In Vitro Profiling of Antitubercular Compounds by Rapid, Efficient, and Nondestructive Assays Using Autoluminescent Mycobacterium tuberculosis.

Anti-infective drug discovery is greatly facilitated by the availability of in vitro assays that are more proficient at predicting the preclinical success of screening hits. Tuberculosis (TB) drug discovery is hindered by the relatively slow growth rate of Mycobacterium tuberculosis and the use of whole-cell-based in vitro assays that are inherently time-consuming, and for these reasons, rapid, noninvasive bioluminescence-based assays have been widely used in anti-TB drug discovery and development. In this study, in vitro assays that employ autoluminescent M. tuberculosis were optimized to determine MIC, minimum bactericidal concentration (MBC), time-kill curves, activity against macrophage internalized M. tuberculosis (90% effective concentration [EC90]), and postantibiotic effect (PAE) to provide rapid and dynamic biological information. Standardization of the luminescence-based MIC, MBC, time-kill, EC90, and PAE assays was accomplished by comparing results of established TB drugs and two ClpC1-targeting TB leads, ecumicin and rufomycin, to those obtained from conventional assays and/or to previous studies. Cumulatively, the use of the various streamlined luminescence-based in vitro assays has reduced the time for comprehensive in vitro profiling (MIC, MBC, time-kill, EC90, and PAE) by 2 months. The luminescence-based in vitro MBC and EC90 assays yield time and concentration-dependent kill information that can be used for pharmacokinetic-pharmacodynamic (PK-PD) modeling. The MBC and EC90 time-kill graphs revealed a significantly more rapid bactericidal activity for ecumicin than rufomycin. The PAEs of both ecumicin and rufomycin were comparable to that of the first-line TB drug rifampin. The optimization of several nondestructive, luminescence-based TB assays facilitates the in vitro profiling of TB drug leads in an efficient manner.

Pharmacokinetics/Pharmacodynamics Evaluation of Flomoxef against Extended-Spectrum Beta-Lactamase-Producing Escherichia coli In Vitro and In Vivo in a Murine Thigh Infection Model.

PURPOSE: Although flomoxef (FMOX) has attracted substantial attention as an antibiotic against extended-spectrum beta-lactamase-producing Escherichia coli (ESBL-producing E. coli), the pharmacokinetics/pharmacodynamics (PK/PD) characteristics of FMOX against ESBL-producing E. coli is unclear. The aim of this study was to determine the PK/PD index of FMOX against ESBL-producing E. coli. METHODS: In vitro time-kill curve studies and in vivo PK/PD experiments were carried out. RESULTS: Time-kill curves exhibited a unique bactericidal activity: time-dependent activity at low concentrations and concentration-dependent activity at high concentrations. In neutropenic murine thigh infection experiments, the antibacterial activity of FMOX correlated with the time that the free drug concentration remaining above the minimum inhibitory concentration (MIC) (fT>MIC) and the ratio of the area under the free drug concentration-time curve for a 24 h period to the MIC (fAUC24/MIC). However, the burden of ESBL producing E. coli significantly reduced when the time intervals for administration were shorter among three dosage regimens with same magnitude of fAUC24/MIC, indicating that fT>MIC is significant PK/PD index. The target value of fT>MIC for 1 log10 kill reduction was 35.1%. CONCLUSIONS: fT>MIC is the most significant PK/PD index of FMOX against ESBL-producing E. coli and its target value is ≥ 40%.

Advanced Quantification Methods To Improve the 18b Dormancy Model for Assessing the Activity of Tuberculosis Drugs In Vitro.

One of the reasons for the lengthy tuberculosis (TB) treatment is the difficulty to treat the nonmultiplying mycobacterial subpopulation. In order to assess the ability of (new) TB drugs to target this subpopulation, we need to incorporate dormancy models in our preclinical drug development pipeline. In most available dormancy models, it takes a long time to create a dormant state, and it is difficult to identify and quantify this nonmultiplying condition. The Mycobacterium tuberculosis 18b strain might overcome some of these problems, because it is dependent on streptomycin for growth and becomes nonmultiplying after 10 days of streptomycin starvation but still can be cultured on streptomycin-supplemented culture plates. We developed our 18b dormancy time-kill kinetics model to assess the difference in the activity of isoniazid, rifampin, moxifloxacin, and bedaquiline against log-phase growth compared to the nonmultiplying M. tuberculosis subpopulation by CFU counting, including a novel area under the curve (AUC)-based approach as well as time-to-positivity (TTP) measurements. We observed that isoniazid and moxifloxacin were relatively more potent against replicating bacteria, while rifampin and high-dose bedaquiline were equally effective against both subpopulations. Moreover, the TTP data suggest that including a liquid culture-based method could be of additional value, as it identifies a specific mycobacterial subpopulation that is nonculturable on solid media. In conclusion, the results of our study underline that the time-kill kinetics 18b dormancy model in its current form is a useful tool to assess TB drug potency and thus has its place in the TB drug development pipeline.

Clinical Mutations That Partially Activate the Stringent Response Confer Multidrug Tolerance in Staphylococcus aureus.

Antibiotic tolerance is an underappreciated antibiotic escape strategy that is associated with recurrent and relapsing infections, as well as acting as a precursor to resistance. Tolerance describes the ability of a bacterial population to survive transient exposure to an otherwise lethal concentration of antibiotic without exhibiting an elevated MIC. It is detected in time-kill assays as a lower rate of killing than a susceptible strain and can be quantified by the metric minimum duration for killing (MDK). The molecular mechanisms behind tolerance are varied, but activation of the stringent response (SR) via gene knockouts and/or chemical induction has long been associated with tolerance. More recently, two Gram-positive clinical isolates from persistent bacteremias were found to bear mutations in the SR controller, Rel, that caused elevated levels of the alarmone (p)ppGpp. Here, we show that introduction of either of these mutations into Staphylococcus aureus confers tolerance to five different classes of antibiotic as a result of (p)ppGpp-mediated growth defects (longer lag time and/or lower growth rate). The degree of tolerance is related to the severity of the growth defect and ranges from a 1.5- to 3.1-fold increase in MDK. Two classes of proposed SR inhibitor were unable to reverse or reduce this tolerance. Our findings reveal the significance of SR-activating mutations in terms of tolerance and clinical treatment failures. The panel of strains reported here provide a clinically relevant model of tolerance for further investigation of its link to resistance development, as well as potential validation of high-throughput tolerance screens.

Synergistic Activity of Colistin-Containing Combinations against Colistin-Resistant Enterobacteriaceae.

Resistance to colistin, a polypeptide drug used as an agent of last resort for the treatment of infections caused by multidrug-resistant (MDR) and extensively drug-resistant (XDR) Gram-negative bacteria, including carbapenem-resistant Enterobacteriaceae (CRE), severely limits treatment options and may even transform an XDR organism into one that is pan-resistant. We investigated the synergistic activity of colistin in combination with 19 antibiotics against a collection of 20 colistin-resistant Enterobacteriaceae isolates, 15 of which were also CRE. All combinations were tested against all strains using an inkjet printer-assisted digital dispensing checkerboard array, and the activities of those that demonstrated synergy by this method were evaluated against a single isolate in a time-kill synergy study. Eighteen of 19 combinations demonstrated synergy against two or more isolates, and the 4 most highly synergistic combinations (colistin combined with linezolid, rifampin, azithromycin, and fusidic acid) were synergistic against ≥90% of strains. Sixteen of 18 combinations (88.9%) that were synergistic in the checkerboard array were also synergistic in a time-kill study. Our findings demonstrate that colistin in combination with a range of antibiotics, particularly protein and RNA synthesis inhibitors, exhibits synergy against colistin-resistant strains, suggesting that colistin may exert a subinhibitory permeabilizing effect on the Gram-negative bacterial outer membrane even in isolates that are resistant to it. These findings suggest that colistin combination therapy may have promise as a treatment approach for patients infected with colistin-resistant XDR Gram-negative pathogens.

Influence of different peritoneal dialysis fluids on the in vitro activity of fosfomycin against Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, and Pseudomonas aeruginosa.

Peritonitis is still the main infectious complication among patients on peritoneal dialysis. For treatment of peritoneal dialysis-related peritonitis, the intraperitoneal administration of antibiotics admixed to peritoneal dialysis fluids (PDFs) should be preferred. However, the influence of diverse PDFs on the activity of frequently used antibiotics has been investigated insufficiently. Thus, the present study set out to investigate the in vitro activity of fosfomycin against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus epidermidis, and Staphylococcus aureus in commercially available PDFs. Time-kill curves in four different PDFs (Dianeal®, Extraneal®, Nutrineal®, and Physioneal®) were performed over 24 h with two different concentrations of fosfomycin (150 and 400 mg/L) and without antibiotics as control. Cation-adjusted Mueller Hinton broth (CA-MHB) was used as a comparator solution. In blank PDFs, bacterial growth of each organism evaluated was reduced when compared to CA-MHB. For S. aureus in blank Physioneal®, a reduction under the limit of detection was observed within 24 h. The activity of fosfomycin was reduced in all PDFs when compared to CA-MHB except for P. aeruginosa in Nutrineal® where the activity of fosfomycin was increased when investigated at 400 mg/L. Against E.coli, bactericidal activity was demonstrated in Extraneal®, Nutrineal®, and Physioneal®. Fosfomycin resistance (MIC > 1024 mg/L) was observed for P. aeruginosa in CA-MHB at both concentrations and in Nutrineal® at 150 mg/L. Fosfomycin is active in PDFs particularly against the frequently isolated enterobacterium E. coli. The choice of the respective PDF considerably influences the microbiological outcome in vitro. Further studies are warranted to investigate the clinical relevance of these findings.

Human Bile Reduces Antimicrobial Activity of Selected Antibiotics against Enterococcus faecalis and Escherichia coli In Vitro.

It has been known from previous studies that body fluids, such as cerebrospinal fluid, lung surfactant, and urine, have a strong impact on the bacterial killing of many anti-infective agents. However, the influence of human bile on the antimicrobial activity of antibiotics is widely unknown. Human bile was obtained and pooled from 11 patients undergoing cholecystectomy. After sterilization of the bile fluid by gamma irradiation, its effect on bacterial killing was investigated for linezolid (LZD) and tigecycline (TGC) against Enterococcus faecalis ATCC 29212. Further, ciprofloxacin (CIP), meropenem (MEM), and TGC were tested against Escherichia coli ATCC 25922. Time-kill curves were performed in pooled human bile and Mueller-Hinton broth (MHB) over 24 h. Bacterial counts (in CFU per milliliter after 24 h) of bile growth controls were approximately equal to MHB growth controls for E. coli and approximately 2-fold greater for E. faecalis, indicating a promotion of bacterial growth by bile for the latter strain. Bile reduced the antimicrobial activity of CIP, MEM, and TGC against E. coli as well as the activity of LZD and TGC against E. faecalis This effect was strongest for TGC against the two strains. Degradation of TGC in bile was identified as the most likely explanation. These findings may have important implications for the treatment of bacterial infections of the gallbladder and biliary tract and should be explored in more detail.

WCK 5107 (Zidebactam) and WCK 5153 Are Novel Inhibitors of PBP2 Showing Potent "ß-Lactam Enhancer" Activity against Pseudomonas aeruginosa, Including Multidrug-Resistant Metallo-ß-Lactamase-Producing High-Risk Clones.

Zidebactam and WCK 5153 are novel ß-lactam enhancers that are bicyclo-acyl hydrazides (BCH), derivatives of the diazabicyclooctane (DBO) scaffold, targeted for the treatment of serious infections caused by highly drug-resistant Gram-negative pathogens. In this study, we determined the penicillin-binding protein (PBP) inhibition profiles and the antimicrobial activities of zidebactam and WCK 5153 against Pseudomonas aeruginosa, including multidrug-resistant (MDR) metallo-ß-lactamase (MBL)-producing high-risk clones. MIC determinations and time-kill assays were conducted for zidebactam, WCK 5153, and antipseudomonal ß-lactams using wild-type PAO1, MexAB-OprM-hyperproducing (mexR), porin-deficient (oprD), and AmpC-hyperproducing (dacB) derivatives of PAO1, and MBL-expressing clinical strains ST175 (blaVIM-2) and ST111 (blaVIM-1). Furthermore, steady-state kinetics was used to assess the inhibitory potential of these compounds against the purified VIM-2 MBL. Zidebactam and WCK 5153 showed specific PBP2 inhibition and did not inhibit VIM-2 (apparent Ki [Kiapp] > 100 µM). MICs for zidebactam and WCK 5153 ranged from 2 to 32 µg/ml (amdinocillin MICs > 32 µg/ml). Time-kill assays revealed bactericidal activity of zidebactam and WCK 5153. LIVE-DEAD staining further supported the bactericidal activity of both compounds, showing spheroplast formation. Fixed concentrations (4 or 8 µg/ml) of zidebactam and WCK 5153 restored susceptibility to all of the tested ß-lactams for each of the P. aeruginosa mutant strains. Likewise, antipseudomonal ß-lactams (CLSI breakpoints), in combination with 4 or 8 µg/ml of zidebactam or WCK 5153, resulted in enhanced killing. Certain combinations determined full bacterial eradication, even with MDR MBL-producing high-risk clones. ß-Lactam-WCK enhancer combinations represent a promising ß-lactam "enhancer-based" approach to treat MDR P. aeruginosa infections, bypassing the need for MBL inhibition.

Prediction of marbofloxacin dosage for the pig pneumonia pathogens Actinobacillus pleuropneumoniae and Pasteurella multocida by pharmacokinetic/pharmacodynamic modelling.

BACKGROUND: Bacterial pneumonia in pigs occurs widely and requires antimicrobial therapy. It is commonly caused by the pathogens Actinobacillus pleuropneumoniae and Pasteurella multocida. Marbofloxacin is an antimicrobial drug of the fluoroquinolone class, licensed for use against these organisms in the pig. In recent years there have been major developments in dosage schedule design, based on integration and modelling of pharmacokinetic (PK) and pharmacodynamic (PD) data, with the objective of optimising efficacy and minimising the emergence of resistance. From in vitro time-kill curves in pig serum, PK/PD breakpoint Area under the curve (AUC) 24h /minimum inhibitory concentration (MIC) values were determined and used in conjunction with published PK, serum protein binding data and MIC distributions to predict dosages based on Monte Carlo simulation (MCS). RESULTS: For three levels of inhibition of growth, bacteriostasis and 3 and 4log10 reductions in bacterial count, mean AUC24h/MIC values were 20.9, 45.2 and 71.7 h, respectively, for P. multocida and 32.4, 48.7 and 55.5 h for A. pleuropneumoniae. Based on these breakpoint values, doses for each pathogen were predicted for several clinical scenarios: (1) bacteriostatic and bactericidal levels of kill; (2) 50 and 90% target attainment rates (TAR); and (3) single dosing and daily dosing at steady state. MCS for 90% TAR predicted single doses to achieve bacteriostatic and bactericidal actions over 48 h of 0.44 and 0.95 mg/kg (P. multocida) and 0.28 and 0.66 mg/kg (A. pleuropneumoniae). For daily doses at steady state, and 90% TAR bacteriostatic and bactericidal actions, dosages of 0.28 and 0.59 mg/kg (P. multocida) and 0.22 and 0.39 mg/kg (A. pleuropneumoniae) were required for pigs aged 12 weeks. Doses were also predicted for pigs aged 16 and 27 weeks. CONCLUSIONS: PK/PD modelling with MCS approaches to dose determination demonstrates the possibility of tailoring clinical dose rates to a range of bacterial kill end-points.

Time-kill curve analysis and pharmacodynamic modelling for in vitro evaluation of antimicrobials against Neisseria gonorrhoeae.

BACKGROUND: Gonorrhoea is a sexually transmitted infection caused by the Gram-negative bacterium Neisseria gonorrhoeae. Resistance to first-line empirical monotherapy has emerged, so robust methods are needed to evaluate the activity of existing and novel antimicrobials against the bacterium. Pharmacodynamic models describing the relationship between the concentration of antimicrobials and the minimum growth rate of the bacteria provide more detailed information than the MIC only. RESULTS: In this study, a novel standardised in vitro time-kill curve assay was developed. The assay was validated using five World Health Organization N. gonorrhoeae reference strains and a range of ciprofloxacin concentrations below and above the MIC. Then the activity of nine antimicrobials with different target mechanisms was examined against a highly antimicrobial susceptible clinical strain isolated in 1964. The experimental time-kill curves were analysed and quantified with a previously established pharmacodynamic model. First, the bacterial growth rates at each antimicrobial concentration were estimated with linear regression. Second, we fitted the model to the growth rates, resulting in four parameters that describe the pharmacodynamic properties of each antimicrobial. A gradual decrease of bactericidal effects from ciprofloxacin to spectinomycin and gentamicin was found. The beta-lactams ceftriaxone, cefixime and benzylpenicillin showed bactericidal and time-dependent properties. Chloramphenicol and tetracycline were purely bacteriostatic as they fully inhibited the growth but did not kill the bacteria. We also tested ciprofloxacin resistant strains and found higher pharmacodynamic MICs (zMIC) in the resistant strains and attenuated bactericidal effects at concentrations above the zMIC. CONCLUSIONS: N. gonorrhoeae time-kill curve experiments analysed with a pharmacodynamic model have potential for in vitro evaluation of new and existing antimicrobials. The pharmacodynamic parameters based on a wide range of concentrations below and above the MIC provide information that could support improving future dosing strategies to treat gonorrhoea.

Antibacterial Activity of Eravacycline Against Carbapenem-Resistant Gram-Negative Isolates in China: An in vitro Study.

Objective: Eravacycline is a novel, fully synthetic fluorocycline antibiotic being developed for the treatment of serious infections, with a broad-spectrum antimicrobial activity, including against carbapenem-resistant gram-negative bacteria (CRGNB). However, the in vitro activity of eravacycline against CRGNB has not been well known in China. In this study, we analysed the antibacterial activity of eravacycline against CRGNB isolates in order to provide a theoretical basis for the clinical treatment. Methods: A total of 346 isolates of CRGNB were collected from two different tertiary care hospitals in Zhejiang, China. Carbapenem resistance genes of all isolates were detected by polymerase chain reaction. And we analysed the in vitro activity of eravacycline against CRGNB by antimicrobial susceptibility tests. In addition, the time-kill curves were generated to evaluate the antibacterial effect of tigecycline and eravacycline. Results: Four different types of carbapenem-resistant isolates were collected, including 50 Escherichia coli isolates, 160 Klebsiella pneumoniae isolates, 42 Enterobacter cloacae complex isolates, and 94 Acinetobacter baumannii isolates. The carbapenem resistance genes were identified in 346 isolates, including bla KPC-2 (48.0%), bla OXA-23 (27.2%), bla NDM-1 (23.1%), and bla NDM-16 (0.3%). The antimicrobial susceptibility testing results showed that the minimum inhibitory concentration (MIC) values of 346 isolates were within the sensitivity range (≤0.0625~16 mg/L) and that the MIC50 or MIC90 of eravacycline was generally approximately 2-fold lower than tigecycline. In addition, the time-kill curves showed that the bactericidal effect of eravacycline was stronger than that of tigecycline against four different types of isolates. Conclusion: Our research indicated that eravacycline had a good antibacterial effect on CRGNB, which could provide a theoretical basis for the clinical treatment of drug-resistant bacterial infections in the future.

Lower Concentrations of Amphotericin B Combined with Ent-Hardwickiic Acid Are Effective against Candida Strains.

Life-threatening Candida infections have increased with the COVID-19 pandemic, and the already limited arsenal of antifungal drugs has become even more restricted due to its side effects associated with complications after SARS-CoV-2 infection. Drug combination strategies have the potential to reduce the risk of side effects without loss of therapeutic efficacy. The aim of this study was to evaluate the combination of ent-hardwickiic acid with low concentrations of amphotericin B against Candida strains. The minimum inhibitory concentration (MIC) values were determined for amphotericin B and ent-hardwickiic acid as isolated compounds and for 77 combinations of amphotericin B and ent-hardwickiic acid concentrations that were assessed by using the checkerboard microdilution method. Time-kill assays were performed in order to assess the fungistatic or fungicidal nature of the different combinations. The strategy of combining both compounds markedly reduced the MIC values from 16 µg/mL to 1 µg/mL of amphotericin B and from 12.5 µg/mL to 6.25 µg/mL of ent-hardwickiic acid, from isolated to combined, against C. albicans resistant to azoles. The combination of 1 µg/mL of amphotericin B with 6.25 µg/mL of ent-hardwickiic acid killed all the cells of the same strain within four hours of incubation.

Design, synthesis and antibacterial activity of novel pleuromutilin derivatives with thieno[2,3-d]pyrimidine substitution.

A series of novel pleuromutilin derivatives with substituted thienopyrimidines were designed, synthesized, and evaluated for antibacterial act ivity. In this study, the activities of these compounds were investigated using the inhibition circle test, the minimum inhibitory concentration (MIC) test, real-time growth curves, time-kill kinetic assays, cytotoxicity assays, and molecular docking. Most of the tested compounds exhibited moderate antibacterial activity against Staphylococcus aureus, Streptococcus agalactiae, and Escherichia coli. Compound A11 was the most active and displayed bacteriostatic activities against methicillin-resistant S. aureus, with MIC values as low as 0.00191 µg/mL, which is 162 and 32 times lower than that of the marketed antibiotics tiamulin and retapamulin, respectively. Furthermore, the mechanism of action of A11 was confirmed by molecular docking studies.

Pharmacodynamics of Ceftriaxone, Ertapenem, Fosfomycin and Gentamicin in Neisseria gonorrhoeae.

OBJECTIVES: To assess the in vitro effect of select antimicrobials on the growth of N. gonorrhoeae and its pharmacodynamic parameters. METHODS: Time-kill assays were performed on two reference N. gonorrhoeae strains (ceftriaxone-resistant WHO X and ceftriaxone-susceptible WHO F) and one clinical N. gonorrhoeae strain (ceftriaxone-susceptible CS03307). Time-kill curves were constructed for each strain by measuring bacterial growth rates at doubling antimicrobial concentrations of ceftriaxone, ertapenem, fosfomycin and gentamicin. Inputs from these curves were used to estimate minimal bacterial growth rates at high antimicrobial concentrations (ψmin), maximum bacterial growth rates in the absence of antimicrobials (ψmax), pharmacodynamic minimum inhibitory concentrations (zMIC), and Hill's coefficients (κ). RESULTS: Ceftriaxone, ertapenem and fosfomycin showed gradual death overtime at higher antimicrobial concentrations with a relatively high ψmin, demonstrating time-dependent activity. Compared to WHO F, the ψmin for WHO X was significantly increased, reflecting decreased killing activity for ceftriaxone, ertapenem and fosfomycin. At high ceftriaxone concentrations, WHO X was still efficiently killed. CS03307 also showed a high ψmin for ceftriaxone in spite of a low MIC and no difference in ψmin for fosfomycin in spite of significant MIC and zMIC differences. Gentamicin showed rapid killing for all three strains at high concentrations, demonstrating concentration-dependent activity. CONCLUSIONS: Based on time-kill assays, high-dosage ceftriaxone could be used to treat N. gonorrhoeae strains with MIC above breakpoint, with gentamicin as a potential alternative. Whether ertapenem or fosfomycin would be effective to treat strains with a high MIC to ceftriaxone is questionable.

In Vitro Pharmacokinetic/Pharmacodynamic Modelling and Simulation of Amphotericin B against Candida auris.

The aims of this study were to characterize the antifungal activity of amphotericin B against Candida auris in a static in vitro system and to evaluate different dosing schedules and MIC scenarios by means of semi-mechanistic pharmacokinetic/pharmacodynamic (PK/PD) modelling and simulation. A two-compartment model consisting of a drug-susceptible and a drug-resistant subpopulation successfully characterized the time-kill data and a modified Emax sigmoidal model best described the effect of the drug. The model incorporated growth rate constants for both subpopulations, a death rate constant and a transfer constant between both compartments. Additionally, the model included a parameter to account for the delay in growth in the absence or presence of the drug. Amphotericin B displayed a concentration-dependent fungicidal activity. The developed PK/PD model was able to characterize properly the antifungal activity of amphotericin B against C. auris. Finally, simulation analysis revealed that none of the simulated standard dosing scenarios of 0.6, 1 and 1.5 mg/kg/day over a week treatment showed successful activity against C. auris infection. Simulations also pointed out that an MIC of 1 mg/L would be linked to treatment failure for C. auris invasive infections and therefore, the resistance rate to amphotericin B may be higher than previously reported.

Techniques for the Assessment of In Vitro and In Vivo Antifungal Combinations.

Systemic fungal infections are associated with high mortality rates despite adequate treatment. Moreover, acquired resistance to antifungals is increasing, which further complicates the therapeutic management. One strategy to overcome antifungal resistance is to use antifungal combinations. In vitro, several techniques are used to assess drug interactions, such as the broth microdilution checkerboard, agar-diffusion methods, and time-kill curves. Currently, the most widely used technique is the checkerboard method. The aim of all these techniques is to determine if the interaction between antifungal agents is synergistic, indifferent, or antagonistic. However, the interpretation of the results remains difficult. Several methods of analysis can be used, based on different theories. The most commonly used method is the calculation of the fractional inhibitory concentration index. Determination of the usefulness of combination treatments in patients needs well-conducted clinical trials, which are difficult. It is therefore important to study antifungal combinations in vivo, in experimental animal models of fungal infections. Although mammalian models have mostly been used, new alternative animal models in invertebrates look promising. To evaluate the antifungal efficacy, the most commonly used criteria are the mortality rate and the fungal load in the target organs.

Predicting Antimicrobial Activity at the Target Site: Pharmacokinetic/Pharmacodynamic Indices versus Time-Kill Approaches.

Antibiotic dosing strategies are generally based on systemic drug concentrations. However, drug concentrations at the infection site drive antimicrobial effect, and efficacy predictions and dosing strategies should be based on these concentrations. We set out to review different translational pharmacokinetic-pharmacodynamic (PK/PD) approaches from a target site perspective. The most common approach involves calculating the probability of attaining animal-derived PK/PD index targets, which link PK parameters to antimicrobial susceptibility measures. This approach is time efficient but ignores some aspects of the shape of the PK profile and inter-species differences in drug clearance and distribution, and provides no information on the PD time-course. Time-kill curves, in contrast, depict bacterial response over time. In vitro dynamic time-kill setups allow for the evaluation of bacterial response to clinical PK profiles, but are not representative of the infection site environment. The translational value of in vivo time-kill experiments, conversely, is limited from a PK perspective. Computational PK/PD models, especially when developed using both in vitro and in vivo data and coupled to target site PK models, can bridge translational gaps in both PK and PD. Ultimately, clinical PK and experimental and computational tools should be combined to tailor antibiotic treatment strategies to the site of infection.

Efficacy of Fosfomycin and Its Combination With Aminoglycosides in an Experimental Sepsis Model by Carbapenemase-Producing Klebsiella pneumoniae Clinical Strains.

Carbapenemase-producing Klebsiella pneumoniae infections are an increasing global threat with scarce and uncertain treatment options. In this context, combination therapies are often used for these infections. The bactericidal and synergistic activity of fosfomycin plus amikacin and gentamicin was studied trough time-kill assays against four clonally unrelated clinical isolates of carbapenemase-producing K. pneumoniae, VIM-1, VIM-1 plus DHA-1, OXA-48 plus CTXM-15, and KPC-3, respectively. The efficacy of antimicrobials that showed synergistic activity in vitro against all the carbapenemase-producing K. pneumoniae were tested in monotherapy and in combination, in a murine peritoneal sepsis model. In vitro, fosfomycin plus amikacin showed synergistic and bactericidal effect against strains producing VIM-1, VIM-1 plus DHA-1, and OXA-48 plus CTX-M-15. Fosfomycin plus gentamicin had in vitro synergistic activity against the strain producing KPC-3. In vivo, fosfomycin and amikacin and its combination reduced the spleen bacterial concentration compared with controls groups in animals infected by K. pneumoniae producing VIM-1 and OXA-48 plus CTX-M-15. Moreover, amikacin alone and its combination with fosfomycin reduced the bacteremia rate against the VIM-1 producer strain. Contrary to the in vitro results, no in vivo efficacy was found with fosfomycin plus amikacin against the VIM-1 plus DHA-1 producer strain. Finally, fosfomycin plus gentamicin reduced the bacterial concentration in spleen against the KPC-3 producer strain. In conclusion, our results suggest that fosfomycin plus aminoglycosides has a dissimilar efficacy in the treatment of this severe experimental infection, when caused by different carbapenemase-producing K. pneumoniae strains. Fosfomycin plus amikacin or plus gentamycin may be useful to treat infections by OXA-48 plus CTX-M-15 or KPC-3 producer strains, respectively.

Antibacterial Activity of Arbutus pavarii Pamp against Methicillin-Resistant Staphylococcus aureus (MRSA) and UHPLC-MS/MS Profile of the Bioactive Fraction.

Arbutus pavarii Pamp is a medicinal plant commonly used by local tribes in East Libya for the treatment of many diseases, such as gastritis, renal infections, cancer and kidney diseases. In this study, the antibacterial activity of the leaf and stem bark extracts of the plant against methicillin-resistant Staphylococcus aureus (MRSA), as well as the metabolite profiles of the bioactive fractions, was investigated. The antibacterial activity was determined by disc diffusion method, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC), while the microbial reduction by the bioactive fraction was evaluated using time-kill test. The bioactive fraction was further subjected to ultrahigh-performance liquid chromatography-mass spectrometry (UHPLC-ESI-MS/MS) analysis to putatively identify the chemical constituents contained therein. All the extracts and fractions showed different levels of antibacterial activity on the tested MRSA strains. The highest total antibacterial activity, i.e., 4007.6 mL/g, was exhibited by the crude leaf methanolic extract. However, the ethyl acetate fraction of the leaf showed moderate to significant antibacterial activity against MRSA at low MIC (0.08-1.25 mg/mL). Metabolite profiling of this fraction using UHPLC-ESI-MS/MS resulted in the putative identification of 28 compounds, which included phenolic acids, flavan-3-ols and flavonols. The results of this study showed that the ethyl acetate fraction of Arbutus pavarii leaf possessed potential antibacterial activity against MRSA and hence can be further explored for pharmaceutical applications as a natural antibacterial agent.

Susceptibility testing of Candida albicans isolated from oropharyngeal mucosa of HIV(+) patients to fluconazole, amphotericin B and Caspofungin. killing kinetics of caspofungin and amphotericin B against fluconazole resistant and susceptible isolates.

A clear understanding of the pharmacodynamic properties of antifungal agents is important for the adequate treatment of fungal infections like candidiasis. For certain antifungal agents, the determination of Minimal Fungicidal Concentration (MFC) and time kill curve could be clinically more relevant than the determination of the Minimal Inhibitory Concentration (MIC). In this study, MIC and MFC to fluconazole, amphotericin B and caspofungin against C. albicans isolates and the killing patterns obtained with caspofungin and amphotericin B against susceptible and resistant strains to fluconazole were determined. The results of MICs showed that all C. albicans isolates were highly susceptible to amphotericin B, but two isolates were fluconazole resistant. The comparative analysis between MIC and MFC showed that MFC of fluconazole was fourfold higher than MIC in 41.9% of the C. albicans isolates. Same values of MFC and MIC of amphotericin B and caspofungin were found for 71% of the isolates. Correlation between time kill curves and MFC of amphotericin B and caspofungin against all 4 isolates tested was observed. The caspofungin killing effect was more evident at MFC in 6 hours of incubation than at MIC in this time, suggesting dependence of concentration. The similarity of results of time-kill curve and MFC values indicate that determination of MFC is an alternative for the detection of the fungicidal activity of these drugs.