Time-kill Kinetics Research Articles

Acrylonitrile adducts: design, synthesis and biological evaluation as antimicrobial, haemolytic and thrombolytic agent

In this work, five acrylonitrile adducts were screened for antibacterial activity against Gram-positive Bacillus subtilis, Microbial Type Culture Collection and Gene Bank (MTCC 1305) and Gram-negative Escherichia coli (MTCC 443). Synthesis was followed by aza-Michael addition reaction, where the acrylonitrile accepts an electron pair from the respective amines and results in the formation of n-alkyliminobis-propionitrile and n-alkyliminopropionitrile under microwave irradiation. Characterization of the compounds were performed using Fourier Transform Infrared (FTIR), Proton Nuclear Magnetic Resonance (1H NMR) and Electrospray Ionisation Mass Spectrometry (ESI–MS). The particle size characterization was done by Dynamic Light Scattering (DLS) technique. The antibacterial study showed higher inhibition rate for both Gram-positive and Gram-negative bacteria. The antibacterial ability was found to be dose dependent. The minimum inhibitory concentration against both bacteria were found to be 1, 3, 0.4, 1, 3 µl/ml for E. coli and 6, 6, 0.9, 0.5, 5 µl/ml for B. subtilis. Time-kill kinetics evaluation showed that the adducts possess bacteriostatic action. Further it was evaluated for high-throughput in vitro assays to determine the compatibility of the adducts for drug delivery. The haemolytic and thrombolytic activity was analysed against normal mouse erythrocytes. The haemolytic activity showed prominent results, and thereby projecting this acrylonitrile adducts as potent antimicrobial and haemolytic agent.

Antibacterial efficacy of Citrus hystrix (makrut lime) essential oil against clinical multidrug-resistant methicillin-resistant and methicillin-susceptible Staphylococcus aureus isolates

The increasing incidence of methicillin-resistant S. aureus is a major public health concern. Recently, the performance of Citrus hystrix essential oil (CHEO) has been shown to contain broad-spectrum antibacterial activity. Therefore, this study aims to determine the antibacterial activity of CHEO alone and in combination with gentamicin against panels of clinical isolates of methicillin-susceptible S. aureus (MSSA, n = 45) and methicillin-resistant S. aureus (MRSA, n = 40). Antibiotic susceptibility testing revealed multidrug-resistant (MDR) patterns among 3 MSSA isolates and 39 MRSA isolates, indicating that the clinical MRSA isolates were associated with MDR (p < 0.05). For the drug resistant isolates, resistance was observed toward most antibiotics, except for chloramphenicol, trimethoprim-sulfamethoxazole, linezolid, and vancomycin. Antibacterial screening by disk diffusion demonstrated that CHEO alone had certain antibacterial activity toward all MSSA isolates (IZD: 16.0 ± 4.7 mm) and MRSA isolates (IZD: 16.5 ± 4.2 mm) (p > 0.05). The MIC values of CHEO are 18.3 ± 6.1 mg/mL in MSSA isolates and 17.9 ± 6.9 mg/mL in MRSA isolates (p > 0.05). The antibacterial activity of CHEO demonstrated the bactericidal effect with MIC index 1.0–1.4. Time-killing kinetics revealed that CHEO at 1 × MIC completely killed MSSA and MRSA within 12 h. Moreover, the checkerboard titration demonstrated the synergistic and additive interactions of CHEO with gentamicin with FIC index 0.012–0.625. CHEO against human epidermal keratinocyte; HaCaT cell line demonstrated the IC50 value at 2.15 mg/mL. The use of CHEO as an alternative antibacterial agent would reduce the emergence of resistant bacteria, especially MDR MRSA.

Zinc Oxide-Based Nanoformulation Zinkicide Mitigates the Xylem-Limited Pathogen Xylella fastidiosa in Tobacco and Southern Highbush Blueberry.

The xylem-limited pathogen Xylella fastidiosa causes severe economic losses worldwide, and no effective antimicrobial disease management options are available. The goal of this study was to evaluate the efficacy of a novel ZnO-based nanoparticle formulation, Zinkicide TMN110 (ZnK), against X. fastidiosa in vitro and in planta. In vitro, minimum bactericidal concentration (MBC) of ZnK analyzed in Pierce's Disease 2 medium was estimated at approximately 60 ppm. Time-kill kinetics assay showed a 100% reduction of culturable X. fastidiosa in less than 1 h after ZnK treatment. Microfluidic chambers assays showed that ZnK also inhibits X. fastidiosa cell aggregation and growth under flow conditions. Phytotoxicity assessments in the greenhouse demonstrated that ZnK can be applied as a soil drench in 50 ml at 500 ppm/plant/week up to four times to tobacco and blueberry without causing visible damage. ZnK was also evaluated for disease control in the greenhouse using tobacco infected with X. fastidiosa subsp. fastidiosa strain TemeculaL. ZnK soil drench weekly applications at concentrations of 500 followed by 1,000 ppm (500/1,000) and 500/500/1,000 ppm (in 50 ml each), reduced X. fastidiosa populations by >2 to 3 log10 units and disease severity by approximately 57 and 76%, respectively, compared with the untreated control. Similarly, when blueberry plants infected with X. fastidiosa subsp. multiplex strain AlmaEm3 were soil drenched with ZnK at concentrations 1,000/1,000 ppm and 1,000/1,000/500 ppm (in 200 ml each), the bacterial population was reduced by approximately 1 to 2 log10 units, and disease severity decreased by approximately 39 and 43%, respectively. Overall, this study shows antibacterial activity of ZnK against X. fastidiosa and its effectiveness in plants to reduce disease symptoms under controlled conditions.

Comparison of Antimicrobial Activity against Porphyromonas gingivalis between Advanced Platelet-Rich Fibrin and Injectable Platelet-Rich Fibrin.

Platelet-rich fibrin (PRF) obtained via low-speed centrifugation has antimicrobial properties. This study was conducted to evaluate the effectiveness of advanced platelet-rich fibrin plus (A-PRF+) and injectable platelet-rich fibrin (I-PRF), obtained from patients with different periodontal states, against Porphyromonas gingivalis. A-PRF+ and I-PRF samples were obtained from venous blood of 60 subjects divided equally into three groups: periodontitis, gingivitis, and healthy gingiva groups. The antibacterial experiments evaluated biofilm inhibition, mature biofilm impact, and time-kill kinetics. The percent reduction in biofilm-growing and mature biofilm bacteria ranged from 39% to 49% and 3% to 7%, respectively. In the time-kill kinetics assay, PRF from the periodontitis group was more effective as an antimicrobial than that from the gingivitis and healthy gingiva group (p < 0.001); I-PRF was more effective than A-PRF+ (p < 0.05) and both of them showed peak antibacterial activity after 12 h of exposure. Both A-PRF+ and I-PRF exhibited antibacterial properties against P. gingivalis, but I-PRF appeared to be more effective. The PRF obtained from the different groups appeared to have different degrees of antimicrobial efficacy.

Rifampicin Enhanced Carbapenem Activity with Improved Antibacterial Effects and Eradicates Established Acinetobacter baumannii Biofilms.

Biofilm-mediated infections are critical to public health and a leading cause of resistance among pathogens, amounting to a prolonged hospital stay and increased mortality rate in the intensive care unit. In this study, the antibacterial and antibiofilm activities of rifampicin or carbapenem monotherapies were compared with rifampicin and carbapenem combination therapies against rifampicin-resistant and carbapenem-resistant Acinetobacter baumannii isolates. Among 29 CRAB isolates, 24/29 (83%) were resistant to rifampicin, with MIC values between 2-256 µg/mL. Checkerboard assays disclosed that combination therapies at FICIs between 1/8 and 1/4 improved the activity of carbapenems at subinhibitory concentrations. Time-kill kinetics indicated a 2- to 4-log reduction at 1/2 MIC rifampicin + 1/4 MIC carbapenem and 1/4 MIC rifampicin + 1/4 MIC carbapenem against the isolates, with the MIC values ranging from 2-8 µg/mL. The MTT assay revealed a dose-dependent decrease of the cell viability of established bacterial biofilm at 4 MIC rifampicin + 2 MIC carbapenems, with a percentage reduction of 44-75%, compared with monotherapies at 16 MIC. Scanning electron microscopy further confirmed bacterial cell membrane disruption, suggesting a synergism between carbapenem and rifampicin against a representative isolate. The findings demonstrated that the combination of rifampicin with carbapenems could improve antibacterial activities and eradicate established Acinetobacter baumannii biofilm.

Time-kill kinetics and antibacterial activity of ethanolic extract of Allium sativum

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Design, synthesis, antimicrobial screening and molecular modeling of novel 6,7 dimethylquinoxalin-2(1H)-one and thiazole derivatives targeting DNA gyrase enzyme

New 6,7-dimethylquinoxalin-2(1H)-one and hydrazineylidene thiazol-4-one derivatives were synthesized, and evaluated for their in vitro antimicrobial activity. The obtained results revealed marked antimicrobial potential against four bacterial, and two fungal strains. Both 6,7-dimethyl-3-(2-(4-nitrophenyl)-2-oxoethyl)quinoxalin-2(1H)-one (4d), and 2-(2-(9H-fluoren-9-ylidene)hydrazineyl)-5-(2-(p-tolyl)hydrazineylidene)thiazol-4(5H)-one (11b) displayed significant antibacterial and antifungal activities having MIC ranges (1.98–15.6 mg/mL) and (1.98–3.9 mg/mL) compared to Tetracycline and Amphotericin B as standard drugs. In addition, they showed noticeable inhibitory activity against DNA gyrase enzyme. Interestingly the thiazole derivative (11b) showed marked inhibitory activity against DNA gyrase with IC50 = 7.82 ± 0.45 μM better than that of ciprofloxacin. The time-kill kinetics profile of the most active compounds against S. aureus and E. coli microorganisms displayed both concentration dependent and time dependent reduction in the number of viable cells. Furthermore, molecular docking study of both compounds in the DNA gyrase binding site was performed, showing agreement with the in vitro inhibitory activities.

Antibacterial potential and synergistic interaction between natural polyphenolic extracts and synthetic antibiotic on clinical isolates

Emergence of antimicrobial resistance complicates treatment of infections by antibiotics. This has driven research on novel and combination antibacterial therapies. The present study evaluated synergistic antimicrobial activity of plant extracts and cefixime in resistant clinical isolates. Preliminary susceptibility profiling of antibiotics and antibacterial activity of extracts was done by disc diffusion and microbroth dilution assays. Checker-board, time-kill kinetics and protein content studies were performed to validate synergistic antibacterial activity. Results showed noteworthy quantities of gallic acid (0.24–19.7 µg/mg), quercetin (1.57–18.44 µg/mg) and cinnamic acid (0.02–5.93 µg/mg) in extracts of plants assessed by reverse-phase high performance liquid chromatography (RP-HPLC). Gram-positive (4/6) and Gram-negative (13/16) clinical isolates were intermediately susceptible or resistant to cefixime, which was used for synergistic studies. EA and M extracts of plants exhibited total synergy, partial synergy and indifferent characteristics whereas aqueous extracts did not show synergistic patterns. Time-kill kinetic studies showed that synergism was both time and concentration-dependent (2–8-fold decrease in concentration). Bacterial isolates treated with combinations at fractional inhibitory concentration index (FICI) showed significantly reduced bacterial growth, as well as protein content (5–62 %) as compared to extracts/cefixime alone treated isolates. This study acknowledges the selected crude extracts as adjuvants to antibiotics to treat resistant bacterial infections.

Chemical Characterization and Antimicrobial Properties of the Hydroalcoholic Solution of Echinacea purpurea (L.) Moench. and Propolis from Northern Italy.

In this study, for the first time, the chemical composition of Echinacea purpurea (L.) Moench. and propolis (EAP) hydroalcoholic solution from the Trentino Alto Adige region of northern Italy was investigated by using SPME-GC-MS to describe the volatile content and GC-MS after silylation to detect the non-volatile compounds in the extractable organic matter. The antimicrobial activity of EAP hydroalcoholic solution was evaluated by Minimum Inhibitory Concentration (MIC) determination on 13 type strains, food and clinical isolates. Time Kill Kinetics (TKK) assays and the determination on swimming and swarming motility for 48 h gave more details on the mode of action of EAP solution. The results highlighted the presence of some terpenes and a large number of compounds belonging to different chemical classes. Among these, sugars and organic acids excelled. The EAP hydroalcoholic solution exhibited a strong antimicrobial activity in terms of MIC, with a clear decrease in the cellular load after 48 h. However, the bacterial motility may not be affected by the EAP treatment, displaying a dynamic swarming and swimming motility capacity over time. Given the complexity of chemical profile and the strong antimicrobial effectiveness, the EAP hydroalcoholic solution can be considered a source of bioactive molecules, deserving further investigation for the versatility of application.

A Temporin Derived Peptide Showing Antibacterial and Antibiofilm Activities against Staphylococcus aureus.

Temporin is one family of the shortest antimicrobial peptides found in Ranidae frogs. Staphylococcus aureus is one of the main pathogens of suppurative diseases and food contamination, causing severe local or systemic infections in humans. Temporin-GHa (GHa) was previously obtained from Hylarana guentheri, showing weak antibacterial activity against S. aureus. Most temporin peptides are positively charged by arginine and lysine; however, GHa contains histidine. In order to investigate the impact of positively charged amino acid on its antibacterial and antibiofilm activity, GHa4R was designed and synthesized by replacing histidine with arginine in GHa. The antibacterial activity and efficacy against S. aureus were detected by minimum inhibitory concentration, minimum bactericidal concentration, and time-killing kinetics assays. The action mechanism was determined by propidium iodide uptake and scanning electron microscopy assays. The antibiofilm activity was measured by the MTT method. Eradication of biofilm was observed by fluorescence microscope. Compared to GHa, GHa4R had stronger antibacterial activity and bactericidal efficacy against S. aureus. Impressively, GHa4R presented antibacterial activity against methicillin-resistant S. aureus (MRSA). It was barely affected by temperature, pH, and storage period, showing high stability. Furthermore, it increased the permeability of the cell membrane and damaged the membrane integrity, leading to cell death. In addition, GHa4R did not induce antibiotic resistance in S. aureus in 30 days, but the MIC of vancomycin was doubled. It not only inhibited S. aureus biofilm formation but also eradicated 24 h-biofilms. The above-mentioned characteristics make GHa4R a promising candidate for the treatment of S. aureus infections.

Small Molecule IITR00693 (2-Aminoperimidine) Synergizes Polymyxin B Activity against Staphylococcus aureus and Pseudomonas aeruginosa.

The rise of antibiotic resistance among skin-infecting pathogens poses an urgent threat to public health and has fueled the search for new therapies. Enhancing the potency of currently used antibiotics is an alternative for the treatment of infections caused by drug-resistant pathogens. In this study, we aimed to identify a small molecule that can potentiate currently used antibiotics. IITR00693 (2-aminoperimidine), a novel antibacterial small molecule, potentiates the antibacterial activity of polymyxin B against Staphylococcus aureus and Pseudomonas aeruginosa. Herein, we investigated in detail the mode of action of this interaction and the molecule's capability to combat soft-tissue infections caused by S. aureus and P. aeruginosa. A microdilution checkerboard assay was performed to determine the synergistic interaction between polymyxin B and IITR00693 in clinical isolates of S. aureus and P. aeruginosa. Time-kill kinetics, post-antibiotic effect, and resistance generation studies were performed to assess the pharmacodynamics of the combination. Assays based on different fluorescent probes were performed to decipher the mechanism of action of this combination. The in vivo efficacy of the IITR00693-polymyxin B combination was determined in a murine acute wound infection model. IITR00693 exhibited broad-spectrum antibacterial activity. IITR00693 potentiated polymyxin B and colistin against polymyxin-resistant S. aureus. IITR00693 prevented the generation of resistant mutants against multiple antibiotics. The IITR00693-polymyxin B combination decreased the S. aureus count by >3log10 CFU in a murine acute wound infection model. IITR00693 is a potential and promising candidate for the treatment of soft-tissue infections along with polymyxins.

Evaluation of the efficacy of the antimicrobial peptide HJH-3 in chickens infected with Salmonella Pullorum.

As a promising substitute for antibiotics, increasing attention has been given to the clinical application of antimicrobial peptides (AMPs). In this study, the mode of action of the HJH-3 against Salmonella Pullorum was investigated. The structure and properties of HJH-3 were examined in silico, and minimum inhibitory concentrations (MICs) were determined to evaluate its antimicrobial spectrum. The time-kill kinetics of HJH-3 was determined. The hemolytic activity of HJH-3 was determined by measuring the hemoglobin ultraviolet absorption value, and the cytotoxicity was determined using a CCK-8 kit. The protective effect of HJH-3 on chickens infected with S. Pullorum was evaluated in vivo. The results demonstrated that HJH-3 exhibited strong antibacterial activity against Gram-negative pathogens at MIC values of 1.5625-25 μg/mL and against Gram-positive pathogens at MIC values of 25-50 μg/mL. HJH-3 also showed activity against the Candida albicans (100 μg/mL) and Bacillus subtilis (6.25-12.5 μg/mL). HJH-3 at 100 μg/mL completely killed S. Pullorum after co-incubation for 6 h. Likewise, the hemolysis rate of CRBCs treated with 100 μg/mL HJH-3 (7.31%) was lower than that of CRBCs treated with 100 μg/mL pexiganan (40.43%). Although the hemolysis rate of CRBCs treated with 400 μg/mL HJH-3 was increased to 13.37%, it was much lower than that of 400 μg/mL pexiganan (57.27%). In regards to cytotoxicity, HJH-3 had almost no-effect on the CEF proliferation, pexiganan decreased CEFs proliferation from 56.93 to 31.00% when increasing the concentration from 50 to 200 μg/mL. In a chicken infection model, the results showed that the antibiotic prevention and HJH-3 prevention groups exhibited the best treatment effect, with the chickens being protected from the lethal dose of S. Pullorum, a decreased number of bacteria in the blood and spleen, and less pathological changes in intestinal segments. The prevention of infection by HJH-3 was similar to that by Ampicillin; the effect of treatment after infection was lower than that of treatment before infection, and the survival rate of infected chicks treated with HJH-3 was 70%, which was still higher than that of the infected chickens. These results suggest that HJH-3 has good clinical application potential and can be used as a substitute for antibiotics for the prevention and treatment of S. Pullorum infection.

One-pot synthesis of pyrazolo[4,3-d]thiazole derivatives containing α-aminophosphonate as potential Mur A inhibitors against MDR pathogens with radiosterilization and molecular modeling simulation.

The present study involves the synthesis of a new series of α-aminophosphonate derivatives in good yields with a simple workup via the Kabachnik-Fields reaction using lithium perchlorate (LiClO4) as a catalyst to facilitate the reaction. All the newly synthesized compounds were confirmed using various physical, spectroscopic, and analytical data, and the obtained results correlated with the proposed molecular structure. The in vitro antimicrobial activities of each compound were evaluated against different clinical isolates. The results indicated that among these derivatives, two compounds (5a and 5b) were the most active and displayed potent activity with MICs in the range from 0.06 to 0.25 μg mL-1 compared with fosfomycin and fluconazole as standard antibiotics. Moreover, the synthesized phosphonates displayed a broad spectrum of bactericidal and fungicidal activities depending on MICs, MBCs/MFCs, and the time-kill kinetics. In addition, the checkerboard assay showed synergistic and partial synergistic activities between the active compounds combined with fosfomycin and fluconazole. Furthermore, the SEM images showed distinct ruptures of the OM integrity of the FOS-R E. coli at their MICs, which was further indicated by the increased EtBr accumulation within the bacterial cells. Moreover, active derivatives revealed MurA inhibitory activity with IC50 values of 3.8 ± 0.39 and 4.5 ± 0.23 μM compared with fosfomycin (IC50 = 12.7 ± 0.27 μM). To our surprise, exposing 5a and 5b compounds to different gamma radiation doses revealed that 7.0 kGy eradicated the microbial load completely. Finally, the results of quantum chemical study supported the binding mode obtained from the docking study performed inside the active site of MurA (PDB: 1UAE), suggesting that these phosphonates may be promising safe candidates for MDR infection therapy clinical trials with no toxic effects on the normal human cells.

Designing Functionally Substituted Pyridine-Carbohydrazides for Potent Antibacterial and Devouring Antifungal Effect on Multidrug Resistant (MDR) Strains.

The emergence of multidrug-resistant (MDR) pathogens and the gradual depletion of available antibiotics have exacerbated the need for novel antimicrobial agents with minimal toxicity. Herein, we report functionally substituted pyridine carbohydrazide with remarkable antimicrobial effect on multi-drug resistant strains. In the series, compound 6 had potent activity against four MDR strains of Candida spp., with minimum inhibitory concentration (MIC) values being in the range of 16-24 µg/mL and percentage inhibition up to 92.57%, which was exceptional when compared to broad-spectrum antifungal drug fluconazole (MIC = 20 µg/mL, 81.88% inhibition). Substitution of the octyl chain in 6 with a shorter butyl chain resulted in a significant anti-bacterial effect of 4 against Pseudomonas aeruginosa (ATCC 27853), the MIC value being 2-fold superior to the standard combination of ampicillin/cloxacillin. Time-kill kinetics assays were used to discern the efficacy and pharmacodynamics of the potent compounds. Further, hemolysis tests confirmed that both compounds had better safety profiles than the standard drugs. Besides, molecular docking simulations were used to further explore their mode of interaction with target proteins. Overall results suggest that these compounds have the potential to become promising antimicrobial drugs against MDR strains.

SAR investigation and optimization of benzimidazole-based derivatives as antimicrobial agents against Gram-negative bacteria

Antibiotic-resistant bacteria represent a serious threat to modern medicine and human life. Only a minority of antibacterial agents are active against Gram-negative bacteria. Hence, the development of novel antimicrobial agents will always be a vital need. In an effort to discover new therapeutics against Gram-negative bacteria, we previously reported a structure-activity-relationship (SAR) study on 1,2-disubstituted benzimidazole derivatives. Compound III showed a potent activity against tolC-mutant Escherichia coli with an MIC value of 2 μg/mL, representing a promising lead for further optimization. Building upon this study, herein, 49 novel benzimidazole compounds were synthesized to investigate their antibacterial activity against Gram-negative bacteria. Our design focused on three main goals, to address the low permeability of our compounds and improve their cellular accumulation, to expand the SAR study to the unexplored ring C, and to optimize the lead compound (III) by modification of the methanesulfonamide moiety. Compounds (25a-d, 25f-h, 25k, 25l, 25p, 25r, 25s, and 26b) exhibited potent activity against tolC-mutant E. coli with MIC values ranging from 0.125 to 4 μg/mL, with compound 25d displaying the highest potency among the tested compounds with an MIC value of 0.125 μg/mL. As its predecessor, III, compound 25d exhibited an excellent safety profile without any significant cytotoxicity to mammalian cells. Time-kill kinetics assay indicated that 25d exhibited a bacteriostatic activity and significantly reduced E. coli JW55031 burden as compared to DMSO. Additionally, combination of 25d with colistin partially restored its antibacterial activity against Gram-negative bacterial strains (MIC values ranging from 4 to 16 μg/mL against E. coli BW25113, K. pneumoniae, A. baumannii, and P. aeruginosa). Furthermore, formulation of III and 25d as lipidic nanoparticles (nanocapsules) resulted in moderate enhancement of their antibacterial activity against Gram-negative bacterial strains (A. Baumannii, N. gonorrhoeae) and compound 25d demonstrated superior activity to the lead compound III. These findings establish compound 25d as a promising candidate for treatment of Gram-negative bacterial infections and emphasize the potential of nano-formulations in overcoming poor cellular accumulation in Gram-negative bacteria where further optimization and investigation are warranted to improve the potency and broaden the spectrum of our compounds.

Colistin sulfate-sodium deoxycholate sulfate micelle formulations; molecular interactions, cell nephrotoxicity and bioactivity

This study aimed to develop a micelle formulation of colistin with several mole ratios of sodium deoxycholate sulfate (SDCS) carrier to reduce its nephrotoxicity. The molar ratio and surface properties of colistin and SDCS were determined. Colistin was incorporated into SDCS micelles followed by lyophilisation. The formulated micelles were evaluated in terms of particle size, zeta potential, morphology, thermal properties, encapsulation efficiency (EE) and release profile. The interactions were analysed with FTIR, NMR and also in-silico studies using molecular docking. The MIC and MBC were calculated, while time-kill kinetics were observed. Furthermore, a cytotoxicity study of the formulations against kidney cells was carried out and data were analysed. Particle sizes were in the range of 140.9–162.6 nm with spherical shapes, and the zeta potential values were between −35.3 and −22.8 mV. The EE of colistin was between 17.73 and 40.15%. FTIR and DSC data indicated an interaction between the colistin and SDCS, and the NMR analysis revealed hydrogen bonding and electrostatic interaction as the main interaction. Molecular docking simulation showed that multiple hydrogen bonds were present between the hydrophilic ring of colistin and SDCS. The formulations showed good safety profiles with non-cytotoxic effects against the kidney cell while maintaining antibacterial activity. The overall results indicated that micelle formulations with SDCS can be a good option for the delivery of colistin to reduce its nephrotoxicity with better antibacterial activity, while further studies in the animal model are important.

Potential of Pm11 antimicrobial peptide against bovine mastitis pathogens

To investigate an alternative treatment for bovine mastitis by using Pm11 antimicrobial peptide. 5 bovine mastitis pathogens that were previously isolated from cows affected by either clinical or subclinical mastitis. The current study introduces Pm11 antimicrobial peptide as an alternative treatment for bovine mastitis. The antibacterial activity of Pm11 was tested against Escherichia coli strain SCM1249, Klebsiella spp strain SCM1282, Staphylococcus aureus strain CM967, Streptococcus agalactiae strain SCM1084, and Streptococcus uberis strain SCM1310 using minimum bactericidal concentrations (MBCs) and time-kill kinetics. The pathogens' morphological changes were demonstrated using a scanning electron microscope (SEM). The cytotoxicity of Pm11 was assessed using the minimum hemolytic concentration assay. MBCs ranged from 2.5 to 10 μM and IC50 ranged from 0.32 to 2.07 μM. Time-kill kinetics at MBC demonstrated that Pm11 reduced viable cell counts of S agalactiae strain SCM1084 and S uberis strain SCM1310 from 105 to 0 CFU/mL within 1 h. E coli strain SCM1249 and S aureus strain CM967 were reduced from 105 to 0 CFU/mL within 4 h. The average Pm11-induced hemolytic activity was < 10% for all Pm11 concentrations tested except at the maximum concentration tested (160 μM: 10.19 ± 2.29%). Based on SEM, Pm11 induced morphological and cellular changes in S aureus and E coli. Pm11 antimicrobial peptide demonstrated in vitro antibacterial activity against the common bovine mastitis pathogens E coli, S aureus, S agalactiae, and S uberis, except Klebsiella spp, and should be further investigated in vivo.

Pharmacokinetics and pharmacodynamics of anti-tuberculosis drugs: An evaluation of in vitro, in vivo methodologies and human studies.

There has been an increased interest in pharmacokinetics and pharmacodynamics (PKPD) of anti-tuberculosis drugs. A better understanding of the relationship between drug exposure, antimicrobial kill and acquired drug resistance is essential not only to optimize current treatment regimens but also to design appropriately dosed regimens with new anti-tuberculosis drugs. Although the interest in PKPD has resulted in an increased number of studies, the actual bench-to-bedside translation is somewhat limited. One of the reasons could be differences in methodologies and outcome assessments that makes it difficult to compare the studies. In this paper we summarize most relevant in vitro, in vivo, in silico and human PKPD studies performed to optimize the drug dose and regimens for treatment of tuberculosis. The in vitro assessment focuses on MIC determination, static time-kill kinetics, and dynamic hollow fibre infection models to investigate acquisition of resistance and killing of Mycobacterium tuberculosis populations in various metabolic states. The in vivo assessment focuses on the various animal models, routes of infection, PK at the site of infection, PD read-outs, biomarkers and differences in treatment outcome evaluation (relapse and death). For human PKPD we focus on early bactericidal activity studies and inclusion of PK and therapeutic drug monitoring in clinical trials. Modelling and simulation approaches that are used to evaluate and link the different data types will be discussed. We also describe the concept of different studies, study design, importance of uniform reporting including microbiological and clinical outcome assessments, and modelling approaches. We aim to encourage researchers to consider methods of assessing and reporting PKPD of anti-tuberculosis drugs when designing studies. This will improve appropriate comparison between studies and accelerate the progress in the field.

α-Terpineol synergizes with gentamicin to rescue Caenorhabditis elegans from Pseudomonas aeruginosa infection by attenuating quorum sensing-regulated virulence

AimsThis study scrutinized α-Terpineol (α-T) for its anti-virulence and anti-fouling potential against P. aeruginosa PAO1 in conjunction with gentamicin (GeN) using in-vitro, in-silico, and in-vivo approaches. Main methodsThe quorum quenching (QQ) potential of the drug combination was studied using a quorum sensing (QS) biosensor strain and tested for synergy using chequerboard and time-kill kinetics assays. The effect of α-T and GeN on bacterial motility, QS-regulated virulence factor production, and biofilm formation was assessed in P. aeruginosa PAO1 along with molecular docking analysis. The protective effects of α-T-GeN combination were also examined in a Caenorhabditis elegans infection model through slow-killing (SK) assays. Key findingsThe drug combination displayed synergy, enhanced QQ activity, and suppressed AHL production in PAO1. At sub-inhibitory concentrations, the drug combination suppressed the expression of genes regulating QS and pseudomonal virulence, thereby inhibiting the production of virulence factors in PAO1. The drug combination compromised all forms of pseudomonal motility, strongly inhibited biofilm formation, and successfully eradicated preformed biofilms. Based on these findings, it is concluded that GeN (alone) does not harbor any QQ properties, but enhances the QQ potential of α-T. Moreover, combinational treatment protected C. elegans from pseudomonal infection and improved survival rates by 73 % at 96 h. SignificanceFor the first time, the molecular mechanism responsible for the anti-QS activity of α-T was unraveled through a comprehensive investigation, thereby asserting its potential as an anti-virulent drug against P. aeruginosa.

Comparative In Vitro Activity of New Lipoglycopeptides and Vancomycin Against Ocular Staphylococci and Their Toxicity on the Human Corneal Epithelium.

The purpose of this study was to assess the potential of new lipoglycopeptides as novel topical therapies for improved treatment of recalcitrant ocular infections. We evaluated the in vitro antimicrobial activity of oritavancin, dalbavancin, and telavancin compared with vancomycin (VAN) against a large collection of ocular staphylococcal isolates and their cytotoxicity on human corneal epithelial cells (HCECs). Antimicrobial susceptibility testing was performed by broth microdilution against 223 Staphylococcus spp. clinical isolates. Time-kill kinetics were determined for methicillin-resistant strains of Staphylococcus aureus (MRSA) (n = 2) and Staphylococcus epidermidis (MRSE) (n = 1). In vitro cytotoxicity assays were performed with AlamarBlue and live/dead staining on HCECs. All new lipoglycopeptides showed strong in vitro potency against ocular staphylococci, including multidrug-resistant MRSA strains, with dalbavancin showing a slightly higher potency overall [minimum inhibitory concentration (MIC) 90 0.06 μg/mL] compared with telavancin and oritavancin (MIC 90 0.12 μg/mL), whereas VAN had the lowest potency (MIC 90 2 μg/mL). Oritavancin exerted rapid bactericidal activity within 1 h for MRSA and 2 h for MRSE. All other drugs were bactericidal within 24 h. At a concentration commonly used for topical preparations (25 mg/mL), cytotoxicity was observed for VAN after 5 min of incubation, whereas reduction in HCEC viability was not seen for telavancin and was less affected by oritavancin and dalbavancin. Cytotoxicity at 25 mg/mL was seen for all drugs at 30 and 60 min but was significantly reduced or undetected for lower concentrations. Our study demonstrates that new lipoglycopeptides have substantially better in vitro antimicrobial activity against ocular staphylococcal isolates compared with VAN, with a similar or improved toxicity profile on HCECs.