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• The most susceptible pathogens tested were Candida glabrata and C. tropicalis. • The n-hexane fraction had the highest antimicrobial activity (0.06 – 0.25 mg/mL). • Cidal effects against C. glabrata (of 0.06 mg/mL hexane fraction, at 5 mins). • Major active compounds include methyl linolenate, n-hexadecanoic acid and phytol. • Time-kill results were favorably comparative to the antifungal Nystatin. Species of the genus Leonotis (Lamiaceae) are commonly used in Africa as traditional herbal remedies for various ailments such as bronchial illness, epilepsy, stomach aches, and skin diseases. Most of these species are found in South Africa, and Leonotis ocymifolia (Burm.f.) Iwarsson (klipdagga) is one of them. The recorded medicinal uses of L. ocymifolia include treating diabetes, hypertension, anemia, nerve weakness, high blood pressure, poor blood circulation, rheumatism, snakebites, eczema, and several skin infections among others. Little is known about the antimicrobial activity. Therefore, this study aimed to investigate the antimicrobial potential of L. ocymifolia by reviewing the ethnomedicinal importance and antimicrobial potential (Minimum Inhibitory Concentration, Minimum bactericidal concentration, and Time-kill assessment) as well as the toxicity of L. ocymifolia 's extracts (DCM: Meth) and fractions (ethyl acetate, hexane, and butanol) against six bacteria and four Candida species. The results demonstrated that all the Candida pathogens tested were susceptible to the extracts, with moderate to noteworthy activity. The n -hexane fraction exhibited the highest antimicrobial activity against all tested pathogens, with mean MIC values between 0.06 – 0.25 mg/mL. Moderate activity was observed for the extracts against the bacteria tested, except for Klebsiella pneumoniae , where noteworthy activity (MIC value of 0.13 mg/mL) was found for the n -butanol fraction. A cidal effect on C. glabrata was recorded by the n -butanol fraction at 1xMIC (0.13 mg/mL) after 30 min of contact time and after 60 min at 1/2xMIC which is better than the positive control (Nystatin) where the cidal effect was only recorded after 4 h of contact time at 0.13 mg/mL. The hexane fraction was subjected to chemical analysis (gas chromatography-mass spectrometry) because of the noteworthy anticandidal activity. Nineteen compounds were identified with major compounds including methyl linolenate (33.31 %), n -hexadecanoic acid (21.32 %), phytol (21.35 %), and octadecenoic acid methyl ester (4.87 %). Leonotis ocymifolia has demonstrated promising antimicrobial properties, particularly against Candida species where the cidal effects link to the possible traditional use for Candida -acquired skin infections. The findings of this study, as determined by the brine shrimp lethality assay, indicate that usage of this plant extract poses minimal risk. [ABSTRACT FROM AUTHOR]

Helicobacter pylori is associated with gastrointestinal diseases, and its treatment is challenging due to antibiotic-resistant strains, necessitating alternative therapies. Brazilian red propolis (BRP), known for its diverse bioactive compounds with pharmaceutical properties, was investigated for its anti-H. pylori activity, focusing on biofilm formation inhibition and eradication. BRP was tested against H. pylori (ATCC 43526) using several assays: time–kill, nucleotide leakage, biofilm formation inhibition (determining the minimum inhibitory concentration of biofilm of 50%—MICB50, and cell viability), and biofilm eradication (determining the minimum eradication concentration of biofilm of 99.9%—MBEC). Standardization of H. pylori biofilm formation was also conducted. In the time–kill assay, BRP at 50 µg/mL eliminated all H. pylori cells after 24 h. The nucleotide leakage assay showed no significant differences between control groups and BRP-treated groups at 25 µg/mL and 50 µg/mL. H. pylori formed biofilms in vitro at 109 CFU/mL after 72 h. The MICB50 of BRP was 15.6 µg/mL, and at 500, 1000, and 2000 µg/mL, BRP eradicated all bacterial cells. The MBEC was 2000 µg/mL. These findings suggest that BRP has promising anti-H. pylori activity, effectively inhibiting and eradicating biofilms. Further studies are necessary to elucidate BRP's mechanisms of action against H. pylori. [ABSTRACT FROM AUTHOR]

Bacterial growth in food sources after harvesting and during food manufacturing has emerged as a major agricultural concern. Currently, several chemical preservatives have been added to foods to prevent or inhibit bacterial growth. These phenomena, however, become a concern when consumers begin to demand the safety of the food they consume, thus resulting in a search for natural plant extracts as an alternative. The antimicrobial activity of Cosmos caudatus leaf extract against six food pathogens was thus investigated in the present work. The disc-diffusion test, the minimum inhibitory concentration (MIC), and the minimum bactericidal concentration (MBC) tests were used to determine the antibacterial potential. Time-kill kinetics were used to determine their killing endpoints at various incubation times. Thereafter, Bacillus cereus was selected as a representative bacterium to be viewed under the light sheet electron microscope (LSEM) to observe the morphological changes. For the application as a sanitiser, different concentrations of C. caudatus extract were applied to chicken meat and oyster mushroom to determine bacterial reduction and sensory acceptability. The toxicity test using brine shrimp lethality assay was also performed to determine the toxicity level of C. caudatus extract. Results showed that C. caudatus extract contain five major peaks of flavonoids. In the antibacterial test, all tested pathogens were inhibited, with B. cereus being the most susceptible, with a significant reduction time of more than 3 log10 at 3.125 mg/mL. Under LSEM, the population of B. cereus was reduced, and the shape was wrinkled. In the application, C. caudatus extract at 0.05% for 5 min treatment time was chosen as the best formulation, whereas the brine shrimp tests showed that C. caudatus extract was safe for human consumption (LC50: 3.54 mg/mL). In conclusion, C. caudatus extract could have the potential to be further explored as a natural food sanitiser. [ABSTRACT FROM AUTHOR]

Antibacterial applications of graphene derivatives have been very highlighted during the last decade. In this study, graphene-based materials, i.e., graphene oxide (GO), reduced graphene oxide (RGO), and carboxylated graphene oxide (GO-COOH), were synthesized and characterized by UV–vis spectrophotometry, energy dispersive X-ray spectroscopy (EDX), and TEM. The main difference in these nanomaterials is the content of their oxygen-containing functional groups. According to EDX elemental analysis, GO-COOH has the highest ratio of carbon to oxygen and the highest oxygen-containing functional groups. Cytotoxicity of GO, RGO, and GO-COOH in eight concentrations at two times (24 and 48 h) on 3T3 cell lines showed concentration-dependence cytotoxicity for these three nanomaterials. The antimicrobial and antibiofilm properties of these three materials against gram-negative (Pseudomonas aeruginosa) and gram-positive (Staphylococcus aureus) bacteria, as well as a fungus (Candida albicans), were evaluated by MIC, MBC, anti-biofilm, and Time-Kill tests. Our data demonstrated that the GO-COOH has the highest antimicrobial properties, which can result from increasing the oxygen-containing functional groups. To the best of our knowledge, comparing all cytotoxic, antibacterial, antifungal, and anti-biofilm properties of these three graphene derivatives in one study has not been reported yet. [ABSTRACT FROM AUTHOR]

Helicobacter pylori is associated with gastrointestinal diseases, and its treatment is challenging due to antibiotic-resistant strains, necessitating alternative therapies. Brazilian red propolis (BRP), known for its diverse bioactive compounds with pharmaceutical properties, was investigated for its anti-H. pylori activity, focusing on biofilm formation inhibition and eradication. BRP was tested against H. pylori (ATCC 43526) using several assays: time–kill, nucleotide leakage, biofilm formation inhibition (determining the minimum inhibitory concentration of biofilm of 50%—MICB50, and cell viability), and biofilm eradication (determining the minimum eradication concentration of biofilm of 99.9%—MBEC). Standardization of H. pylori biofilm formation was also conducted. In the time–kill assay, BRP at 50 µg/mL eliminated all H. pylori cells after 24 h. The nucleotide leakage assay showed no significant differences between control groups and BRP-treated groups at 25 µg/mL and 50 µg/mL. H. pylori formed biofilms in vitro at 109 CFU/mL after 72 h. The MICB50 of BRP was 15.6 µg/mL, and at 500, 1000, and 2000 µg/mL, BRP eradicated all bacterial cells. The MBEC was 2000 µg/mL. These findings suggest that BRP has promising anti-H. pylori activity, effectively inhibiting and eradicating biofilms. Further studies are necessary to elucidate BRP’s mechanisms of action against H. pylori.

Aim Candida auris , fast evolving drug-resistant fungus, poses an imminent global health threat. Alternative drug-resistance nonevoking treatment options are necessary. This study explored the antifungal and antibiofilm efficacies of Withania somnifera seed oil extracted using super critical CO2 (WSSO) against clinically isolated Fluconazole-resistant C. auris and its putative mode-of-action. Methods and Results Effects of WSSO on C. auris were tested by broth microdilution method, with observed IC50 at 5.96 mg ml−1. Time-kill assay revealed that WSSO is fungistatic. Mechanistically, ergosterol binding and sorbitol protection assays showed that C. auris cell membrane and cell wall are the targets for WSSO. Lactophenol: Cotton-Blue: Trypan-Blue staining confirmed loss of intracellular contents by WSSO treatment. Candida auris biofilm formation was disrupted by WSSO (BIC50: 8.52 mg ml−1). Additionally, WSSO exhibited dose and time-dependent mature biofilm eradication property with 50% efficacies at 23.27, 19.28, 18.18, and 7.22 mg ml−1 over 24, 48, 72, and 96 h, respectively. Biofilm eradication by WSSO was further substantiated through scanning electron microscopy. Standard-of-Care Amphotericin B, at its break-point concentration, (2 μg ml−1) was found to be inefficient as an antibiofilm agent. Conclusions WSSO is a potent antifungal agent effective against planktonic C. auris and its biofilm. [ABSTRACT FROM AUTHOR]

Introduction: The environment of the infection site affects bacterial growth and antibiotic activity.When bacterial growth and antibiotic activity are studied in body fluids, samples of multiple subjects are usually pooled, averaging out potentially relevant differences in composition. The ascitic fluid (AF) environment is frequently associated with spontaneous bacterial peritonitis (SBP) in cirrhotic patients. In this study, bacterial growth and ceftriaxone activity were evaluated in individual AF using an in vitro model of SBP, reflecting the environment and pharmacokinetics at the infection site. Methods: AF was obtained from nine cirrhotic patients with non-infected ascites. Growth of nine bacterial strains (three Escherichia coli, four Staphylococcus aureus, one Enterococcus faecalis, and one Klebsiella pneumoniae)in individual AF was assessed and correlated with biomarkers including potential risk factors for SBP. Ceftriaxone time-kill experiments, in which the pharmacokinetic profile observed in AF following a 1 g intravenous infusion was replicated, were performed with two E. coli and two S. aureus isolates with minimum inhibitory concentrations around the ceftriaxone resistance breakpoint. Results: Significant correlations were found between bacterial growth and AF levels of protein (Spearman's rank correlation coefficient ρ = -0.35), albumin (ρ = -0.31), and complement C3c (ρ = -0.28), and serum levels of bilirubin (ρ = 0.39) and aspartate aminotransferase (ρ = 0.25). Ceftriaxone was active in AF, even against resistant isolates, generally resulting in ≥2 log reductions in bacterial count within 24 h. Conclusion: Ascites patients may be predisposed to or protected against SBP based on the antimicrobial capacity of their AF. Ceftriaxone at clinical AF concentrations is active in the AF environment. [ABSTRACT FROM AUTHOR]

Purpose: To determine the effect of the ethanol leaf extract of M. calabura (EEMC) on cell leakage and time-kill against S. aureus. Methods: The leaves were macerated with 96 % ethanol (1:8; w/v) for 27 h to produce EEMC. Chemical compounds of EEMC were analyzed using liquid chromatography with tandem mass spectrometry (LC-MS/MS). Various concentrations of EEMC (12.5; 25; 50; 100 mg/mL) were tested to determine the Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against S. aureus. Furthermore, EEMC was tested for its effect on cell leakage, changes in extracellular electrical conductivity, and time-kill against S. aureus. UV-spectrophotometer was used to test for leakages of nucleic acid, protein, DNA, and RNA, while atomic absorption spectrophotometer was used to evaluate leakage of potassium ion (K+). Results: The MIC and MBC of EEMC against S. aureus were 10 % w/v (100 mg/mL). The highest cell leakage occurred in S. aureus exposed to 2x MIC, with leakages of protein, DNA, RNA, and K+ reaching 137.79 ± 58.99, 2298 ± 263.26, 1839 ± 210.61 and 770.86 ± 40.11 µg/mL respectively. The EEMC (1x MBC and 1.5x MBC) killed S. aureus in 24 h. Analysis of LC-MS/MS of EEMC showed that flavonoids (48.33 %) followed by anthraquinones (16.10 %) were the major classes of compounds present in the extract. Conclusion: The ethanol leaf extract of M. calabura kills S. aureus by inducing cell leakage possibly due to flavonoids and anthraquinones contained in it. The extract should be further isolated and its active principles with potent antibacterial properties developed for therapeutic applications. [ABSTRACT FROM AUTHOR]

The antimicrobial effect of chitosan and synthetic chitosan derivatives has been confirmed on many Gram-positive and Gram-negative bacteria and fungi. The tests were carried out on pathogenic microorganisms, so the mechanism and concentration dependence of the inhibitory effect of chitosan were revealed. We conducted our tests on a probiotic strain, Lactobacillus plantarum. Commercially available chitosan derivatives of different molecular weights were added to L. plantarum suspension in increasing concentrations. The minimum inhibitory concentration (MIC) value of chitosan was determined and confirmed the viability decreasing effect at concentrations above the MIC with a time-kill assay. The release of bacterium cell content was measured at 260 nm after treatment with 0.001–0.1% concentration chitosan solution. An increase in the permeability of the cell membrane was observed only with the 0.1% treatment. The interaction was also investigated by zeta potential measurement, and the irreversible interaction and concentration dependence were established in all concentrations. The interaction of fluorescein isothiocyanate (FITC) labeled low molecular weight chitosan and bacterial cells labeled with membrane dye (FM® 4–64) was confirmed by confocal microscopy. In conclusion, the inhibitory effect of chitosan was verified on a probiotic strain, which is an undesirable effect in probiotic preparations containing chitosan additives, while the inhibitory effect experienced with pathogenic strains is beneficial. [ABSTRACT FROM AUTHOR]

Introduction: The environment of the infection site affects bacterial growth and antibiotic activity. When bacterial growth and antibiotic activity are studied in body fluids, samples of multiple subjects are usually pooled, averaging out potentially relevant differences in composition. The ascitic fluid (AF) environment is frequently associated with spontaneous bacterial peritonitis (SBP) in cirrhotic patients. In this study, bacterial growth and ceftriaxone activity were evaluated in individual AF using an in vitro model of SBP, reflecting the environment and pharmacokinetics at the infection site.Methods: AF was obtained from nine cirrhotic patients with non-infected ascites. Growth of nine bacterial strains (three Escherichia coli, four Staphylococcus aureus, one Enterococcus faecalis, and one Klebsiella pneumoniae) in individual AF was assessed and correlated with biomarkers including potential risk factors for SBP. Ceftriaxone time-kill experiments, in which the pharmacokinetic profile observed in AF following a 1 g intravenous infusion was replicated, were performed with two E. coli and two S. aureus isolates with minimum inhibitory concentrations around the ceftriaxone resistance breakpoint.Results: Significant correlations were found between bacterial growth and AF levels of protein (Spearman’s rank correlation coefficient ρ = −0.35), albumin (ρ = −0.31), and complement C3c (ρ = −0.28), and serum levels of bilirubin (ρ = 0.39) and aspartate aminotransferase (ρ = 0.25). Ceftriaxone was active in AF, even against resistant isolates, generally resulting in ≥2 log reductions in bacterial count within 24 h.Conclusion: Ascites patients may be predisposed to or protected against SBP based on the antimicrobial capacity of their AF. Ceftriaxone at clinical AF concentrations is active in the AF environment.

S. aureus and A. baumannii are among the main bacterial species responsible for several infections worldwide due to the different resistance and virulence mechanisms, which can interfere with treatment. The use of antimicrobial combination is one of the therapeutic options; however, they may exhibit potential for nephrotoxicity and hepatotoxicity. Therefore, the use of natural compounds and antimicrobial combinations has been studied, considering the effectiveness of antimicrobial activity, and reduction of toxic effects. The aim of this study was to analyze the synergistic effect between riparin III and antimicrobials against S. aureus and A. baumannii, as well as possible in vitro ultrastructural changes induced for the synergistic combination. Riparin III and antimicrobial (ciprofloxacin, oxacillin, meropenem and colistin) against multidrug resistant (MDR) clinical isolates and MIC determination using microbroth dilution assay was performed. The checkerboard method and the Fractional Inhibitory Concentration Index (FICI) were used to determine the type of interaction between the different antibiotics and riparin III, and the time-kill assays confirmed the results. Through Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM) possible morphological changes in bacterial cells can be observed. S. aureus exhibited resistance to oxacillin, ciprofloxacin, and A. baumannii were both described as resistant to meropenem and isolate 54 was resistant to colistin. FICI for A. baumannii indicated synergism between riparin III and colistin, and MIC reversal revealed a maximal-fold significant reduction in MIC colistin in synergistic combinations. Checkerboard and time-kill demonstrated a potential for synergy in this combination against A. baumannii and showed bactericidal activity. Several ultrastructural changes in A. baumannii treated with the synergistic mixture were visualized for SEM and TEM, such as reduction in the cell number, rough appearance, in addition to indefinite morphologies and total defragmentation of the bacterial cell wall. Therefore, riparin III and colistin combination showed synergism against MDR A. baumannii and decrease in the MIC of colistin, as well as several ultrastructural changes in bacterial cells.

Background: Carbapenems are the treatment of choice for multidrug-resistant (MDR) and extensively drug-resistant (XDR) Acinetobacter baumannii infections, but the emergence of carbapenem-resistant A. baumannii (CRAB) has rendered it ineffective in the vast majority of cases. Combination therapy has grown in popularity over the last decade; this study aims to analyze A.baumannii growth kinetics after exposure to meropenem and ampicillin-sulbactam compared with meropenem and amikacin antibiotic combinations in clinically relevant concentrations. Methods: This experimental laboratory study was conducted on the A. baumannii ATCC 19606 isolate and three clinical isolates that were intermediate or resistant to tested antibiotics. Meropenem and ampicillin-sulbactam, as well as meropenem and amikacin, were tested at four different concentrations against isolates. Turbidity measurements were taken at predetermined time points of 0, 1, 2, 4, 6, 8, and 24 hours following exposure; bacterial concentration was enumerated using the agar plate method, with the results plotted in a time-kill curve. Results: A bactericidal effect was achieved in isolates that were intermediate to ampicillin-sulbactam and resistant to meropenem after the administration of meropenem and ampicillin-sulbactam combination with a concentration of 4 µg/ml and 16/8 µg/ml, respectively. The combination of meropenem and ampicillin-sulbactam demonstrated bacteriostatic activity against isolates that were resistant to both antibiotics. Isolates treated with resistant antibiotics showed an increased growth rate compared to the growth control. Conclusion: The combination of meropenem and ampicillin-sulbactam could be a promising combination therapy in treating CRAB infections. The mechanism and degree of antibiotic resistance in the isolates affect the efficacy of antibiotic combinations; further research is needed to corroborate the findings of this study.

Developing novel antimicrobial agents has become a necessitate due to the increasing rate of microbial resistance to antibiotics. All the newly adamantane derivatives were evaluated for their antimicrobial activities against six MDR clinical pathogenic isolates. The results exhibited that 13 compounds have from potent to good activity. Among those, five derivatives (6, 7, 9, 14a, and 14b) displayed the potent activities against the different isolates tested (MIC < 0.25 µg/ml with bacteria and <8 µg/ml with fungi) compared with Ciprofloxacin (CIP) and Fluconazole (FCA). Additionally, the potent adamantanes showed bactericidal and fungicidal effects based on (MBCs and MFCs) and the time‐kill assay. The most active adamantane derivatives 7 and 14b exhibited a synergistic effect of ΣFIC ≤ 0.5 with CIP and FCA against the bacterial and fungal isolates. Moreover, no antagonistic effect appeared for the tested derivatives. Additionally, the interaction of DNA gyrase and topoisomerase IV enzymes with the compounds 6, 7, 9, 14a, and 14b exhibited potent antimicrobial activity using in vitro biochemical assays and gel‐based DNA‐supercoiling inhibition method. The activity of DNA gyrase and topoisomerase IV enzymes showed inhibitory activity (IC50) of 6.20 µM and 9.40 µM with compound 7 and 10.14 µM and 13.28 µM with compound 14b, respectively. Surprisingly, exposing compound 7 to gamma irradiation sterilized and increased its activity. Finally, the in‐silico analysis predicted that the most active derivatives had good drug‐likeness and safe properties. Besides, molecular docking and quantum chemical studies revealed several important interactions inside the active sites and showed the structural features necessary for activity. [ABSTRACT FROM AUTHOR]

(1) Background: Three antimicrobial absorbable sutures have different triclosan (TS) loads, triclosan release kinetics and hydrolysis times. This in vitro study aims to analyse and compare their antimicrobial pharmacodynamics. (2) Methods: Time-kill assays were performed with eight triclosan-susceptible microorganisms common in surgical site infections (SSIs) and a segment of each TS. Microbial concentrations were measured at T0, T4, T8 and T24 h. Similar non-triclosan sutures (NTS) were used as controls. Microbial concentrations were plotted and analysed with panel analysis. They were predicted over time with a double-exponential model and four parameters fitted to each TS × microorganism combination. (3) Results: The microbial concentration was associated with the triclosan presence, timeslot and microorganism. It was not associated with the suture material. All combinations shared a common pattern with an early steep concentration reduction from baseline to 4–8 h, followed by a concentration up to a 24-h plateau in most cases with a mild concentration increase. (4) Conclusions: Microorganisms seem to be predominantly killed by contact or near-contact killing with the suture rather than the triclosan concentration in the culture medium. No significant in vitro antimicrobial pharmacodynamic difference between the three TS is identified. Triclosan can reduce the suture microbial colonisation and SSI risk. [ABSTRACT FROM AUTHOR]

Multidrug-resistant (MDR) Klebsiella pneumoniae is a top-prioritized Gramnegative pathogen with a high incidence in hospital-acquired infections. Polymyxins have resurged as a last-line therapy to combat Gram-negative "superbugs", including MDR K. pneumoniae. However, the emergence of polymyxin resistance has increasingly been reported over the past decades when used as monotherapy, and thus combination therapy with non-antibiotics (e.g., metabolites) becomes a promising approach owing to the lower risk of resistance development. Genome-scale metabolic models (GSMMs) were constructed to delineate the altered metabolism of New Delhi metallo-ß-lactamase- or extended spectrum β-lactamase-producing K. pneumoniae strains upon addition of exogenous metabolites in media. The metabolites that caused significant metabolic perturbations were then selected to examine their adjuvant effects using in vitro static time-kill studies. Metabolic network simulation shows that feeding of 3-phosphoglycerate and ribose 5-phosphate would lead to enhanced central carbon metabolism, ATP demand, and energy consumption, which is converged with metabolic disruptions by polymyxin treatment. Further static time-kill studies demonstrated enhanced antimicrobial killing of 10 mM 3-phosphoglycerate (1.26 and 1.82 log10 CFU/ml) and 10 mM ribose 5-phosphate (0.53 and 0.91 log10 CFU/ml) combination with 2 mg/L polymyxin B against K. pneumoniae strains. Overall, exogenous metabolite feeding could possibly improve polymyxin B activity via metabolic modulation and hence offers an attractive approach to enhance polymyxin B efficacy. With the application of GSMM in bridging the metabolic analysis and time-kill assay, biological insights into metabolite feeding can be inferred from comparative analyses of both results. Taken together, a systematic framework has been developed to facilitate the clinical translation of antibiotic-resistant infection management. [ABSTRACT FROM AUTHOR]

Background: Carbapenems are the treatment of choice for multidrug-resistant (MDR) and extensively drug-resistant (XDR) Acinetobacter baumannii infections, but the emergence of carbapenem-resistant A. baumannii (CRAB) has rendered it ineffective in the vast majority of cases. Combination therapy has grown in popularity over the last decade; this study aims to analyze A.baumannii growth kinetics after exposure to meropenem and ampicillin-sulbactam compared with meropenem and amikacin antibiotic combinations in clinically relevant concentrations. Methods: This experimental laboratory study was conducted on the A.baumannii ATCC 19606 isolate and three clinical isolates that were intermediate or resistant to tested antibiotics. Meropenem and ampicillin-sulbactam, as well as meropenem and amikacin, were tested at four different concentrations against isolates. Turbidity measurements were taken at predetermined time points of 0, 1, 2, 4, 6, 8, and 24 hours following exposure; bacterial concentration was enumerated using the agar plate method, with the results plotted in a time-kill curve. Results: A bactericidal effect was achieved in isolates that were intermediate to ampicillin sulbactam and resistant to meropenem after the administration of meropenem and ampicillin-sulbactam combination with a concentration of 4 µg/ml and 16/8 µg/ml, respectively. The combination of meropenem and ampicillin-sulbactam demonstrated bacteriostatic activity against isolates that were resistant to both antibiotics. Isolates treated with resistant antibiotics showed an increased growth rate compared to the growth control. Conclusion: The combination of meropenem and ampicillin-sulbactam could be a promising combination therapy in treating CRAB infections. The mechanism and degree of antibiotic resistance in the isolates affect the efficacy of antibiotic combinations; further research is needed to corroborate the findings of this study.

Metallo-beta-lactamase (MBL)-producing carbapenem-resistant Enterobacteriaceae (CRE) infections continue to pose a serious threat to healthcare. Due to their unique active site, MBLs evade the activity of many novel beta-lactam/beta-lactamase inhibitor combinations, which have been specifically targeted toward those carbapenemases with serine active sites. Furthermore, resistance to most, if not all, other clinically relevant antimicrobial classes leaves few reliable therapeutic options. Combination therapy has thus played a vital role in the treatment of MBL-producing CRE infections. In this study, we utilized the static time-kill assay to investigate clinically relevant concentrations of cefepime, piperacillin-tazobactam, and meropenem alone and in combination with either amikacin or the novel plazomicin to determine if combinations of routinely used beta-lactam therapy with an aminoglycoside would achieve bactericidal activity against eight clinically isolated Verona integron-encoded MBL (VIM)-producing CRE. Furthermore, we compared this activity to the combination of aztreonam/avibactam, which has shown potent activity against MBL-producing CRE. Both aztreonam/avibactam and meropenem with either aminoglycoside were rapidly bactericidal within 4 hours and remained bactericidal through 24 hours against all isolates with few exceptions. Combinations including cefepime and piperacillin-tazobactam were also rapidly bactericidal, but activity after 24 hours was inconsistent depending upon the partner aminoglycoside and isolate. Further investigation is warranted to elucidate optimal antibiotic exposures against MBL-producing CRE, including novel agents in the pipeline.IMPORTANCECarbapenem-resistant Enterobacterales (CRE) are one of the most pressing antimicrobial-resistant threats at present. In addition to exhibiting resistance to many, if not all, commonly used antimicrobial agents, CRE achieves these resistant phenotypes through a variety of mechanisms, each of which can uniquely affect available treatment options. The present study is an in vitro investigation of several Verona integron-encoded metallo-beta-lactamase (VIM)-producing CRE isolated from patients at our academic medical center. Because metallo-beta-lactamases (MBLs) are inherently resistant to many of the novel treatments designed to treat CRE due to their different active site composition, we tested several antimicrobial combinations containing routinely utilized broad-spectrum beta-lactams and aminoglycosides. Our results further our understanding of combination therapy options against VIM-producing CRE, including with non-carbapenem-beta-lactams cefepime and piperacillin. By optimizing combinations of existing antimicrobial agents, we hope to expand the available armamentarium against these resistant pathogens.

Tuberculosis (TB) is still a global emergency, thus, there is need for new approaches to discovery more effective therapeutic agents. Natural products suggest a fascinating alternative in the search for novel antimycobacterial compounds. The indigenous people of Endau Rompin, Johor, Malaysia used the sap from Musa gracilis to treat cough. The present study was carried out to evaluate the antimycobacterial activity of M. gracilis extracts and to investigate its effects on cell growth, as well as its mode of action on Mycobacterium smegmatis. M. gracilis pseudostem was sequentially macerated in hexane, ethyl acetate, methanol, and water. Furthermore, micro broth dilution assay was used to evaluate the antimycobacterial activity of the crude extracts. Time-kill analysis and electron microscopy were carried out to assess the effect on cell growth and potential mode of action. Gas Chromatography-Mass Spectrometry (GC-MS) was used to identify the phyto-constituents. This study revealed that ethyl acetate extract had the best Minimum Inhibitory Concentration (MIC) value of 0.39 mg/mL and Minimum Bactericidal Concentration (MBC) values of 3.13 mg/mL. At just 8 hours after treatment with 3X MIC of the ethyl acetate extract, the viable cells count was reduced by about 99.9%. The images from the scanning electron microscopy revealed that the extract caused a disruption of M. smegmatis cells. Some phyto-compounds including phytol acetate, Vitamin E, and hexadecanoic acid assumed to be responsible for the biological activity were identified. [ABSTRACT FROM AUTHOR]

This study investigated the chemical constituents of Gaultheria procumbens essential oil and is the first to relate cytogenotoxicity with oxidative metabolism and antimicrobial activity. Chromatographic analysis of the essential oil showed methyl salicylate (99.96%) and linalool (0.04%) as the major compounds. The essential oil showed no signs of cytogenotoxicity at different concentrations (1.82 to 58.34 mg mL−1). Furthermore, G. procumbens essential oil and methyl salicylate were used to evaluate the minimal inhibitory concentrations (MIC) and minimal microbicidal concentrations (MMC). The results showed efficacy against several microorganisms, including Aeromonas caviae, Candida albicans, and Mycobacterium fortuitum with MIC values ranging from 1.82 to 3.64 mg mL−1 and MMC values ranging from 3.64 to 12.67 mg mL−1, which were confirmed by time-kill kinetics. Based on our results, the essential oil is a promising alternative to developing future formulations to treat infections caused by microorganisms. [ABSTRACT FROM AUTHOR]

Abstract Background The use of combined antibiotic therapy has become an option for infections caused by multidrug-resistant (MDR) bacteria. The time-kill (TK) assay is considered the gold standard method for the evaluation of in vitro synergy, but it is a time-consuming and expensive method. The purpose of this study was to evaluate two methods for testing in vitro antimicrobial combinations: the disk diffusion method through disk approximation (DA) and the agar gradient diffusion method via the MIC:MIC ratio. The TK assay was included as the gold standard. MDR Gram-negative clinical isolates (n = 62; 28 Pseudomonas aeruginosa, 20 Acinetobacter baumannii, and 14 Serratia marcescens) were submitted to TK, DA, and MIC:MIC ratio synergy methods. Results Overall, the agreement between the DA and TK assays ranged from 20 to 93%. The isolates of A. baumannii showed variable results of synergism according to TK, and the calculated agreement was statistically significant in this species against fosfomycin with meropenem including colistin-resistant isolates. The MIC:MIC ratiometric agreed from 35 to 71% with TK assays. The kappa test showed good agreement for the combination of colistin with amikacin (K = 0.58; P = 0.04) among the colistin-resistant A. baumannii isolates. Conclusions The DA and MIC:MIC ratiometric methods are easier to perform and might be a more viable tool for clinical microbiology laboratories.

Objectives: Treatment of infections caused by carbapenemase-producing Klebsiella pneumoniae (CPKP) frequently involves combination therapy with various antimicrobial agents in the hope of achieving synergistic effects. Routine laboratory antimicrobial synergy testing is a service that is currently unavailable owing to the laborious nature of the reference time–kill assay (TKA) as well as the widely used chequerboard method. In this study, we explored whether easier methods, based on the Etest technique, might offer a suitable alternative. Methods: In vitro interactions of tigecycline combination with colistin, gentamicin, fosfomycin or meropenem against 26 CPKP isolates were evaluated employing the TKA, chequerboard method and three Etest methodologies (the MIC/MIC ratio, the cross formation and the agar/Etest method). Rates of consequent synergy and concordance of the studied methods were determined. Results: All antimicrobial combinations demonstrated some degree of synergy against the CPKP isolates tested. No antagonism was observed for any of the combinations. All methods showed poor synergy concordance with the TKA, producing non-significant kappa (κ) results. Etest methods (MIC/MIC ratio and agar/Etest) exhibited fair agreement (κ = 0.29 and 0.38, respectively) with the chequerboard method. Conclusion: There is a poor correlation between synergy testing methods of tigecycline combinations, which may be associated with their different endpoints. To elucidate method comparability and reliability, their correlation with clinical outcomes appears important.

The antimicrobial effect of chitosan and synthetic chitosan derivatives has been confirmed on many Gram-positive and Gram-negative bacteria and fungi. The tests were carried out on pathogenic microorganisms, so the mechanism and concentration dependence of the inhibitory effect of chitosan were revealed. We conducted our tests on a probiotic strain, Lactobacillus plantarum. Commercially available chitosan derivatives of different molecular weights were added to L. plantarum suspension in increasing concentrations. The minimum inhibitory concentration (MIC) value of chitosan was determined and confirmed the viability decreasing effect at concentrations above the MIC with a time-kill assay. The release of bacterium cell content was measured at 260 nm after treatment with 0.001–0.1% concentration chitosan solution. An increase in the permeability of the cell membrane was observed only with the 0.1% treatment. The interaction was also investigated by zeta potential measurement, and the irreversible interaction and concentration dependence were established in all concentrations. The interaction of fluorescein isothiocyanate (FITC) labeled low molecular weight chitosan and bacterial cells labeled with membrane dye (FM® 4–64) was confirmed by confocal microscopy. In conclusion, the inhibitory effect of chitosan was verified on a probiotic strain, which is an undesirable effect in probiotic preparations containing chitosan additives, while the inhibitory effect experienced with pathogenic strains is beneficial.

Background: The emergence of multi-drug resistant bacterial strains worldwide has necessitated the scientific search for novel, potent, and affordable antimicrobial agents including medicinal plants and non-antibiotic drugs for therapy of infectious diseases. The objective of this study is to assess in vitro antibacterial activities and time kill kinetics of some non-antibiotic drugs against pathogenic clinical bacterial isolates. Methodology: In vitro antibacterial activities including minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and time kill kinetics of Amlodipine (AML), Thioridazine (THI) and Promethazine (PRO) against Staphylococcus aureus, coagulase negative staphylococci (CoNS), Streptococcus spp, Escherichia coli, Enterobacter spp, Klebsiella pneumoniae and Pseudomonas aeruginosa clinical isolates were determined using disc diffusion, broth microdilution and plate count techniques. Results: The mean growth inhibition zones by the disc diffusion assay of AML, THI and PRO against the isolates were ≤15.1±1.0 mm with MIC and MBC values ranging from 12.5 to 50μg/ml and 25 to 100μg/ml respectively. The time-kill assay revealed bactericidal effect of AML, THI and PRO on Gram positive bacteria evidenced by mean log reductions in viable bacterial cell counts ranging from 0.13 Log10 to 2.41 Log10 CFU/ml for S. aureus, 0.88 Log10 to 2.08 Log10 CFU/ml for Streptococcus spp, and 0.26 Log10 to 2.34 Log10 CFU/ml for CoNS after ≤30hrs post inoculation at 1xMIC. The range of log reduction in viable cell counts of Gram-negative bacteria exposed to AML, THI and PRO were E. coli (0.11 to 3.23 Log10 CFU/ml), P. aeruginosa (0.52 to 2.56 Log10 CFU/ml), K. pneumoniae (0.85 to 3.0 Log10 CFU/ml) and Enterobacter spp (0.38 to 2.08 Log10 CFU/ml) after ≤30 hrs post inoculation at 1x MIC. Conclusion: These findings demonstrate in vitro antibacterial efficacies and time kill kinetics of AML, THI and PRO against pathogenic clinical bacterial isolates, which indicate that these non-antibiotic drugs may be useful therapeutic alternatives in the bid to reduce the burden of infectious diseases associated with antibiotic resistant pathogens. [ABSTRACT FROM AUTHOR]

(1) Background: Three antimicrobial absorbable sutures have different triclosan (TS) loads, triclosan release kinetics and hydrolysis times. This in vitro study aims to analyse and compare their antimicrobial pharmacodynamics. (2) Methods: Time-kill assays were performed with eight triclosan-susceptible microorganisms common in surgical site infections (SSIs) and a segment of each TS. Microbial concentrations were measured at T0, T4, T8 and T24 h. Similar non-triclosan sutures (NTS) were used as controls. Microbial concentrations were plotted and analysed with panel analysis. They were predicted over time with a double-exponential model and four parameters fitted to each TS × microorganism combination. (3) Results: The microbial concentration was associated with the triclosan presence, timeslot and microorganism. It was not associated with the suture material. All combinations shared a common pattern with an early steep concentration reduction from baseline to 4–8 h, followed by a concentration up to a 24-h plateau in most cases with a mild concentration increase. (4) Conclusions: Microorganisms seem to be predominantly killed by contact or near-contact killing with the suture rather than the triclosan concentration in the culture medium. No significant in vitro antimicrobial pharmacodynamic difference between the three TS is identified. Triclosan can reduce the suture microbial colonisation and SSI risk.

Background: Design, identification, and synthesis of new antimicrobial agents along with preventive proceedings are essential to confront antibiotic-resistant pathogenic bacteria. Heterocyclic Schiff bases are biologically important compounds whose antimicrobial potentials have been proven to bacterial and fungal pathogens. Objectives: In this study, some quinoline Schiff bases were synthesized from condensation of 2-chloro3-quinolinecarboxaldehyde and aniline derivatives. Their inhibitory activities were evaluated against 6 gram-positive and 2 gram-negative bacterial pathogens. Methods: Disc diffusion, broth microdilution, and time-kill tests were applied according to the CLSI guidelines to determine IZD, MIC, and MBC values. Results: 2-Chloro-3-quinolinecarboxaldehyde Schiff bases could inhibit the growth of bacteria with IZDs of 7.5-19.8 mm, MICs of 256-2048 μg mL-1, and MBCs of 512 to ≥2048 μg mL-1. Conclusion: Moderate antibacterial effects were observed with heterocyclic Schiff bases. Complexation and structural changes can improve their antimicrobial properties.

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Background: Enterococcus faecalis and Enterococcus faecium can cause community-acquired and nosocomial infections. Combination antibiotic therapies have a distinct advantage over monotherapies in terms of their synergistic effect. In the study, it was aimed to investigate in vitro activity of vancomycin combined with fosfomycin by agar dilution method against VRE strains. Methods: A total of 30 clinical VRE strains were included in the study. Bacterial identifications of the strains were undertaken using conventional routine methods. The resistance to vancomycin was investigated using the broth microdilution method compared to fosfomycin by agar dilution method, and the results were interpreted in accordance with the CLSI guidelines. All experiments contained 25 mg/mL glucose-6-phosphate for fosfomycin. The fractional inhibitory concentration (FIC) indexes (FICI) were interpreted as synergism, FICI ≤ 0.5. Additionally, two strains in 30 VRE were studied to determine the time-kill curves to verify the synergistic results. Both antibiotics were studied at 1 x MIC in the tests. Viable counts were determined at 0, 4, 8, 12, and 24-hour intervals. Time-kill curves were constructed by plotting mean colony forming unit counts versus time. Results: Susceptibility rate to fosfomycin was found at 16.6% (5/30). The MIC50,90 and MICrange values of antimicrobials were 512, 512, and 512 - 1,024 mg/L for vancomycin, and 128, 128, and 64 - 160 mg/L for fosfomycin. The rate of synergism was found as 100%. Conclusions: The result shows that the combination of vancomycin with fosfomycin gives hope that it may be an option in the treatment of infections caused by VRE. [ABSTRACT FROM AUTHOR]

Background: The objective of this study was to study the effect of Klebsiella-specific phage isolated from sewage with and without the combination of antibiotics (imipenem) on the growth of clinical isolates of multidrug-resistant Klebsiella pneumoniae by time-kill assay and also to study the effect of bacteriophage and bacteriophage-antibiotic (imipenem) combination on biofilm production., Methods: A total of 40 MDR K. pneumoniae isolates were used. Klebsiella-specific phage was isolated using K. pneumoniae subspp. pneumoniae ATCC 33495 as the host from sewage. In vitro time kill curve assays were performed to evaluate the effect of Klebsiella-specific phage with and without the combination of antibiotics on the viable cell counts of MDR K. pneumoniae isolates. Microtiter plate method of O'Toole and Kolter was used to study the effect of Klebsiella-specific phage with and without the combination of antibiotics on biofilm production. For the Time kill assay, results were analyzed for significant differences using Friedman test. Tests for significant differences between the different groups were found using the Mann-Whitney U test. The correlation between the formation of biofilm was analyzed using Karl Pearson's coefficient of correlation. P value of <0.05 was considered to be statistically significant., Results: In vitro time-kill assay showed a 0.4 log decline and a 0.5 log decline in K. pneumoniae colony counts at 4 h, when phage was administered individually and in combination with imipenem, respectively (p < 0.001). Phage and phage-imipenem combinations reduced the ability of K. pneumoniae to produce biofilm by 38 % and 53 %, respectively., Conclusion: In conclusion, this study suggests that phage therapy has inhibiting activity against MDR K. pneumoniae. It is found to reduce bacterial cell count and biofilm formation but does not have a total cidal effect. However, in order to get a notable result, a phage cocktail or combination of phage with other antibiotic(s) is suggested., (Copyright © 2023 Indian Association of Medical Microbiologists. Published by Elsevier B.V. All rights reserved.)

The global health concern of antimicrobial resistance has harnessed research interest to find new classes of antibiotics to combat disease-causing pathogens. In our studies, 3-halobenzo[b]thiophene derivatives were synthesized and tested for their antimicrobial activities using the broth microdilution susceptibility method. The 3-halo substituted benzo[b]thiophenes were synthesized starting from 2-alkynyl thioanisoles using a convenient electrophilic cyclization methodology that utilizes sodium halides as the source of electrophilic halogens when reacted along with copper(II) sulfate. This environmentally benign methodology is facile, uses ethanol as the solvent, and results in 3-halo substituted benzo[b]thiophene structures in very high yields. The cyclohexanol-substituted 3-chloro and 3-bromobenzo[b]thiophenes resulted in a low MIC of 16 µg/mL against Gram-positive bacteria and yeast. Additionally, in silico absorption, distribution, metabolism, and excretion (ADME) properties of the compounds were determined. The compounds with the lowest MIC values showed excellent drug-like properties with no violations to Lipinski, Veber, and Muegge filters. The time-kill curve was obtained for cyclohexanol-substituted 3-chlorobenzo[b]thiophenes against Staphylococcus aureus, which showed fast bactericidal activity at MIC.

Phloem-limited bacterial "Candidatus Liberibacter" species are associated with incurable plant diseases worldwide. Antimicrobial treatments for these pathogens are challenging due to the difficulty of reaching the vascular tissue they occupy at bactericidal concentrations. Here, in vitro antimicrobial mechanisms of Zinkicide TMN110 (ZnK), a nonphytotoxic zinc oxide (ZnO)-based nanoformulation, were compared to those of bulk ZnO (b-ZnO) using as a model the only culturable species of the genus, Liberibacter crescens. Minimum bactericidal concentration (MBC) determination and time-kill assays showed that ZnK has a bactericidal effect against L. crescens, whereas b-ZnO is bacteriostatic. When ZnK was used at the MBC (150 ppm), its antimicrobial mechanisms included an increase in Zn solubility, generation of intracellular reactive oxygen species, lipid peroxidation, and cell membrane disruption; all of these were of greater intensity than those of b-ZnO. Inhibition of biofilms, which are important during insect vector colonization, was stronger by ZnK than by b-ZnO at concentrations between 2.5 and 10 ppm in batch cultures; however, neither ZnK nor b-ZnO removed L. crescens preformed biofilms when applied between 100 and 400 ppm. In microfluidic chambers simulating source-to-sink phloem movement, ZnK significantly outperformed b-ZnO in Zn mobilization and bactericidal activity against L. crescens planktonic cells in sink reservoirs. In microfluidic chamber assays assessing antibiofilm activity, ZnK displayed a significantly enhanced bactericidal activity against L. crescens individual attached cells as well as preformed biofilms compared to that of b-ZnO. The superior mobility and antimicrobial activity of ZnK in microenvironments make this formulation a promising product to control plant diseases caused by "Candidatus Liberibacter" species and other plant vascular pathogens. [ABSTRACT FROM AUTHOR]

Background: The use of combined antibiotic therapy has become an option for infections caused by multidrug-resistant (MDR) bacteria. The time-kill (TK) assay is considered the gold standard method for the evaluation of in vitro synergy, but it is a time-consuming and expensive method. The purpose of this study was to evaluate two methods for testing in vitro antimicrobial combinations: the disk diffusion method through disk approximation (DA) and the agar gradient diffusion method via the MIC:MIC ratio. The TK assay was included as the gold standard. MDR Gram-negative clinical isolates (n = 62; 28 Pseudomonas aeruginosa, 20 Acinetobacter baumannii, and 14 Serratia marcescens) were submitted to TK, DA, and MIC:MIC ratio synergy methods. Results: Overall, the agreement between the DA and TK assays ranged from 20 to 93%. The isolates of A. baumannii showed variable results of synergism according to TK, and the calculated agreement was statistically significant in this species against fosfomycin with meropenem including colistin-resistant isolates. The MIC:MIC ratiometric agreed from 35 to 71% with TK assays. The kappa test showed good agreement for the combination of colistin with amikacin (K = 0.58; P = 0.04) among the colistin-resistant A. baumannii isolates. Conclusions: The DA and MIC:MIC ratiometric methods are easier to perform and might be a more viable tool for clinical microbiology laboratories. [ABSTRACT FROM AUTHOR]

Aims: This study aimed to identify the most effective antimicrobial agent from a selection of essential oils (EO) and investigate its bactericidal properties against Pseudomonas aeruginosa. Methods and Results: The disc diffusion assay and minimal inhibitory/bactericidal concentration tests were used to identify antimicrobial potential. Several oils exhibited antimicrobial effects at concentrations as low as 0·03% (v/v). Significantly, cinnamon (Cinnamomum zeylanicum) bark EO exhibited a broad‐spectrum activity against Gram‐negative and Gram‐positive bacteria and showed bacteriostatic and bactericidal effects against P. aeruginosa PAO1 at 0·125% (v/v) and all other tested organisms, including known multidrug resistant species. Time‐kill assays and metabolic activity tests showed cinnamon oil to exhibit rapid killing, with bactericidal activity observed in ≤6 min at ≥0·5% (v/v). Furthermore, scanning electron microscopy and a membrane permeability assay indicated damage to membrane integrity, loss of turgor and cell collapse. Conclusion: Cinnamon bark EO is a broad‐spectrum antimicrobial agent capable of rapid killing at low concentrations. Significance and Impact of the Study: This study provides a sound basis for further investigation of the potential of cinnamon bark EO as an alternative to conventional antimicrobial products due to its fast‐acting bactericidal properties at low concentrations. [ABSTRACT FROM AUTHOR]

Aims: Global spread of carbapenem‐resistant Pseudomonas aeruginosa (CRPsA) and Acinetobacter baumannii (CRAB) is an emerging clinical problem. Since, quercetin is a well‐known antibacterial agent; it would be relevant to demonstrate synergy between quercetin and meropenem and elucidate molecular basis of effective bactericidal activity of quercetin‐meropenem against CRPsA and CRAB. Methods and Results: Hence, minimum inhibitory concentration (MIC), checkerboard, time‐kill and Baclight assays were performed to determine the antibacterial and synergistic activity of quercetin with meropenem against CRPsA and CRAB. To corroborate synergism, fractional inhibitory concentration index, combination index calculation and isobologram analysis were performed on selected isolates. Finally, to elucidate molecular basis of effective bactericidal activity of quercetin‐meropenem against CRPsA and CRAB, effect of meropenem, quercetin and their combinations on cellular morphology and levels of blaNDM, mexB and adeB expressions were evaluated on selected isolates by scanning electron microscope and qRT‐PCR. Quercetin exhibited synergy with meropenem. It inhibited blaNDM and AdeB expression. Half‐MIC combination of quercetin‐meropenem exerted bactericidal activity by disrupting cell wall/membrane integrity and altering cellular morphology. Conclusion: Quercetin could potentiate meropenem activity against CRPsA and CRAB. Significance and Impact of the Study: This study had demonstrated bactericidal and synergistic activity of quercetin with meropenem against CRPsA and CRAB. [ABSTRACT FROM AUTHOR]

Tuberculosis has been described as a global health crisis since the 1990s, with an estimated 1.4 million deaths in the last year. Herein, a series of 20 1H-indoles were synthesized and evaluated as in vitro inhibitors of Mycobacterium tuberculosis (Mtb) growth. Furthermore, the top hit compounds were active against multidrug-resistant strains, without cross-resistance with first-line drugs. Exposing HepG2 and Vero cells to the molecules for 72 h showed that one of the evaluated structures was devoid of apparent toxicity. In addition, this 3-phenyl-1H-indole showed no genotoxicity signals. Finally, time-kill and pharmacodynamic model analyses demonstrated that this compound has bactericidal activity at concentrations close to the Minimum Inhibitory Concentration, coupled with a strong time-dependent behavior. To the best of our knowledge, this study describes the activity of 3-phenyl-1H-indole against Mtb for the first time.

Candida auris is an emerging multiresistant yeast against which amphotericin B (AMB) is still the first therapeutic choice in certain clinical situations (i.e., meningitis, endophthalmitis, and urinary tract infections). As data about the in vitro killing activity of AMB against C. auris clades are lacking, we determined MICs, minimum fungicidal concentrations (MFCs), and killing activity of AMB against 22 isolates representing the 4 major C. auris clades (South Asian n = 6; East Asian n = 4; South African n = 6, and South American n = 6). MIC values were ≤1 mg/L regardless of clades; MFC ranges were, 1–4 mg/L, 2–4 mg/L, 2 mg/L, and 2–8 mg/L for South Asian, East Asian, South African, and South American clades, respectively. AMB showed concentration-, clade-, and isolate-dependent killing activity. AMB was fungicidal at 1 mg/L against two of six, two of four, three of six, and one of six isolates from the South Asian, East Asian, South African, and South American clades, respectively. Widefield fluorescence microscopy showed cell number decreases at 1 mg/L AMB in cases of the South Asian, East Asian, and South African clades. These data draw attention to the weak killing activity of AMB against C. auris regardless of clades, even when MICs are low (≤1 mg/L). Thus, AMB efficacy is unpredictable in treatment of invasive C. auris infections.

Pseudomonas aeruginosa is responsible for nosocomial and chronic infections in healthcare settings. The major challenge in treating P. aeruginosa-related diseases is its remarkable capacity for antibiotic resistance development. Bacteriophage (phage) therapy is regarded as a possible alternative that has, for years, attracted attention for fighting multidrug-resistant infections. In this work, we characterized five phages showing different lytic spectrums towards clinical isolates. Two of these phages were isolated from the Russian Microgen Sextaphage formulation and belong to the Phikmvviruses, while three Pbunaviruses were isolated from sewage. Different phage formulations for the treatment of P. aeruginosa PAO1 resulted in diversified time–kill outcomes. The best result was obtained with a formulation with all phages, prompting a lower frequency of resistant variants and considerable alterations in cell motility, resulting in a loss of 73.7% in swimming motility and a 79% change in swarming motility. These alterations diminished the virulence of the phage-resisting phenotypes but promoted their growth since most became insensitive to a single or even all phages. However, not all combinations drove to enhanced cell killings due to the competition and loss of receptors. This study highlights that more caution is needed when developing cocktail formulations to maximize phage therapy efficacy. Selecting phages for formulations should consider the emergence of phage-resistant bacteria and whether the formulations are intended for short-term or extended antibacterial application.

Background: Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of skin and soft tissue infections and their recurrences. Although traditionally not considered for use against MRSA, cefazolin presents a possible option when administered using ultrasonic drug dispersion (UD2). This novel technique localizes delivery of drug into the subcutaneous tissue and achieves concentrations that exceed the minimum inhibitory concentrations (MICs) of most clinical MRSA isolates. The purpose of this study was to evaluate the impact of achievable cefazolin concentrations on the rate and extent of bactericidal activity using time-kill methodologies Materials and Methods: The cefazolin MICs of the four MRSA isolates selected for this in vitro time-kill study were 64, 128, 256, and 512 mg/L. Duplicates of drug-free control and cefazolin experiments were carried out using the average UD2-achievable cefazolin concentration (1,300 mg/L). Experiments were incubated at 37°C throughout each run. Samples were plated and incubated for 18 to 24 hours. The lower limit of detection of colony forming units per milliliter (CFU/mL) was 1.7 log10 CFU/mL. Cefazolin was considered bactericidal when it decreased bacterial density by ≥3 log10 CFU/mL from the initial inoculum after 24 hours of incubation. Results: Cefazolin produced mean 24-hour CFU changes of -4.39 to -4.89 log10 CFU/mL against MRSA isolates with MICs from 64 to 512 mg/L. Cefazolin demonstrated bactericidal activity against all studied MRSA isolates and no regrowth was observed at 24 hours. Conclusions: The mean cefazolin tissue concentration achieved by UD2 was bactericidal against four MRSA isolates. Further investigation is warranted to assess the utility of UD2-administered cefazolin against MRSA skin and soft tissue infections. [ABSTRACT FROM AUTHOR]

Abstract Little is known regarding the appropriate timing and sequencing of a carbapenem and polymyxin in combination against carbapenem-resistant Enterobacteriaceae. Meropenem and polymyxin B were administered simultaneously or 1 agent 2 h prior to the other, in vitro. The carbapenem should be administered prior to the polymyxin when used in combination. Highlights • Polymyxin B administered before meropenem against carbapenem-resistant Enterobacteriaceae (CRE) is not bactericidal at 24 h. • Meropenem given before polymyxin B against CRE is bactericidal and synergistic. • Simultaneous administration is not as active as meropenem first against CRE. [ABSTRACT FROM AUTHOR]

Aims: To investigate the mechanisms of action of natural products with bactericidal (cinnamon root powder, peppermint oil, trans‐cinnamaldehyde, menthol and zingerone) or bacteriostatic (fresh garlic bulb extract, garlic clove powder, Leptospermum honey and allicin) activity against two Clostridium difficile strains. Methods and Results: Bactericidal products significantly reduced intracellular ATP after 1 h (P ≤ 0·01), quantified using the BacTiter‐Glo reagent, and damaged the cell membrane, shown by the leakage of both 260‐nm‐absorbing materials and protein, and the uptake of propidium iodide. Bacteriolysis was not observed, determined by measuring optical density of treated cell suspensions at 620‐nm. The effect of three bacteriostatic products on protein synthesis was quantified using an Escherichia coli S30 extract system, with Leptospermum honey (16% w/v) showing significant inhibition (P < 0·01). Lastly, no products showed elevated minimum inhibitory concentrations against antimicrobial‐resistant C. difficile, determined by broth microdilution. Conclusions: Cytoplasmic membrane damage was identified as a mechanism of action that may contribute to the activity of several natural products against C. difficile. Significance and Impact of the Study: This study describes the possible mechanisms of action of natural products against C. difficile, yet the efficacy in vivo to be determined. [ABSTRACT FROM AUTHOR]

Background: Bacterial persistence to antibiotics relates to the phenotypic ability to survive lethal concentrations of otherwise bactericidal antibiotics. The quantitative nature of the time–kill assay, which is the sector’s standard for the study of antibiotic bacterial persistence, is an invaluable asset for global, unbiased, and cross-species analyses. Methods: We compiled the results of antibiotic persistence from antibiotic-sensitive bacteria during planktonic growth. The data were extracted from a sample of 187 publications over the last 50 years. The antibiotics used in this compilation were also compared in terms of structural similarity to fluorescent molecules known to accumulate in Escherichia coli. Results: We reviewed in detail data from 54 antibiotics and 36 bacterial species. Persistence varies widely as a function of the type of antibiotic (membrane-active antibiotics admit the fewest), the nature of the growth phase and medium (persistence is less common in exponential phase and rich media), and the Gram staining of the target organism (persistence is more common in Gram positives). Some antibiotics bear strong structural similarity to fluorophores known to be taken up by E. coli, potentially allowing competitive assays. Some antibiotics also, paradoxically, seem to allow more persisters at higher antibiotic concentrations. Conclusions: We consolidated an actionable knowledge base to support a rational development of antipersister antimicrobials. Persistence is seen as a step on the pathway to antimicrobial resistance, and we found no organisms that failed to exhibit it. Novel antibiotics need to have antipersister activity. Discovery strategies should include persister-specific approaches that could find antibiotics that preferably target the membrane structure and permeability of slow-growing cells.

Mycobacterium abscessus complex consist of three rapidly growing subspecies: M. abscessus, M. massiliense, and M. bolletii. They are clinically important human pathogens responsible for opportunistic pulmonary and skin and soft tissue infections. Treatment of M. abscessus infections is difficult due to in vitro resistance to most antimicrobial agents. Tedizolid (TZD) is a next-generation oxazolidinone antimicrobial with a wide spectrum of activity even against multidrug resistant Gram-positive bacteria. In this study, the in vitro activity of TZD against the M. abscessus complex (n = 130) was investigated. Susceptibility testing by broth microdilution showed lower TZD minimum inhibitory concentrations (MICs) when compared to linezolid. The MIC50 and MIC90 was 1 mg/L and 4 mg/L, respectively across all M. abscessus complex members, reflecting no difference in subspecies response to TZD. Pre-exposure of M. abscessus complex to subinhibitory concentrations of TZD did not trigger any inducible drug resistance. Single-drug time kill assays and bactericidal activity assays demonstrated bacteriostatic activity of TZD in all three M. abscessus subspecies, even at high drug concentrations of 4 to 8x MIC. Combination testing of TZD with clarithromycin, doxycycline and amikacin using the checkerboard approach showed no antagonistic interactions. TZD may be an effective therapeutic antimicrobial agent for the treatment of M. abscessus infections.

Mycobacterium abscessus complex consist of three rapidly growing subspecies: M. abscessus, M. massiliense , and M. bolletii. They are clinically important human pathogens responsible for opportunistic pulmonary and skin and soft tissue infections. Treatment of M. abscessus infections is difficult due to in vitro resistance to most antimicrobial agents. Tedizolid (TZD) is a next-generation oxazolidinone antimicrobial with a wide spectrum of activity even against multidrug resistant Gram-positive bacteria. In this study, the in vitro activity of TZD against the M. abscessus complex (n = 130) was investigated. Susceptibility testing by broth microdilution showed lower TZD minimum inhibitory concentrations (MICs) when compared to linezolid. The MIC50 and MIC90 was 1 mg/L and 4 mg/L, respectively across all M. abscessus complex members, reflecting no difference in subspecies response to TZD. Pre-exposure of M. abscessus complex to subinhibitory concentrations of TZD did not trigger any inducible drug resistance. Single-drug time kill assays and bactericidal activity assays demonstrated bacteriostatic activity of TZD in all three M. abscessus subspecies, even at high drug concentrations of 4 to 8x MIC. Combination testing of TZD with clarithromycin, doxycycline and amikacin using the checkerboard approach showed no antagonistic interactions. TZD may be an effective therapeutic antimicrobial agent for the treatment of M. abscessus infections. [ABSTRACT FROM AUTHOR]

We compared killing activity of micafungin in time-kill experiments in RPMI-1640 with and without 50% serum against Candida albicans, Candida dubliniensis and Candida africana reference strains and clinical isolates. Killing rates ( k values) were determined for each strain and concentration. In RPMI-1640 MIC ranges were 0.015-0.03, 0.015-0.03 and 0.015 mg/L against C. albicans, C. dubliniensis and C. africana, respectively. In 50% serum MIC values for the three species increased 16- to 64-fold. In RPMI-1640 micafungin was fungicidal against two of three C. albicans isolates at 16 and 32 mg/L within 14.54 h and fungistatic against all C. africana and C. dubliniensis. Fifty per cent serum significantly decreased the growth rate of C. africana, but not of the other two species; weak in vivo replication ability of C. africana was confirmed in murine model. In 50% serum micafungin at 0.25 and 1 mg/L did not inhibit any of the three species ( k values were always negative). Micafungin killing rate in 50% serum at 4, 16 and 32 mg/L was significantly decreased for C. albicans, but increased for C. dubliniensis compared to RPMI-1640. Killing activity of micafungin against C. africana was comparable or higher in 50% serum than in RPMI-1640. Although micafungin is a highly protein-bound drug, it was equally effective against the species of the C. albicans complex in 50% serum at therapeutic trough concentration (4 mg/L). Both in vitro and in vivo data confirmed the low virulence of C. africana compared to the two sibling species. [ABSTRACT FROM AUTHOR]

As the number of hospitalized and immunocompromised patients continues to rise, invasive fungal infections, such as invasive candidiasis and aspergillosis, threaten the life of millions of patients every year. The azole antifungals are currently the most prescribed drugs clinically that display broad-spectrum antifungal activity and excellent oral bioavailability. Yet, the azole antifungals have their own limitations and are unable to meet the challenges associated with increasing fungal infections and the accompanied development of resistance against azoles. Exploring combination therapy that involves the current azoles and another drug has been shown to be a promising strategy. Haloperidol and its derivative, bromperidol, were originally discovered as antipsychotics. Herein, we synthesize and report a series of bromperidol derivatives and their synergistic antifungal interactions in combination with a variety of current azole antifungals against a wide panel of fungal pathogens. We further select two representative combinations and confirm the antifungal synergy by performing time-kill assays. Furthermore, we evaluate the ability of selected combinations to destroy fungal biofilm. Finally, we perform mammalian cytotoxicity assays with the representative combinations against three mammalian cell lines. [ABSTRACT FROM AUTHOR]

Propolis is a natural mixture produced by bees from plant resin substances. This study focuses on the general characteristics of five samples of Polish extract propolis originating from agricultural areas. Chemical composition with high performance liquid chromatography‒diode array detector method, total content of flavonoids and polyphenols, and antioxidative activity were determined in the ethanol extracts of propolis (EEP) samples. Minimum inhibitory concentration (MIC), minimum bactericidal/fungicidal concentration (MBC/MFC) and time-kill curves were studied for foodborne pathogens and food spoilage microorganisms. In EEPs the predominant flavonoid compounds were pinocembrin, chrysin, pinobanksin, apigenin, and kaempferol and the predominant phenolic acids were p-coumaric acid, ferulic acid, and caffeic acid. A strong antioxidative action of propolis in vitro was observed (IC50 for DPPH radical was at the level of 0.9−2.1 µg/mL). EEPs had MIC values for bacteria in the range of 1−16 mg/mL, whereas MIC for fungi ranged from 2 to 32 mg/mL. Extract of propolis originating from southern Poland was distinguished by higher content of bioactive components, and stronger antioxidative and antimicrobial activity than EPPs from the remaining areas of Poland. The results indicate the possibility of applying ethanol extracts from Polish propolis to protect food against microbiological spoilage.

Human African trypanosomiasis (HAT) is a neglected tropical disease, with a population of 70 million at risk. Current treatment options are limited. In the search for new therapeutics, the repurposing of the broad-spectrum antiprotozoal drug fexinidazole has completed Phase III trials with the anticipation that it will be the first oral treatment for HAT. This study used the recently validated bioluminescence imaging model to assess the dose and rate of kill effect of fexinidazole in infected mice, and the dose-dependent effect of fexinidazole on trypanosome infection. Pharmacokinetics of fexinidazole in plasma and central nervous system (CNS) compartments were similar in both infected and uninfected mice. Drug distribution within the CNS was further examined by microdialysis, showing similar levels in the cortex and hippocampus. However, high variability in drug distribution and exposure was found between mice. [ABSTRACT FROM AUTHOR]

Bactericidal effect against non-dividing bacteria is a very advantageous, but rare characteristic among antimicrobial agents, mostly possessed by those affecting the cell membrane. These kinds of agents can kill bacterial cells without lysis. We assessed these characteristics on primycin, a topical anti-staphylococcal agent highly effective against prevalent multiresistant strains, as it also acts on the cell membrane. In time-kill studies, primycin preserved its bactericidal activity against growth-arrested Staphylococcus aureus cultures. The bactericidal action was slower against growth-arrested cultures compared to the exponentially growing ones to different extents depending on the manner of arrest. The bactericidal effect was less influenced by stringent response and by protein synthesis inhibition, proving that it does not depend on metabolic activity. In contrast, uncoupling of the membrane potential predominantly slowed, and low temperature almost stopped killing of bacteria. In consideration of published data, these facts suggest that the antibacterial action of primycin involves disrupting of the membrane potential, and is predominantly influenced by the membrane fluidity. Optical density measurements and transmission electron microscopy verified that primycin kills bacterial cells without lysis. These results reveal favorable characteristics of primycin and point to, and broaden the knowledge on its membrane-targeted effect. [ABSTRACT FROM AUTHOR]