides an actual overview of approved antimicrobial peptides used in clinical practice and covers current trends in their analysis by convenient and advanced methodologies used for their identification and/or quantitation in biological matrices for therapeutic drug monitoring purposes. Special emphasis is given to the methods with perspective clinical outcomes.

A rapid microwave approach for ‘one-pot’ synthesis of antibiotic conjugated silver nanoparticles with antimicrobial activity against multi-drug resistant bacterial pathogens

Deaths directly attributable to drug-resistant infections reached 1.27 million in 2019 and continue to rise. This escalating resistance to antibiotics has driven a resurgence in the exploration of ancient antimicrobials to develop efficacious alternatives. The modern field of nanomaterials is a promising area of research with silver nanoparticles performing well as antimicrobial agents due to their large surface area and multiple bacterial targets. In the current study antibiotic conjugated silver nanoparticles (3–35 nm) were synthesized using β-lactam antibiotic, ampicillin. The method of heating during synthesis either microwave (4 min) or convection (4 h) influenced the physical characteristics of the ampicillin coated silver nanoparticles, however both approaches produced nanomaterials with antimicrobial activity against a variety of multi-drug resistant (MDR) clinical isolates in physiologically relevant media (when present at <0.2–2.28 mg L−1 in defined media). Critically, the microwave method is five times faster than the traditional water bath method, allowing rapid synthesis of ampicillin-conjugated nanoparticles, which supports scale up processes for industry. We suggest that the combination of antibiotic and silver in these nanoparticles produces a synergistic effect that circumvents resistance mechanisms and has the potential to provide a new line of combinatorial agents able to treat multi-drug resistant infections.

Targeting the phosphatidylglycerol lipid: An amphiphilic dendrimer as a promising antibacterial candidate.

The rapid emergence and spread of multidrug-resistant bacterial pathogens require the development of antibacterial agents that are robustly effective while inducing no toxicity or resistance development. In this context, we designed and synthesized amphiphilic dendrimers as antibacterial candidates. We report the promising potent antibacterial activity shown by the amphiphilic dendrimer AD1b, composed of a long hydrophobic alkyl chain and a tertiary amine-terminated poly(amidoamine) dendron, against a panel of Gram-negative bacteria, including multidrug-resistant Escherichia coli and Acinetobacter baumannii. AD1b exhibited effective activity against drug-resistant bacterial infections in vivo. Mechanistic studies revealed that AD1b targeted the membrane phospholipids phosphatidylglycerol (PG) and cardiolipin (CL), leading to the disruption of the bacterial membrane and proton motive force, metabolic disturbance, leakage of cellular components, and, ultimately, cell death. Together, AD1b that specifically interacts with PG/CL in bacterial membranes supports the use of small amphiphilic dendrimers as a promising strategy to target drug-resistant bacterial pathogens and addresses the global antibiotic crisis.

Biogenic silver nanoparticles synthesized using rose petals: Potential to inhibit multidrug-resistant bacterial pathogens in disabled patients

BackgroundDrug resistant bacterial infections can be fatal for disabled people needing multiple medications and new effective antibacterial drugs such as biosynthesized nanoparticles are needed. A cost-effective and green method was studied for the biosynthesis of silver nanoparticles using pink rose petals. Materials and methodsSilver nanoparticles (AgNP) were synthesized using the petals of pink rose flowers. The AgNPs were characterized using FITR, SEM, XRD, and EDS analyses. The antibacterial activity of the AgNPs against multidrug-resistant bacterial pathogens Staphylococcus aureus, Escherichia coli, Acinetobacter baumannii, and colistin-resistant Klebsiella pneumoniae were studied using the microplate method. ResultsTh AgNPs had a spherical shape and an average size of 26 nm. XRD analysis showed that the nanoparticles were Ag0. Antibacterial activity was the highest against the multidrug-resistant S. aureus the minimum inhibitory concentration (MIC) being 400 µg/ml AgNPs. The multidrug-resistant E. coli, multidrug-resistant A. baumannii, and colistin-resistant K. pneumoniae MICs were 800 µg/ml, 3200 µg/ml, and 1600 µg/ml, respectively. ConclusionThe green synthesized of AgNPS using pink rose petals and their antibacterial effects offer material for the treatments of common multidrug-resistant bacterial pathogens.

Valnemulin restores colistin sensitivity against multidrug-resistant gram-negative pathogens

Colistin is one of the last-resort antibiotics in treating infections caused by multidrug-resistant (MDR) pathogens. Unfortunately, the emergence of colistin-resistant gram-negative strains limit its clinical application. Here, we identify an FDA-approved drug, valnemulin (Val), exhibit a synergistic effect with colistin in eradicating both colistin-resistant and colistin-susceptible gram-negative pathogens both in vitro and in the mouse infection model. Furthermore, Val acts synergistically with colistin in eliminating intracellular bacteria in vitro. Functional studies and transcriptional analysis confirm that the combinational use of Val and colistin could cause membrane permeabilization, proton motive force dissipation, reduction in intracellular ATP level, and suppression in bacterial motility, which result in bacterial membrane disruption and finally cell death. Our findings reveal the potential of Val as a colistin adjuvant to combat MDR bacterial pathogens and treat recalcitrant infections.

Antimicrobial resistance profile and associated factors of hospital-acquired gram-negative bacterial pathogens among hospitalized patients in northeast Ethiopia

BackgroundAntimicrobial resistance is a major global public health issue. Infections caused by resistant species are associated with higher mortality rates, longer hospital stays, medication failure, and rising medical costs. The World Health Organisation has declared multidrug resistance-associated infections as an epidemic of public health concern.ObjectiveThis study aimed to evaluate the antimicrobial resistance profile and associated factors of hospital-acquired Gram-negative bacterial pathogens among hospitalized patients in Northeast Ethiopia.Materials and methodsA health facility-based cross-sectional study was conducted among hospitalized patients from March 2021 to February 2022. About 810 clinical specimens were collected, transported, and processed from admitted patients following the standard bacteriological procedures. The clinical samples were inoculated onto blood agar, MacConkey agar, and chocolate agar. Furthermore, the species identification was done using gram reactions, colony morphology, and color and biochemical tests. Antimicrobial susceptibility tests, extended-spectrum beta-lactamase, and carbapenemase production were performed as per the clinical laboratory standard institute guidelines. For analysis, the information was entered into Epi-data and exported to SPSS. A P value of < 0.05 with a 95% confidence interval was considered as a statistically significant association.ResultsOut of 810 clinical specimens, 285/810 (35.2%) developed bacterial infections. From the isolated bacteria, E. coli was the predominant bacteria accounting for 78/285 (27.4%) followed by K. pneumoniae, 69/285(24.42%), whereas P. vulgaris accounted for the least, 7/285 (2.5%). Overall, 132/285 (46.3%) and 99/285 (34.7%) of culture-positive patients were infected by extended-spectrum beta-lactamase and carbapenemase-producing bacteria. The overall multidrug resistance rate of the isolated bacteria was 89.4%. The highest antibiotic resistance rates were detected for doxycycline (92.9%), amoxicillin-clavulanic acid (83.9%), and ampicillin (93%). The least antibiotic resistance rate was observed for meropenem at 41.1% and amikacin at 1.7%, respectively.Conclusions and recommendationsIn the study area, significant health concerns include a range of hospital-acquired bacterial infections associated with elevated rates of multidrug resistance, Extended-spectrum beta-lactamase (ESBL), and carbapenemase-producing bacterial pathogens. Consequently, it is recommended to conduct drug-susceptibility testing of isolates and molecular detection at a national level to optimize antibiotic usage for treating prevalent bacterial infections in this area.

Fatty Acid Uptake in Klebsiella pneumoniae and the Landscape of Its Infectious Niches.

Klebsiella pneumoniae is consistently ranked among the most problematic multidrug-resistant bacterial pathogens in healthcare systems. Developing novel treatments requires a better understanding of its interaction with the host environment. Although bacteria can synthesize fatty acids, emerging findings suggest a potential preference for their acquisition from the host. Fatty acid profiling of mice revealed a dramatic increase in the level of hepatic lipids during K. pneumoniae infection. The K. pneumoniae fatty acid composition and uptake capabilities were found to be largely clonally conserved. Correlations between fatty acid uptake, outer membrane vesicle production, and cell permeability were observed, but this did not translate to alterations in cell morphology, capsule production, or antimicrobial susceptibility. Importantly, hyper-capsulation did not prevent the uptake of hydrophobic fatty acids. The uptake of a saturated fatty acid by hypervirulent K. pneumoniae isolate may provide insights into the clinical association of K. pneumoniae infections with hyperlipidemic and/or obese individuals.

The Biological Characteristics of Mycobacterium Phage Henu3 and the Fitness Cost Associated with Its Resistant Strains.

Tuberculosis (TB), caused by Mycobacterium tuberculosis, is an infectious disease that seriously affects human life and health. Despite centuries of efforts to control it, in recent years, the emergence of multidrug-resistant bacterial pathogens of M. tuberculosis due to various factors has exacerbated the disease, posing a serious threat to global health. Therefore, a new method to control M. tuberculosis is urgently needed. Phages, viruses that specifically infect bacteria, have emerged as potential biocontrol agents for bacterial pathogens due to their host specificity. In this study, a mycobacterium phage, Henu3, was isolated from soil around a hospital. The particle morphology, biological characteristics, genomics and phylogeny of Henu3 were characterized. Additionally, to explore the balance between phage resistance and stress response, phage Henu3-resistant strains 0G10 and 2E1 were screened by sequence passage and bidirectional validation methods, which significantly improved the sensitivity of phage to antibiotics (cefotaxime and kanamycin). By whole-genome re-sequencing of strains 0G10 and 2E1, 12 genes involved in cell-wall synthesis, transporter-encoded genes, two-component regulatory proteins and transcriptional regulatory factor-encoded genes were found to have mutations. These results suggest that phage Henu3 has the potential to control M. tuberculosis pathogens, and phage Henu3 has the potential to be a new potential solution for the treatment of M. tuberculosis infection.

Bacterial outer membrane vesicles increase polymyxin resistance in Pseudomonas aeruginosa while inhibiting its quorum sensing

The persistent emergence of multidrug-resistant bacterial pathogens is leading to a decline in the therapeutic efficacy of antibiotics, with Pseudomonas aeruginosa (P. aeruginosa) emerging as a notable threat. We investigated the antibiotic resistance and quorum sensing (QS) system of P. aeruginosa, with a particular focused on outer membrane vesicles (OMVs) and polymyxin B as the last line of antibiotic defense. Our findings indicate that OMVs increase the resistance of P. aeruginosa to polymyxin B. The overall gene transcription levels within P. aeruginosa also reveal that OMVs can reduce the efficacy of polymyxin B. However, both OMVs and sublethal concentrations of polymyxin B suppressed the transcription levels of genes associated with the QS system. Furthermore, OMVs and polymyxin B acted in concert on the QS system of P. aeruginosa to produce a more potent inhibitory effect. This suppression was evidenced by a decrease in the secretion of virulence factors, impaired bacterial motility, and a notable decline in the ability to form biofilms. These results reveal that OMVs enhance the resistance of P. aeruginosa to polymyxin B, yet they collaborate with polymyxin B to inhibit the QS system. Our research contribute to a deeper understanding of the resistance mechanisms of P. aeruginosa in the environment, and provide new insights into the reduction of bacterial infections caused by P. aeruginosa through the QS system.

Bacillus subtilis-derived peptides disrupt quorum sensing and biofilm assembly in multidrug-resistant Staphylococcus aureus.

The formation of biofilms by multidrug-resistant bacterial pathogens, such as Staphylococcus aureus, increases these microorganisms' virulence and decreases the efficacy of common antibiotic regimens. Probiotics possess a variety of strain-specific strategies to reduce biofilm formation in competing organisms; however, the mechanisms and compounds responsible for these phenomena often go uncharacterized. In this study, we identified a mixture of small probiotic-derived peptides capable of Agr quorum sensing interference as one of the mechanisms driving antibiofilm activity against S. aureus. This collection of peptides also improved antibiotic killing and protected human gut epithelial cells from S. aureus-induced toxicity by stimulating an adaptive cytokine response. We conclude that purposeful strain screening and selection efforts can be used to identify unique probiotic strains that possess specially desired mechanisms of action. This information can be used to further improve our understanding of the ways in which probiotic and probiotic-derived compounds can be applied to prevent bacterial infections or improve bacterial sensitivity to antibiotics in clinical and agricultural settings.

Purification of Potential Antimicrobial Metabolites from Endophytic Fusarium oxysporum Isolated from Myrtus communis.

The rise of microbial resistance and emerging infections pose significant health threats. Natural products from endophytic fungi offer a promising source of novel compounds with the potential as major drug leads. This research aims to screen Myrtus communis and Moringa oleifera for endophytic fungi and screen their metabolites for antibacterial and antifungal potential. Six endophytic fungal strains were isolated using a potato dextrose agar (PDA) medium. The M. communis isolates were designated MC1, MC2, and MC3, and the M. oleifera isolates were named MO1, MO2, and MO3. Preliminary bioactivity testing revealed that the MC3 isolate exhibited significant growth inhibition against multidrug-resistant bacterial and fungal pathogens, including Staphylococcus aureus, Enterococcus faecalis, Bacillus subtilis, Pseudomonas aeruginosa, Escherichia coli, Candida albicans, and Candida glabrata. The MC3 isolate was identified as Fusarium oxysporum through morphological and microscopic methods. For metabolite production, the fungal strain was cultured in potato dextrose broth (PDB) medium at 28°C for 14days in a shaking incubator. The metabolites were purified using various chromatographic techniques, HPLC and GC-MS. The GC-MS analysis of the bioactive compound containing fungal strain (F. oxysporum) revealed multiple compounds at different retention times using the NIST-20 Library. Based on RSI values and probability indices, two compounds were targeted for further purification. Structure elucidation was performed using 1D and 2D nuclear magnetic resonance (NMR) experiments on a Varian 500 NMR machine. The compounds identified were ethyl iso-allocholate (C26H44O5, exact mass 436.32) and 1-monolinoleoyl glycerol trimethylsilyl ether (C27H56O4Si2, exact mass 500.37). The MS (NIST-20) library facilitated the investigation of the in silico antimicrobial activity of these compounds against the elastase virulence protein of P. aeruginosa and protease Sapp1p from C. parapsilosis. Both the compounds were docked with druggable proteins using the Glide induced fit docking (IFD) algorithm. The ethyl iso-allocholate and 1-monolinoleoyl glycerol trimethylsilyl ether compounds showed binding scores - 10.07kcalmol-1 and - 7.47kcalmol-1 against elastase, and - 8.16kcalmol-1 and - 6.89kcalmol-1 against aspartic protease, respectively. In vitro studies confirmed the inhibitory activity of these compounds against multidrug-resistant P. aeruginosa and E. faecalis. Ethyl iso-allocholate exhibited higher bioactivity against P. aeruginosa with inhibition rates of 41%, 27%, and 35% at concentrations of 1000, 500, and 250µgmL-1, respectively. These results suggest that bioactive compounds from F. oxysporum have the potential as antimicrobial agents, warranting further research.

Membrane-targeting, ultrashort lipopeptide acts as an antibiotic adjuvant and sensitizes MDR gram-negative pathogens toward narrow-spectrum antibiotics

Globally, infections due to multi-drug resistant (MDR) Gram-negative bacterial (GNB) pathogens are on the rise, negatively impacting morbidity and mortality, necessitating urgent treatment alternatives. Herein, we report a detailed bio-evaluation of an ultrashort, cationic lipopeptide ‘SVAP9I’ that demonstrated potent antibiotic activity and acted as an adjuvant to potentiate existing antibiotic classes towards GNBs. Newly synthesized lipopeptides were screened against ESKAPE pathogens and cytotoxicity assays were performed to evaluate the selectivity index (SI). SVAP9I exhibited broad-spectrum antibacterial activity against critical MDR-GNB pathogens including members of Enterobacteriaceae (MIC 4–8 mg/L), with a favorable CC50 value of ≥100 mg/L and no detectable resistance even after 50th serial passage. It demonstrated fast concentration-dependent bactericidal action as determined via time-kill analysis and also retained full potency against polymyxin B-resistant E. coli, indicating distinct mode of action. SVAP9I targeted E. coli's outer and inner membranes by binding to LPS and phospholipids such as cardiolipin and phosphatidylglycerol. Membrane damage resulted in ROS generation, depleted intracellular ATP concentration and a concomitant increase in extracellular ATP. Checkerboard assays showed SVAP9I's synergism with narrow-spectrum antibiotics like vancomycin, fusidic acid and rifampicin, potentiating their efficacy against MDR-GNB pathogens, including carbapenem-resistant Acinetobacter baumannii (CRAB), a WHO critical priority pathogen. In a murine neutropenic thigh infection model, SVAP9I and rifampicin synergized to express excellent antibacterial efficacy against MDR-CRAB outcompeting polymyxin B. Taken together, SVAP9I's distinct membrane-targeting broad-spectrum action, lack of resistance and strong in vitro andin vivopotency in synergism with narrow spectrum antibiotics like rifampicin suggests its potential as a novel antibiotic adjuvant for the treatment of serious MDR-GNB infections.

Identification of Potential Therapeutics of Mentha Essential Oil Content as Antibacterial MDR Agents against AcrAB-TolC Multidrug Efflux Pump from Escherichia coli: An In Silico Exploration.

Multidrug-resistant bacterial pathogens, such as E. coli, represent a major human health threat. Due to the critical need to overcome this dilemma, since the drug efflux pump has a vital function in the evolution of antimicrobial resistance in bacteria, we have investigated the potential of Mentha essential oil major constituents (1-19) as antimicrobial agents via their ability to inhibit pathogenic DNA gyrase and, in addition, their potential inhibition of the E. coli AcrB-TolC efflux pump, a potential target to inhibit MDR pathogens. The ligand docking approach was conducted to analyze the binding interactions of Mentha EO constituents with the target receptors. The obtained results proved their antimicrobial activity through the inhibition of DNA gyrase (1kzn) with binding affinity ΔG values between -4.94 and -6.49 kcal/mol. Moreover, Mentha EO constituents demonstrated their activity against MDR E. coli by their ability to inhibit AcrB-TolC (4dx7) with ΔG values ranging between -4.69 and -6.39 kcal/mol. The antimicrobial and MDR activity of Mentha EOs was supported via hydrogen bonding and hydrophobic interactions with the key amino acid residues at the binding site of the active pocket of the targeted receptors.

Lipid A in outer membrane vesicles shields bacteria from polymyxins.

The continuous emergence of multidrug-resistant bacterial pathogens poses a major global healthcare challenge, with Klebsiella pneumoniae being a prominent threat. We conducted a comprehensive study on K. pneumoniae's antibiotic resistance mechanisms, focusing on outer membrane vesicles (OMVs) and polymyxin, a last-resort antibiotic. Our research demonstrates that OMVs protect bacteria from polymyxins. OMVs derived from Polymyxin B (PB)-stressed K. pneumoniae exhibited heightened protective efficacy due to increased vesiculation, compared to OMVs from unstressed Klebsiella. OMVs also shield bacteria from different bacterial families. This was validated ex vivo and in vivo using precision cut lung slices (PCLS) and Galleria mellonella. In all models, OMVs protected K. pneumoniae from PB and reduced the associated stress response on protein level. We observed significant changes in the lipid composition of OMVs upon PB treatment, affecting their binding capacity to PB. The altered binding capacity of single OMVs from PB stressed K. pneumoniae could be linked to a reduction in the lipid A amount of their released vesicles. Although the amount of lipid A per vesicle is reduced, the overall increase in the number of vesicles results in an increased protection because the sum of lipid A and therefore PB binding sites have increased. This unravels the mechanism of the altered PB protective efficacy of OMVs from PB stressed K. pneumoniae compared to control OMVs. The lipid A-dependent protective effect against PB was confirmed in vitro using artificial vesicles. Moreover, artificial vesicles successfully protected Klebsiella from PB ex vivo and in vivo. The findings indicate that OMVs act as protective shields for bacteria by binding to polymyxins, effectively serving as decoys and preventing antibiotic interaction with the cell surface. Our findings provide valuable insights into the mechanisms underlying antibiotic cross-protection and offer potential avenues for the development of novel therapeutic interventions to address the escalating threat of multidrug-resistant bacterial infections.