Concentration-dependent Bactericidal Effect Research Articles

Surface Immobilization of Oxidized Carboxymethyl Cellulose on Polyurethane for Sustained Drug Delivery.

Polyurethane (PU) has a diverse array of customized physical, chemical, mechanical, and structural characteristics, rendering it a superb option for biomedical applications. The current study involves modifying the polyurethane surface by the process of aminolysis (aminolyzed polyurethane; PU-A), followed by covalently immobilizing Carboxymethyl cellulose (CMC)polymer utilizing Schiff base chemistry. Oxidation of CMC periodically leads to the creation of dialdehyde groups along the CMC chain. When the aldehyde groups on the OCMC contact the amine group on a modified PU surface, they form an imine bond. Scanning electron microscopy (SEM), contact angle, and X-ray photoelectron spectroscopy (XPS) techniques are employed to analyze and confirm the immobilization of OCMC on aminolyzed PU film (PU-O). The OCMC gel incorporates Nitrofurantoin (NF) and immobilizes it on the PU surface (PU-ON), creating an antibacterial PU surface. The confirmation of medication incorporation is achieved using EDX analysis. The varying doses of NF have demonstrated concentration-dependent bacteriostatic and bactericidal effects on both Gram-positive and Gram-negative bacteria, in addition to sustained release. The proposed polyurethane (PU-ON) surface exhibited excellent infection resistance in in vivo testing. The material exhibited biocompatibility and is well-suited for biomedical applications.

Antibacterial Activity and Mechanism of Candesartan Cilexetil against Enterococcus faecalis.

Enterococcus faecalis infections pose a significant clinical challenge due to their multidrug resistance and propensity for biofilm formation. Exploring alternative treatment options, such as repurposing existing drugs, is crucial in addressing this issue. This study investigates the antibacterial activity of candesartan cilexetil against E. faecalis and elucidates its mechanism of action. Candesartan cilexetil exhibited notable antibacterial activity against both E. faecalis and Enterococcus faecium, with minimum inhibitory concentration (MIC) of ≤25 μM. Time-kill curves demonstrated concentration-dependent bactericidal effects. Candesartan cilexetil could significantly inhibited biofilm formation at the concentration of 1/4× MIC and induced alterations in biofilm structure. Permeability assays revealed compromised bacterial membranes, accompanied by the dissipation of membrane potential in E. faecalis cells after treatment with candesartan cilexetil. Checkerboard analysis showed that bacterial membrane phospholipids phosphatidylglycerol and cardiolipin could neutralize the antibacterial activity of candesartan cilexetil in a dose-dependent manner. Biolayer interferometry (BLI) assay indicated specific interactions between candesartan cilexetil and phosphatidylglycerol or cardiolipin. This study demonstrates the promising antibacterial and antibiofilm activities of candesartan cilexetil against multidrug-resistant E. faecalis. The mechanism of action involves disruption of bacterial membranes, possibly by interacting with membrane phospholipids. These findings underscore the potential utility of candesartan cilexetil as an effective therapeutic agent for combating E. faecalis infections, offering a valuable strategy in the battle against antibiotic-resistant pathogens.

Secapin: a promising antimicrobial peptide against multidrug-resistant Acinetobacter baumannii.

Acinetobacter baumannii, renowned for its exceptional multidrug resistance and its role as a prevalent nosocomial pathogen, poses a formidable challenge to conventional antibiotic therapies. The primary objective of this investigation was to evaluate the efficacy of Secapin, an antimicrobial peptide, against multidrug-resistant (MDR) baumannii. Furthermore, the mechanisms underlying Secapin's antibacterial and antibiofilm activities were elucidated. The antimicrobial and antibiofilm effectiveness of Secapin against MDR A. baumannii was assessed through a series of experiments. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of Secapin were determined using established protocols. Time-kill kinetic analysis was performed to assess the concentration-dependent bactericidal effect of Secapin. Additionally, the capacity of Secapin to impede biofilm formation and eradicate A. b aumannii biofilms was investigated. Hemolytic potential was evaluated using human red blood cells, while mammalian cell viability was examined at varying Secapin concentrations. Secapin exhibited robust bactericidal activity at minimal concentrations, with an MIC of 5 µg/mL and an MBC of 10 µg/mL against MDR A. baumannii. The time-kill kinetic analysis confirmed the concentration-dependent efficacy of Secapin in diminishing bacterial viability. Moreover, Secapin demonstrated the ability to prevent biofilm formation and eliminate established A. baumannii biofilms. Notably, Secapin exhibited no hemolytic activity and preserved mammalian cell viability up to a concentration of 100 µg/mL. These findings underscore the substantial potential of Secapin as a potent agent against multidrug-resistant A. baumannii, showcasing its efficacy in both antibacterial and antibiofilm capacities. The favorable attributes of Secapin, characterized by its minimal hemolytic effects and high mammalian cell viability, position it as a promising contender in the fight against antibiotic resistance.

In vitro bioevaluation and docking study of dihydrosphingosine and ethambutol analogues against sensitive and multi-drug resistant Mycobacterium tuberculosis

Tuberculosis remains a major public health problem and one of the top ten causes of death worldwide. The alarming increase in multidrug-resistant and extensively resistant variants (MDR, pre-XDR, and XDR) makes the disease more difficult to treat and control. New drugs that act against MDR/XDR strains are needed for programs to contain this major epidemic. The present study aimed to evaluate new compounds related to dihydro-sphingosine and ethambutol against sensitive and pre-XDR Mycobacterium strains, as well as to characterize the pharmacological activity through in vitro and in silico approaches in mmpL3 protein. Of the 48 compounds analyzed, 11 demonstrated good to moderate activity on sensitive and MDR Mycobacterium tuberculosis (Mtb), with a Minimum Inhibitory Concentration (MIC) ranging from 1.5 to 8 μM. They presented 2 to 14 times greater potency of activity when compared to ethambutol in pre-XDR strain, and demonstrated a selectivity index varying between 2.21 and 82.17. The substance 12b when combined with rifampicin, showed a synergistic effect (FICI = 0.5) on sensitive and MDR Mtb. It has also been shown to have a concentration-dependent intracellular bactericidal effect, and a time-dependent bactericidal effect in M. smegmatis and pre-XDR M. tuberculosis. The binding mode of the compounds in its cavity was identified through molecular docking and using a predicted structural model of mmpL3. Finally, we observed by transmission electron microscopy the induction of damage to the cell wall integrity of M. tuberculosis treated with the substance 12b. With these findings, we demonstrate the potential of a 2-aminoalkanol derivative to be a prototype substance and candidate for further optimization of molecular structure and anti-tubercular activity in preclinical studies.

The therapeutic effect of florfenicol-loaded carboxymethyl chitosan-gelatin shell nanogels against Escherichia coli infection in mice

To improve the therapeutic effect of Escherichia coli infections, the florfenicol-loaded CMCh-gelatin double-shell nanogels have been formulated by inclusion action and electrostatic interaction. The optimal formula of florfenicol nanogels was 100 mg polyethylene glycol, 150 mg gelatin, 100 mg carboxymethyl chitosan, and 1.0 mg/mL sodium tripolyphosphate, meanwhile, the loading capacity and encapsulation efficiency of the optimal formula were 29.7%±1.2% and 52.1%±1.7%, respectively. The mean particle diameter, polydispersity index, and zeta potential were 161.7±2.2 nm, 161.7±2.2 nm, and -23.8±1.6 mV, respectively. Scanning electron microscopy images showed uniformly distributed spherical florfenicol nanogels particles. The X-ray diffraction and fourier transform infrared of the powder showed that florfenicol nanogels were successfully formulated by complexation and electrostatic interaction. The minimum inhibitory concentration of florfenicol standard, commercial florfenicol solution, and florfenicol nanogels against Escherichia coli were 2, 4, and 2 μg/ml. In vitro time-killing curves and Live/Dead bacterial staining suggested that the florfenicol standard, commercial florfenicol solution, and florfenicol nanogels against Escherichia coli had a concentration-dependent bactericidal effect. With the help of H&E staining, florfenicol nanogels were demonstrated to be more effective than commercialized florfenicol solution for the treatment of Escherichia coli infections.

Antibacterial activity of enrofloxacin loaded gelatin-sodium alginate composite nanogels against intracellular Staphylococcus aureus small colony variants.

BackgroundThe poor intracellular concentration of enrofloxacin might lead to treatment failure of cow mastitis caused by Staphylococcus aureus small colony variants (SASCVs).ObjectivesIn this study, enrofloxacin composite nanogels were developed to increase the intracellular therapeutic drug concentrations and enhance the efficacy of enrofloxacin against cow mastitis caused by intracellular SASCVs.MethodsEnrofloxacin composite nanogels were formulated by an electrostatic interaction between gelatin (positive charge) and sodium alginate (SA; negative charge) with the help of CaCl2 (ionic crosslinkers) and optimized by a single factor test using the particle diameter, zeta potential (ZP), polydispersity index (PDI), loading capacity (LC), and encapsulation efficiency (EE) as indexes. The formation mechanism, structural characteristics, bioadhesion ability, cellular uptake, and the antibacterial activity of the enrofloxacin composite nanogels against intracellular SASCVs strain were studied systematically.ResultsThe optimized formulation was comprised of 10 mg/mL (gelatin), 5 mg/mL (SA), and 0.25 mg/mL (CaCl2). The size, LC, EE, PDI, and ZP of the optimized enrofloxacin composite nanogels were 323.2 ± 4.3 nm, 15.4% ± 0.2%, 69.6% ± 1.3%, 0.11 ± 0.02, and −34.4 ± 0.8 mV, respectively. Transmission electron microscopy showed that the enrofloxacin composite nanogels were spherical with a smooth surface and good particle size distributions. In addition, the enrofloxacin composite nanogels could enhance the bioadhesion capacity of enrofloxacin for the SASCVs strain by adhesive studies. The minimum inhibitory concentration, minimum bactericidal concentration, minimum biofilm inhibitory concentration, and minimum biofilm eradication concentration were 2, 4, 4, and 8 μg/mL, respectively. The killing rate curve had a concentration-dependent bactericidal effect as increasing drug concentrations induced swifter and more radical killing effects.ConclusionsThis study provides a good tendency for developing enrofloxacin composite nanogels for treating cow mastitis caused by intracellular SASCVs and other intracellular bacterial infections.

Antimicrobial activity of two novel antimicrobial peptides AA139 and SET-M33 against clinically and genotypically diverse Klebsiella pneumoniae isolates with differing antibiotic resistance profiles

Colistin is an antimicrobial peptide (AMP) used as a drug of last resort, although plasmid-mediated colistin resistance (MCR) has been reported. AA139 and SET-M33 are novel AMPs currently in development for the treatment of multidrug-resistant (MDR) Gram-negative bacterial infections. As many AMPs have a similar mode of action to colistin, potentially leading to cross-resistance, the antimicrobial activity of AA139 and SET-M33 was investigated against a collection of 50 clinically and genotypically diverse Klebsiella pneumoniae isolates with differing antibiotic resistance profiles, including colistin-resistant strains. The collection was genotypically characterised and susceptibility to clinically relevant antibiotics was determined. Susceptibility to AA139 and SET-M33 did not differ among the collection despite differences in underlying mechanisms of resistance or susceptibility to colistin. For three colistin-susceptible and three colistin-resistant strains with distinct MDR profiles as well as an additional MCR-producing strain, the bactericidal activity of AA139, SET-M33 and colistin during 24 h of exposure was examined. Following 24 h of exposure to AA139, SET-M33 or colistin, the seven strains were tested for changes in susceptibility to the respective AMPs. AA139 and SET-M33 showed a concentration-dependent bactericidal effect irrespective of bacterial susceptibility to colistin. Exposure to low colistin concentrations resulted in the development of colistin resistance in colistin-susceptible strains, whereas susceptibility to AA139 and SET-M33 following exposure to the respective AMPs was maintained. The two novel AMPs remained effective against colistin-resistant strains and may be promising novel drugs for the treatment of clinically and genotypically diverse MDR K. pneumoniae infections, including infections associated with colistin-resistant bacteria.

Time-kill assay and post-antibiotic effect of acetone extract from the stem bark of Canarium odontophyllum against Methicillin-resistant Staphylococcus aureus Mu50 strain

Canarium odontophyllum Miq. is an indigenous fruit found in Sarawak, Malaysia. Methicillin-resistant Staphylococcus aureus (MRSA) is a deadly pathogen that causes to hospital (HA-MRSA) and community (CA-MRSA) infections worldwide. Vancomycin has been the therapeutic drug of choice against MRSA but unfortunately this pathogen has developed some degree of resistance to vancomycin. This research aimed to evaluate the antimicrobial activity of the stem bark extract of C. odontophyllum against MRSA Mu50 strain. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of extract and vancomycin against MRSA were determined using broth microdilution method and streak plate method. The rate of killing by the extract against was determined using time-kill assay (TKA) at 1X MIC, 2X MIC, 4X MIC and 8X MIC of the extract. The post-antibiotic effect (PAE) time of extract 10X MIC against MRSA was also investigated. The extract exhibited bacteriostatic effect against MRSA Mu50 strain with MIC and MBC values of 1.563 mg/ml and 3.125 mg/ml, respectively. From TKA analysis, the extract was not capable of killing the Mu50 strain at 1XMIC and 2XMIC but it displayed bactericidal activity at higher concentrations tested. Interestingly, the acetone stem back extract of C. odontophyllum at 4XMIC showed comparable time-killing kinetic with the standard antibiotic in the study. The PAE time of the extract was 3.6 ± 0.51 hr against MRSA Mu50 compared to vancomycin at 2.4 ± 0.68 hr. In conclusion, the stem bark acetone extract from C. odontophyllum demonstrated concentration-dependent bactericidal effect with prolonged PAE time against MRSA Mu50 strain.

Efficacy of enrofloxacin against transmissible resistance gene (qnrS and aac (6’)-Ib-cr) - containing Escherichia coli isolates and clones

Aim of this study was to investigate the effects of qnrS1 and aac (6’)-Ib-cr on susceptibility, some pharmacodynamics of enrofloxacin against molecularly constructed and natural isolates of E. coli from animals. The MPCs and mutation frequencies of the drug were determined using the agar dilution method. Time-kill assays were used to determine the antimicrobial activity of the drug against E. coli strains. MPCs of enrofloxacin for qnrS1- and aac(6´)-Ib-cr-containing transformants increased from 0.128 µg/ml to 2 and 4 µg/ml. MPC:MIC ratio of enrofloxacin for transformants and E. coli isolates were 4 and 8, respectively. Mutation frequencies were markedly higher for transformants and isolates compared to control strain. Mutation frequencies diminished up to 6.2x10-18 by increasing the concentrations used in the assay. The results of this study showed that enrofloxacin is less active against E. coli strains in case of additional mutations in QRDR. The concentration-dependent bactericidal effect of enrofloxacin is only observable by the eight times the MIC and increasing concentrations did not alter the bactericidal activity. The combination therapy can be considered to fight with resistant E. coli instead of monotherapy and artificially constructed E. coli strains, which exhibit similar reactions against ENR with natural E. coli isolates, can be used to test the pharmacological efficacy of the combinations.

Antimicrobial Studies Using the Therapeutic Tissue Cross-Linking Agent, Sodium Hydroxymethylglycinate: Implication for Treating Infectious Keratitis.

PurposeOur recent studies raise the possibility of using sodium hydroxymethylglycinate (SMG), for pharmacologic therapeutic tissue cross-linking (TXL) of the cornea. The present study was performed to evaluate the antimicrobial effects of SMG for potential use in treating infectious keratitis.MethodsIn initial (group 1) experiments, methicillin-sensitive Staphylococcus aureus (MSSA), methicillin-resistant Staphylococcus aureus (MRSA), and Pseudomonas aeruginosa (PA) were treated with SMG (10–40 mM) for 10 to 120 minutes. In group 2 experiments, MRSA, PA, Candida albicans (CA), and vancomycin-resistant Enterococcus (VRE) were treated with SMG (20–200 mM) for 30 minutes. In group 2 experiments, BSA and neutralizing buffer were added to provide a proteinaceous medium, and to ensure precise control of SMG exposure times, respectively. SMG effectiveness was quantitated based on pathogen growth following a 24- to 48-hour incubation period.ResultsIn group 1 experiments, as expected, time- and concentration-dependent bactericidal effects were noted using MSSA. In addition, the effect of SMG (40 mM) was greatest against MSSA (99.3%), MRSA (96.0%), and PA (97.4%) following a 2-hour exposure with lesser effects following 30- and 10-minute exposures. In group 2 experiments, concentration-dependent bactericidal effects were confirmed for MRSA (91%), PA (99%), and VRE (55%) for 200-mM SMG with 30-minute treatment. SMG was not as effective against CA, with a maximum kill rate of 37% at 80 mM SMG.ConclusionsSMG solution exhibits a dose-dependent bactericidal effect on MSSA, MRSA, and PA, with milder effects on VRE and CA. These studies raise the possibility of using SMG TXL for the treatment of infectious keratitis.

Telavancin: a novel semisynthetic lipoglycopeptide agent to counter the challenge of resistant Gram-positive pathogens.

Telavancin (TD-6424), a semisynthetic lipoglycopeptide vancomycin-derivative, is a novel antimicrobial agent developed by Theravance for overcoming resistant Gram-positive bacterial infections, specifically methicillin-resistant Staphylococcus aureus (MRSA). The US Food and Drug Administration (USFDA) had approved telavancin in 2009 for the treatment of complicated skin and skin structure infections (cSSSIs) caused by Gram-positive bacteria, including MRSA (S. aureus, Streptococcus agalactiae, Streptococcus pyogenes, Streptococcus anginosus group, or Enterococcus faecalis). Telavancin has two proposed mechanisms of action. In vitro, telavancin has a rapid, concentration-dependent bactericidal effect, due to disruption of cell membrane integrity. Telavancin has demonstrable in vitro activity against aerobic and anaerobic Gram-positive bacteria. Telavancin and vancomycin have similar spectra of activity. Gram-negative bacteria are usually non-susceptible to telavancin. Telavancin has been successfully tested in various animal models of bacteremia, endocarditis, meningitis, and pneumonia. Phase II Telavancin versus Standard Therapy for Treatment of Complicated Skin and Soft-Tissue Infections due to Gram-Positive Bacteria (FAST 1 and FAST 2) and phase III [Assessment of Telavancin in Complicated Skin and Skin Structure Infections 1 (ATLAS 1 and ATLAS 2)] clinical trials have been conducted for evaluating telavancin's efficacy and safety in cSSSIs. Phase III clinical trials have been carried out for evaluating telavancin's safety and efficacy in nosocomial pneumonia [Assessment of Telavancin for Treatment of Hospital acquired Pneumonia 1 and 2 (ATTAIN 1 and ATTAIN 2)]. A phase II randomized, double-blind, clinical trial has been carried out for evaluating telavancin's safety and efficacy in uncomplicated S. aureus bacteremia [Telavancin for Treatment of Uncomplicated S. aureus Bacteremia (ASSURE)]. Pacemaker lead-related infective endocarditis due to a vancomycin intermediate S. aureus (VISA) strain (non-daptomycin susceptible) was successfully treated with parenteral telavancin for 8 weeks. Telavancin extensively binds to serum albumin (~93%) and has a relatively small volume of distribution. Telavancin is not biotransformed by any cytochrome P450 microsomal enzymes and excreted mainly in the urine. Though well-tolerated, worrisome adverse effects, including renal dysfunction and QTc prolongation are of potential concern. Given its extensive binding to plasma proteins, long half-life, and a long post-antibiotic effect, it represents a promising addition to the therapeutic armamentarium in combating infections caused by resistant Gram-positive pathogens, namely, MRSA.

Alkaloid extracts from Combretum zeyheri inhibit the growth of Mycobacterium smegmatis

BackgroundCurrent tuberculosis regimens have failed to combat the issue of drug resistance and ethno medicines may represent a possible source of antimycobacterial agents. Combretum species are well known in African traditional medicines and used for various ailments including pneumonia, venereal diseases like syphilis, mental problems, relief of sore throats and colds, fever, and chest coughs associated with tuberculosis. Alkaloids function as either hydrogen-acceptor or hydrogen-donor in hydrogen bonding critical for the interaction between targets thus, potentiating effects of curative agents on diseases. Alkaloid extracts from leaves of Combretum zeyheri, Combretum platypetalum, Combretum molle and Combretum apiculatum, were assessed for antimycobacterial activity to establish rationale for their use in traditional medicines for various ailments including pneumonia, relief of sore throats and colds, fever, and chest coughs associated with tuberculosis.MethodsAlkaloids were extracted from the leaves of Combretum zeyheri, Combretum platypetalum, Combretum molle and Combretum apiculatum. The broth microdilution method was used for the screening of growth inhibitory activity. The standard drug rifampicin was used as the positive control. Alkaloid extracts from the most potent plant species, Combretum zeyheri were further investigated for time-kill dependency effects on drug transport in Mycobacterium smegmatis.ResultsUsing the broth microdilution susceptibility method, C. zeyheri alkaloid extract, was found to have the most antimycobacterial effects with an MIC value of 125 μg/ml whilst MICs for C. molle and C. platypetalum were above 1000 μg/ml. An MBC value of 250 μg/ml was observed with alkaloid extracts from Combretum zeyheri whilst the remaining three Combretum species showed no bactericidal activity. It was also shown that C. zeyheri had potential efflux pump inhibitory activity. Determination of the time-kill kinetics of extracts from C. zeyheri showed not only a concentration-dependent activity but time-dependent bactericidal effect as well.ConclusionsAlkaloid extracts from the leaves of C. zeyheri have potential as a source of lead compounds that may be developed further into antimycobacterial compounds. The mechanism of action of may be due to inhibition of transport across the cell membrane. Further work needs to be done to isolate the active components in these extracts.

Chamaecyparis obtusa Suppresses Virulence Genes in Streptococcus mutans

Chamaecyparis obtusa (C. obtusa) is known to have antimicrobial effects and has been used as a medicinal plant and in forest bathing. This study aimed to evaluate the anticariogenic activity of essential oil of C. obtusa on Streptococcus mutans, which is one of the most important bacterial causes of dental caries and dental biofilm formation. Essential oil from C. obtusa was extracted, and its effect on bacterial growth, acid production, and biofilm formation was evaluated. C. obtusa essential oil exhibited concentration-dependent inhibition of bacterial growth over 0.025 mg/mL, with 99% inhibition at a concentration of 0.2 mg/mL. The bacterial biofilm formation and acid production were also significantly inhibited at the concentration greater than 0.025 mg/mL. The result of LIVE/DEAD® BacLight™ Bacterial Viability Kit showed a concentration-dependent bactericidal effect on S. mutans and almost all bacteria were dead over 0.8 mg/mL. Real-time PCR analysis showed that gene expression of some virulence factors such as brpA, gbpB, gtfC, and gtfD was also inhibited. In GC and GC-MS analysis, the major components were found to be α-terpinene (40.60%), bornyl acetate (12.45%), α-pinene (11.38%), β-pinene (7.22%), β-phellandrene (3.45%), and α-terpinolene (3.40%). These results show that C. obtusa essential oil has anticariogenic effect on S. mutans.

In vitro bactericidal activity of 4- and 5-chloro-2-hydroxy-N-[1-oxo-1-(phenylamino)alkan-2-yl]benzamides against MRSA.

A series of nine substituted 2-hydroxy-N-[1-oxo-1-(phenylamino)alkan-2-yl]benzamides was assessed as prospective bactericidal agents against three clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) and S. aureus ATCC 29213 as the reference and quality control strain. The minimum bactericidal concentration was determined by subculturing aliquots from MIC determination onto substance-free agar plates. The bactericidal kinetics of compounds 5-chloro-2-hydroxy-N-[(2S)-3-methyl-1-oxo-1-{[4-(trifluoromethyl)phenyl]amino}butan-2-yl]benzamide (1f), N-{(2S)-1-[(4-bromophenyl)amino]-3-methyl-1-oxobutan-2-yl}-4-chloro-2-hydroxybenzamide (1g), and 4-chloro-N-{(2S)-1-[(3,4-dichlorophenyl)amino]-3-methyl-1-oxobutan-2-yl}-2-hydroxybenzamide (1h) was established by time-kill assay with a final concentration of the compound equal to 1x, 2x, and 4x MIC; aliquots were removed at 0, 4, 6, 8, and 24 h time points. The most potent bactericidal agent was compound 1f exhibiting remarkable rapid concentration-dependent bactericidal effect even at 2x MIC at 4, 6, and 8 h (with a reduction in bacterial count ranging from 3.08 to 3.75 log10 CFU/mL) and at 4x MIC at 4, 6, 8, and 24 h (5.30 log10 CFU/mL reduction in bacterial count) after incubation against MRSA 63718. Reliable bactericidal effect against other strains was maintained at 4x MIC at 24 h.

PHARMACOKINETIC BEHAVIOR OF ENROFLOXACIN AND ITS METABOLITE CIPROFLOXACIN IN URUTU PIT VIPERS(BOTHROPS ALTERNATUS)AFTER INTRAMUSCULAR ADMINISTRATION

Enrofloxacin is widely used in veterinary medicine and is an important alternative to treating bacterial infections, which play an important role as causes of disease and death in captive snakes. Its extralabel use in nontraditional species has been related to its excellent pharmacokinetic and antimicrobial characteristics. This can be demonstrated by its activity against gram-negative organisms implicated in serious infectious diseases of reptile species with a rapid and concentration-dependent bactericidal effect and a large volume of distribution. Pharmacokinetic parameters for enrofloxacin were investigated in seven urutu pit vipers (Bothrops alternatus), following intramuscular injections of 10 mg/kg. The plasma concentrations of enrofloxacin and its metabolite, ciprofloxacin, were measured using high-performance liquid chromatography. Blood samples were collected from the ventral coccygeal veins at 0.5, 1, 2, 4, 8, 12, 24, 36, 48, 72, 96, 108, and 168 hr. The kinetic behavior was characterized by a relatively slow absorption (time of maximal plasma concentration = 4.50 +/- 3.45 hr) with peak plasma concentration of 4.81 +/- 1.12 microg/ml. The long half-life during the terminal elimination phase (t1/2 lambda = 27.91 +/- 7.55 hr) of enrofloxacin after intramuscular administration, calculated in the present study, could suggest that the antibiotic is eliminated relatively slowly and/or the presence of a slow absorption in urutu pit vipers. Ciprofloxacin reached a peak plasma concentration of 0.35 microg/ml at 13.45 hr, and the fraction of enrofloxacin metabolized to ciprofloxacin was 13.06%. If enrofloxacin's minimum inhibitory concentration (MIC90) values of 0.5 microg/ml were used, the ratios AUC(e+c): MIC90 (276 +/- 67 hr) and Cmax(e+c): MIC90 (10 +/- 2) reach the proposed threshold values (125 hr and 10, respectively) for optimized efficacy and minimized resistance development when treating infections caused by Pseudomonas. The administration of 10 mg/kg of enrofloxacin by the i.m. route should be considered to be a judicious choice in urutu pit vipers against infections caused by microorganisms with MIC values < or = 0.5 microg/ml. For less susceptible bacteria, a dose increase and/or an interval reduction should be evaluated.

Antibacterial and Antiinflammatory Kinetics of Curcumin as a Potential Antimucositis Agent in Cancer Patients

The antiinflammatory agent curcumin (diferuloylmethane) has a potential to mitigate cancer therapy-induced mucositis. We assessed the in vitro extent of its bactericidal activity and determined the kinetics of its antiinflammatory effect on pharyngeal cells. Bactericidal activity was assessed using the LIVE/DEAD® Kit after 4 h of exposure to curcumin (50–200 μM) in 18 oropharyngeal species commonly associated with bacteremia in febrile neutropenia. Moraxella catarrhalis or its outer membrane vesicles were used to determine the inhibitory effect of curcumin on bacteria-induced proinflammatory activity as determined by cytokine release into the supernatant of Detroit 562 pharyngeal cells using the Luminex® xMAP® technology. Curcumin exerted a concentration-dependent bactericidal effect on all 18 species tested. After 4 h at 200 μM, 12 species tested were completely killed. Preincubation of Detroit cells with 200 μM curcumin for 5 to 60 min resulted in complete suppression of the release of tumor necrosis factor-α, interleukin (IL)-6, IL-8, monocyte chemoattractant protein 1, granulocyte macrophage-colony stimulating factor, and vascular endothelial growth factor. Fibroblast growth factor-2 and interferon-γ were not affected. Repetitive exposure to curcumin resulted in repetitive suppression of cytokine/chemokine expression lasting from 4 to 6 h. Through reduction of oral microbial density as well as suppression of inflammation cascades curcumin may prevent cancer therapy-induced oral mucositis, e.g., when applied as multiple daily mouth washes.

Powerful bacterial killing by buckwheat honeys is concentration-dependent, involves complete DNA degradation and requires hydrogen peroxide.

Exposure of bacterial cells to honey inhibits their growth and may cause cell death. Our previous studies showed a cause-effect relationship between hydroxyl radical generated from honey hydrogen peroxide and growth arrest. Here we explored the role of hydroxyl radicals as inducers of bacterial cells death. The bactericidal effect of ·OH on antibiotic-resistant clinical isolates of MRSA and VRE and standard bacterial strains of E. coli and B. subtiles was examined using a broth microdilution assay supplemented with 3′-(p-aminophenyl) fluorescein (APF) as the ·OH trap, followed by colony enumeration. Bactericidal activities of eight honeys (six varieties of buckwheat, blueberry and manuka honeys) were analyzed. The MBC/MIC ratio ≤4 and the killing curves indicated that honeys exhibited powerful, concentration-dependent bactericidal effect. The extent of killing depended on the ratio of honey concentration to bacterial load, indicating that honey dose was critical for its bactericidal efficacy. The killing rate and potency varied between honeys and ranged from over a 6-log10 to 4-log10 CFU/ml reduction of viable cells, equivalent to complete bacterial eradication. The maximal killing was associated with the extensive degradation of bacterial DNA. Honey concentration at which DNA degradation occurred correlated with cell death observed in the concentration-dependent cell-kill on agar plates. There was no quantitative relationship between the ·OH generation by honey and bactericidal effect. At the MBC, where there was no surviving cells and no DNA was visible on agarose gels, the ·OH levels were on average 2–3x lower than at Minimum Inhibitory Concentration (MICs) (p < 0.0001). Pre-treatment of honey with catalase, abolished the bactericidal effect. This raised possibilities that either the abrupt killing prevented accumulation of ·OH (dead cells did not generate ·OH) or that DNA degradation and killing is the actual footprint of ·OH action. In conclusion, honeys of buckwheat origin exhibited powerful, concentration-dependent bactericidal effect. The killing and DNA degradation showed a cause-effect relationship. Hydrogen peroxide was an active part of honey killing mechanism.

Telavancin, a new lipoglycopeptide antimicrobial, in complicated skin and soft tissue infections

Telavancin, a novel lipoglycopeptide with rapid concentration-dependent bactericidal effects, is a semisynthetic derivative of the glycopeptide, vancomycin. Telavancin has a dual mechanism of action, ie, inhibition of peptidoglycan polymerization and disruption of the bacterial membrane. It has linear pharmacokinetics, rapid bactericidal killing, and broad spectrum activity against Gram positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant S. aureus. Phase II and III clinical trials for complicated skin and skin structure infections have shown telavancin to have similar efficacy and tolerability to that of vancomycin and standard anti-staphylococcal β-lactams plus vancomycin. In Phase II trials, there was a significant difference in eradication of MRSA between groups, ie, telavancin therapy 92% and standard therapy (vancomycin, nafcillin, oxacillin, or cloxacillin) 68% (P < 0.05). In Phase III trials, among clinically evaluable patients who had MRSA isolated at baseline, the overall therapeutic response was higher in patients treated with telavancin than in patients treated with vancomycin (89.9% versus 84.7%; 95% CI −0.3, 10.5). Also, the efficacy of telavancin was not inferior to that of vancomycin for the treatment of complicated skin and skin structure infections in the clinical trials.

Dimers of Nostocarboline with Potent Antibacterial Activity

Objectives: In this study, the in vitro antimicrobial activity and spectrum of new dimeric compounds derived from the cyanobacterial alkaloid nostocarboline were investigated. The mechanism of action and selectivity to bacteria were studied and compared to the cationic antiseptic chlorhexidine. Methods:Minimal inhibitory concentrations were determined against clinical isolates and against a panel of microbial reference strains using the CLSI microdilution method. Bacterial membrane damage was addressed by measuring ATP leakage and the mode of action was investigated in Escherichia coli reporter strains. Selectivity was tested by a cytotoxicity assay using MTS. Results: The antimicrobial potency of dimers varied with length of the hydrophobic linker. The most potent compounds, NCD9 and NCD10, had a C10 and C12 linker, respectively, and showed strong activity against Gram-positive bacteria, notably methicillin-resistant Staphylococcus aureus strains. Similar to chlorhexidine, these compounds showed a rapid concentration-dependent bactericidal effect, which correlated with membrane damage as indicated by ATP leakage. NCD9, in contrast to NCD10 and chlorhexidine, lacked activity against yeast strains and showed low cytotoxicity in CHO cells indicating a high degree of selectivity. In E. coli reporter strains, NCD9 induced the DegP response pathway as well as the SOS response, suggesting interaction with both the cell envelope and DNA metabolism. Conclusions: The results presented in this report indicate the potential of this new class of cationic antimicrobial compounds for the design of potent and selective antibacterials with low cytotoxicity.

Functional whole-colony screening method to identify antimicrobial peptides

A high throughput method for screening cDNA libraries has been developed to identify putative antimicrobial peptides (AMPs). It is based on a rapid dye inclusion assay for assessing antagonism of bacterial viability. Colonies are grown on a membrane on a permissive medium until full colony size is reached. The membrane, supporting the array of colonies, is transferred onto an inductive medium containing a vital dye. Upon expression of any antagonizing peptides, the cell membrane becomes compromised allowing dye infusion to permit visual identification of deleterious peptides. Our approach was validated by screening a synthetic oligonucleotide library expressed in Escherichia coli. A random oligonucleotide library, containing inserts of up to 75 nucleotides in length was constructed and expressed in E. coli. From a potential pool of 100 000 peptides, in a single round of screening, three were found to be antimicrobial: L1, L3, and L8. Peptide L1 was shown to have a concentration-dependent bactericidal effect against Gram-negative E. coli and moderate biostatic activity against the Gram-positive bacteria Listeria monocytogenes. L8 was found to have bacteriostatic, and possibly bactericidal effect against E. coli, Pseudomonas aeruginosa and Salmonella typhimurium. These results validated this high throughput AMP identification assay based on filter bound colony array libraries and vital dye inclusion.