A randomized placebo-controlled trial of nicotinamide riboside and pterostilbene supplementation in experimental muscle injury in elderly individuals.

BACKGROUNDDuring aging, there is a functional decline in the pool of muscle stem cells (MuSCs) that influences the functional and regenerative capacity of skeletal muscle. Preclinical evidence has suggested that nicotinamide riboside (NR) and pterostilbene (PT) can improve muscle regeneration, e.g., by increasing MuSC function. The objective of this study was to investigate if supplementation with NR and PT (NRPT) promotes skeletal muscle regeneration after muscle injury in elderly individuals by improved recruitment of MuSCs.METHODSThirty-two elderly individuals (55-80 years of age) were randomized to daily supplementation with either NRPT (1,000 mg NR and 200 mg PT) or matched placebo. Two weeks after initiation of supplementation, skeletal muscle injury was induced by electrically induced eccentric muscle work. Skeletal muscle biopsies were obtained before, 2 hours after, and 2, 8, and 30 days after injury.RESULTSA substantial skeletal muscle injury was induced by the protocol and associated with release of myoglobin and creatine kinase, muscle soreness, tissue edema, and a decrease in muscle strength. MuSC content, proliferation, and cell size revealed a large demand for recruitment after injury, but this was not affected by NRPT. Furthermore, histological analyses of muscle fiber area, central nuclei, and embryonic myosin heavy chain showed no NRPT supplementation effect.CONCLUSIONDaily supplementation with 1,000 mg NR and 200 mg PT is safe but does not improve recruitment of the MuSC pool or other measures of muscle recovery in response to injury or subsequent regeneration in elderly individuals.TRIAL REGISTRATIONClinicalTrials.gov NCT03754842.FUNDINGNovo Nordisk Foundation (NNF17OC0027242) and Novo Nordisk Foundation CBMR.

ß-Lactoglobulin Is Insulinotropic Compared with Casein and Whey Protein Ingestion during Catabolic Conditions in Men in a Double-Blinded Randomized Crossover Trial.

BACKGROUND: Muscle loss during acute infectious disease is mainly triggered by inflammation, immobilization, and malnutrition. OBJECTIVE: The objective was to compare muscle protein kinetics and metabolism following ingestion of the dairy protein supplements ß-lactoglobulin (BLG), casein (CAS), and whey (WHE) during controlled catabolic conditions. METHODS: We used a randomized crossover design (registered at clinicaltrials.gov as NCT03319550) to investigate 9 healthy male participants [age: 20-40 y; BMI (in kg/m2) 20-30] who were randomly assigned servings of BLG, CAS, or WHE (0.6 g protein/kg, one-third as bolus and two-thirds as sip every 20 min) on 3 separate occasions separated by ∼6-8 wk. The participants received an infusion of lipopolysaccharide (1 ng/kg) combined with 36 h of fasting and bed rest before each study day, mimicking a clinical catabolic condition. The forearm model and isotopic tracer techniques were used to quantify muscle protein kinetics. Muscle biopsy specimens were obtained and intramyocellular signaling investigated using Western blot. RESULTS: BLG, CAS, and WHE improved the net balance of phenylalanine (NBphe) from baseline with ∼75% (P < 0.001) with no difference between interventions (primary outcome, P < 0.05). No difference in rates of appearance and disappearance of phenylalanine or in intramyocellular signaling activation was found between interventions (secondary outcomes). The incremental AUC for serum insulin was 62% higher following BLG compared with CAS (P < 0.001) and 30% higher compared with WHE (P = 0.002), as well as 25% higher in WHE compared with CAS (P = 0.006). Following BLG consumption, plasma concentrations of glucose-dependent insulinotropic peptide (GIP) increased 70% compared with CAS (P = 0.001) and increased 34% compared with WHE (P = 0.06). No significant difference was found between WHE and CAS (P = 0.12). CONCLUSION: BLG, WHE, and CAS have similar effects on muscle in young male participants during catabolic conditions. BLG showed specific, possibly GIP-dependent, insulinotropic properties, which may have future clinical implications.

Effective antimicrobial combination in vivo treatment predicted with microcalorimetry screening.

OBJECTIVES: The worldwide emergence of antibiotic resistance calls for effective exploitation of existing antibiotics. Antibiotic combinations with different modes of action can synergize for successful treatment. In the present study, we used microcalorimetry screening to identify synergistic combination treatments against clinical MDR isolates. The synergistic effects were validated in a murine infection model. METHODS: The synergy of meropenem combined with colistin, rifampicin or amikacin was tested on 12 isolates (1 Escherichia coli, 5 Klebsiella pneumoniae, 3 Pseudomonas aeruginosa and 3 Acinetobacter baumannii) in an isothermal microcalorimeter measuring metabolic activity. One A. baumannii strain was tested with two individual pairings of antibiotic combinations. The microcalorimetric data were used to predict in vivo efficacy in a murine peritonitis/sepsis model. NMRI mice were inoculated intraperitoneally and after 1 h treated with saline, drug X, drug Y or X+Y. Bacterial load was determined by cfu in peritoneal fluid and blood after 4 h. RESULTS: In vitro, of the 13 combinations tested on the 12 strains, 3 of them exhibited a synergistic reduction in MIC (23% n = 3/13), 5 showed an additive effect (38.5% n = 5/13) and 5 had indifferent or antagonistic effects (38.5% n = 5/13). There was a significant correlation (P = 0.024) between microcalorimetry-screening FIC index values and the log reduction in peritoneal fluid from mice that underwent combination treatment compared with the most effective mono treatment. No such correlation could be found between chequerboard and in vivo results (P = 0.16). CONCLUSIONS: These data support microcalorimetic metabolic readout to predict additive or synergistic effects of combination treatment of MDR infections within hours.

Oral D/L-3-Hydroxybutyrate Stimulates Cholecystokinin and Insulin Secretion and Slows Gastric Emptying in Healthy Males.

BACKGROUND: D-3-hydroxybutyrate (D-3-OHB) is a ketone body that serves as an alternative nutritional fuel but also as an important signaling metabolite. Oral ketone supplements containing D/L-3-OHB are becoming a popular approach to achieve ketosis. AIM: To explore the gut-derived effects of ketone supplements. METHODS: Eight healthy lean male volunteers were investigated on 2 separate occasions:An acetaminophen test was performed to evaluate gastric emptying and blood samples were obtained consecutively throughout the study period. RESULTS: We show that oral consumption of D/L-3-OHB stimulates cholecystokinin release (P = 0.02), elevates insulin (P = 0.03) and C-peptide (P < 0.001) concentrations, and slows gastric emptying (P = 0.01) compared with matched intravenous D/L-3-OHB administration. Measures of appetite and plasma concentrations of glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) were unaffected by interventions. CONCLUSION: Our findings show that D/L-3-OHB exert incretin effects and indicate luminal sensing in the gut endothelium. This adds to our understanding of ketones as signaling metabolites and displays the important difference between physiological ketosis and oral ketone supplements.

Mutational change of CTX-M-15 to CTX-M-127 resulting in mecillinam resistant Escherichia coli during pivmecillinam treatment of a patient.

Pivmecillinam (amdinocillin pivoxil) is the recommended first-choice antibiotic used to treat urinary tract infections (UTIs) in Denmark. The frequency of mutation to mecillinam (MEC) resistance is described as high in vitro; however, treatment of UTI has a good clinical response and prevalence of mecillinam resistance in Escherichia coli remains low despite many years of use. We describe occurrence of in vivo mecillinam resistance in a clinical isolate of ESBL-producing E. coli following pivmecillinam treatment. The identified phenotypic differences in the mecillinam resistant isolate compared with the original mecillinam susceptible isolate were a full-length LPS with O-antigen (O25), mecillinam resistance and a lower MIC for ceftazidime. Regarding genotype, the resistant isolate differed with a mutation in blaCTX-M-15 to blaCTX-M-127 , loss of a part of a plasmid and a genomic island, respectively, and insertion of a transposase in wbbL, causing the rough phenotype. The observed mecillinam resistance is expected to be caused by the mutation in blaCTX-M-15 with additional contribute from the serotype shift. We continue to recommend the use of pivmecillinam as first-line treatment for UTI.

Effects of Nicotinamide Riboside on Endocrine Pancreatic Function and Incretin Hormones in Nondiabetic Men With Obesity.

OBJECTIVE: Augmenting nicotinamide adenine dinucleotide (NAD+) metabolism through dietary provision of NAD+ precursor vitamins translates to improved glucose handling in rodent models of obesity and diabetes. Preclinical evidence suggests that the NAD+/SIRT1 axis may be implicated in modulating important gut-related aspects of glucose regulation. We sought to test whether NAD+ precursor supplementation with nicotinamide riboside (NR) affects ß-cell function, α-cell function, and incretin hormone secretion as well as circulating bile acid levels in humans. DESIGN: A 12-week randomized, double-blind, placebo-controlled, parallel-group trial in 40 males with obesity and insulin resistance allocated to NR at 1000 mg twice daily (n = 20) or placebo (n = 20). Two-hour 75-g oral glucose tolerance tests were performed before and after the intervention, and plasma concentrations of glucose, insulin, C-peptide, glucagon, glucagon-like peptide 1 (GLP-1), and glucose-dependent insulinotropic polypeptide (GIP) were determined. ß-Cell function indices were calculated based on glucose, insulin, and C-peptide measurements. Fasting plasma concentrations of bile acids were determined. RESULTS: NR supplementation during 12 weeks did not affect fasting or postglucose challenge concentrations of glucose, insulin, C-peptide, glucagon, GLP-1, or GIP, and ß-cell function did not respond to the intervention. Additionally, no changes in circulating adipsin or bile acids were observed following NR supplementation. CONCLUSION: The current study does not provide evidence to support that dietary supplementation with the NAD+ precursor NR serves to impact glucose tolerance, ß-cell secretory capacity, α-cell function, and incretin hormone secretion in nondiabetic males with obesity. Moreover, bile acid levels in plasma did not change in response to NR supplementation.

Comparative Activity of Ceftriaxone, Ciprofloxacin, and Gentamicin as a Function of Bacterial Growth Rate Probed by Escherichia coli Chromosome Replication in the Mouse Peritonitis Model.

Commonly used antibiotics exert their effects predominantly on rapidly growing bacterial cells; yet, the growth dynamics taking place during infection in a complex host environment remain largely unknown. Hence, a means to measure in situ bacterial growth rate is essential to predict the outcome of antibacterial treatment. We have recently validated chromosome replication as a readout of in situ bacterial growth rate during Escherichia coli infection in the mouse peritonitis model. By the use of two complementary methods (quantitative PCR and fluorescence microscopy) for differential genome origin and terminus copy number quantification, we demonstrated the ability to track bacterial growth rate, both on a population average level and on a single-cell level, from one single biological specimen. Here, we asked whether the in situ growth rate predicts antibiotic treatment effect during infection in the same model. Parallel in vitro growth experiments were conducted as a proof of concept. Our data demonstrate that the activities of the commonly used antibiotics ceftriaxone and gentamicin correlated with pretreatment bacterial growth rate; both drugs performed better during rapid growth than during slow growth. Conversely, ciprofloxacin was less sensitive to bacterial growth rate, both in a homogenous in vitro bacterial population and in a more heterogeneous in vivo bacterial population. The method serves as a platform to test any antibiotic's dependency on active in situ bacterial growth. Improved insight into this relationship in vivo could ultimately prove helpful in evaluating future antibacterial strategies.

A randomized placebo-controlled clinical trial of nicotinamide riboside in obese men: safety, insulin-sensitivity, and lipid-mobilizing effects.

Background: Animal studies suggest a positive role for nicotinamide riboside (NR) on insulin sensitivity and hepatic steatosis in models of obesity and type 2 diabetes. NR, an NAD+ precursor, is a member of the vitamin B-3 family now available as an over-the-counter supplement. Although data from preclinical trials appear consistent, potential effects and safety need to be evaluated in human clinical trials. Objective: The aim of this study was to test the safety of dietary NR supplementation over a 12-wk period and potential to improve insulin sensitivity and other metabolic parameters in obese, insulin-resistant men. Design: In an investigator-initiated randomized, placebo-controlled, double-blinded, and parallel-group designed clinical trial, forty healthy, sedentary men with a body mass index (BMI) > 30 kg/m2, age-range 40-70 y were randomly assigned to 12 wk of NR (1000 mg twice daily) or placebo. We determined the effects of NR supplementation on insulin sensitivity by a hyperinsulinemic euglycemic clamp and substrate metabolism by indirect calorimetry and labeled substrates of tritiated glucose and palmitate. Body composition and fat mass distribution were determined by whole-body dual-energy X-ray absorptiometry (DXA) and MRI scans, and measurements of intrahepatic lipid content were obtained by MR spectroscopy. Results: Insulin sensitivity, endogenous glucose production, and glucose disposal and oxidation were not improved by NR supplementation. Similarly, NR supplementation had no effect on resting energy expenditure, lipolysis, oxidation of lipids, or body composition. No serious adverse events due to NR supplementation were observed and safety blood tests were normal. Conclusion: 12 wk of NR supplementation in doses of 2000 mg/d appears safe, but does not improve insulin sensitivity and whole-body glucose metabolism in obese, insulin-resistant men. This trial was registered at clinicaltrials.gov as NCT02303483.

Pharmacokinetics and Pharmacodynamics of Fosfomycin and Its Activity against Extended-Spectrum-ß-Lactamase-, Plasmid-Mediated AmpC-, and Carbapenemase-Producing Escherichia coli in a Murine Urinary Tract Infection Model.

Fosfomycin has become an attractive treatment alternative for urinary tract infections (UTIs) due to increasing multidrug resistance (MDR) in Escherichia coli In this study, we evaluated the pharmacokinetic (PK) and pharmacodynamic (PD) indices of fosfomycin and its in vivo activity in an experimental murine model of ascending UTI. Subcutaneous administration of fosfomycin showed that the mean peak plasma concentrations of fosfomycin were 36, 280, and 750 mg/liter following administration of a single dose of 0.75, 7.5, and 30 mg/mouse, respectively, with an elimination half-life of 28 min, and urine peak concentrations of 1,100, 33,400, and 70,000 mg/liter expected to be sustained above 1 mg/liter (MIC of the test strain, NU14) for 5, 8, and 9.5 h, respectively. The optimal PK/PD indices for reducing urine colony counts (number of CFU per milliliter) were determined to be the area under the concentration-time curve/MIC from 0 to 72 h and the maximum concentration/MIC on the basis of the dose-dependent bloodstream PK and the results of an evaluation of six dosing regimens. With a dosing regimen of 15 mg/mouse twice (every 36 h), fosfomycin significantly reduced the number of CFU per milliliter of all susceptible strains in urine, including clinical MDR strains, except for one clinical strain (P = 0.062). Variable degrees of reduction were observed in the bladder and kidneys. No significant reductions in the number of CFU per milliliter were observed with the resistant strains. In conclusion, fosfomycin shows concentration-dependent in vivo activity, and the results suggest that fosfomycin is an effective alternative to carbapenems in treating MDR E. coli in uncomplicated UTIs. The data on the effectiveness of fosfomycin against the MDR isolates along with the results of PK/PD modeling should facilitate the further development of improved recommendations for its clinical use.

Effects of 12 weeks high dose vitamin D3 treatment on insulin sensitivity, beta cell function, and metabolic markers in patients with type 2 diabetes and vitamin D insufficiency - a double-blind, randomized, placebo-controlled trial.

OBJECTIVES: Vitamin D insufficiency is common in subjects with type 2 diabetes. Observational studies suggest that vitamin D plays a role in the pathogenesis of type 2 diabetes. However, results of intervention studies have been inconsistent. We investigated the effects of improving vitamin D status on insulin sensitivity, insulin secretion, and inflammatory markers in patients with type 2 diabetes. MATERIALS/METHODS: A double blind, randomized, placebo controlled trial was conducted. Sixteen patients with type 2 diabetes and hypovitaminosis D were recruited. Eight patients received colecalciferol and (280 µg daily for 2 weeks, 140 µg daily for 10 weeks) and 8 patients received identical placebo tablets for 12 weeks. Before and after intervention, patients underwent IVGTT, hyperinsulinemic euglycemic clamp, assessment of baseline high-frequency insulin pulsatility, glucose-entrained insulin pulsatility, DXA scans, 24-hour-ambulatory blood pressure monitorings, and fasting blood samples. RESULTS: Serum-25(OH) vitamin D and serum-1,25(OH)2 vitamin D increased significantly after 12 weeks in the intervention group (p=0.01, p=0.004). Serum-25(OH) vitamin D was also significantly higher in the vitamin D group compared to the placebo group (p=0.02) after intervention. Although no significant changes in insulin sensitivity, inflammation, blood pressure, lipid profile, or HbA1c were found, we observed borderline (p between 0.05 and 0.10) improvements of insulin secretion, in terms of c-peptide levels, first phase incremental AUC insulin and insulin secretory burst mass. CONCLUSIONS: Improvement in vitamin D status does not improve insulin resistance, blood pressure, inflammation or HbA1c, but might increase insulin secretion in patients with established type 2 diabetes.

Efficacy of topical and systemic antibiotic treatment of meticillin-resistant Staphylococcus aureus in a murine superficial skin wound infection model.

Meticillin-resistant Staphylococcus aureus (MRSA) is a rapidly spreading pathogen associated predominantly with skin infections. The lack of clinical evidence indicating the best treatment strategy to combat MRSA skin infections prompted us to investigate the efficacy of available treatment options in an experimental skin wound infection model in mice. Mice were treated either topically with retapamulin (1%), fusidic acid (2%) or mupirocin (2%) or systemically with linezolid (50-100 mg/kg/day) or vancomycin (50-200 mg/kg/day) twice daily for 3 days or 6 days and the total bacterial loads in the skin lesions were determined. Retapamulin, fusidic acid and mupirocin treatment for 3 days reduced the bacterial loads by 2.5, 2.9 and 2.0 log(10) CFU, respectively, and treatment for 6 days by 5.0, 4.2 and 5.1 log(10) CFU, respectively, compared with non-treated controls (P < 0.001). Systemic treatment with linezolid for 6 days reduced the bacterial loads by 1.6 log(10) CFU compared with non-treated mice (P < 0.001), whereas vancomycin treatment showed no effect on reducing the bacterial loads in infected skin lesions. These findings suggest that topical treatment with retapamulin and mupirocin is significantly more effective than systemic treatment with linezolid and vancomycin in eradicating MRSA in skin wounds. Retapamulin and mupirocin may provide an alternative to fusidic acid treatment of MRSA in skin wounds when resistance to fusidic acid is suspected.

High-dose resveratrol supplementation in obese men: an investigator-initiated, randomized, placebo-controlled clinical trial of substrate metabolism, insulin sensitivity, and body composition.

Obesity, diabetes, hypertension, and hyperlipidemia constitute risk factors for morbidity and premature mortality. Based on animal and in vitro studies, resveratrol reverts these risk factors via stimulation of silent mating type information regulation 2 homolog 1 (SIRT1), but data in human subjects are scarce. The objective of this study was to examine the metabolic effects of high-dose resveratrol in obese human subjects. In a randomized, placebo-controlled, double-blinded, and parallel-group design, 24 obese but otherwise healthy men were randomly assigned to 4 weeks of resveratrol or placebo treatment. Extensive metabolic examinations including assessment of glucose turnover and insulin sensitivity (hyperinsulinemic euglycemic clamp) were performed before and after the treatment. Insulin sensitivity, the primary outcome measure, deteriorated insignificantly in both groups. Endogenous glucose production and the turnover and oxidation rates of glucose remained unchanged. Resveratrol supplementation also had no effect on blood pressure; resting energy expenditure; oxidation rates of lipid; ectopic or visceral fat content; or inflammatory and metabolic biomarkers. The lack of effect disagrees with persuasive data obtained from rodent models and raises doubt about the justification of resveratrol as a human nutritional supplement in metabolic disorders.

Impact of low-level fluoroquinolone resistance genes qnrA1, qnrB19 and qnrS1 on ciprofloxacin treatment of isogenic Escherichia coli strains in a murine urinary tract infection model.

OBJECTIVES: To study the impact of qnrA1, qnrB19 and qnrS1 on the ciprofloxacin treatment of urinary tract infection (UTI). METHODS: From a wild-type (wt) Escherichia coli UTI isolate, three isogenic strains were constructed carrying low-level ciprofloxacin resistance genes qnrA1, qnrB19 or qnrS1 (ciprofloxacin MIC range: 0.19-0.38 mg/L). Time-kill studies were performed for all four isogenic strains at the following concentrations: 1×, 2×, 4×, 8× and 16× MIC. Ciprofloxacin serum and urine pharmacokinetics was determined to calculate a murine dose equivalent (AUC(24)) to the standard human dose of 500 mg twice daily, which corresponded to 0.2 mg/mouse four times daily. In the murine UTI model, mice infected with each of the isogenic qnr strains or the wt strain were treated with ciprofloxacin (0.2 mg/mouse) or saline (only the E. coli wt) subcutaneously four times daily for 3 days starting 24 h after bacterial inoculation. RESULTS: In vitro, the strains responded to ciprofloxacin concentrations of 4-16× MIC by several log(10) reductions. In vivo, despite ciprofloxacin reaching urine concentrations far exceeding the MICs for the strains (500 mg/L), ciprofloxacin was significantly less efficient at reducing the urine and bladder bacterial counts of qnrA1-, qnrB19- and qnrS1-positive strains compared with the ciprofloxacin-treated wt strain (P < 0.05). None of the four strains infected the kidneys well, with median cfu counts of <1 log(10). CONCLUSIONS: Although qnr genes only confer low levels of resistance to ciprofloxacin, a reduced bactericidal activity of ciprofloxacin was observed in both urine and bladder in the murine model of UTI.

Effect of generics on price and consumption of ciprofloxacin in primary healthcare: the relationship to increasing resistance.

BACKGROUND: The introduction of generic versions of drugs has often resulted in an increase in the consumption of the agents involved. In December 2001, generic ciprofloxacin was marketed in Denmark. Our objective was to evaluate, in a community setting, the effect of price on consumption of ciprofloxacin and on ciprofloxacin resistance in Escherichia coli urine isolates. METHODS: We conducted a retrospective ecological study collecting monthly national data on the number of marketed versions and primary healthcare (PHC) sales of ciprofloxacin during January 1995-December 2005. Data were compared with a median price per defined daily dose (DDD) of ciprofloxacin during September 1999-December 2005. Yearly PHC consumption data from seven Danish counties were compared with the antimicrobial resistance profiles of PHC E. coli urine isolates. RESULTS: During 2002, the number of marketed versions increased from 3 to 10, and the median price per DDD decreased by 53%. From 2002 to 2005, the total consumption of oral ciprofloxacin in PHC increased significantly from 0.13 DDD/1000 inhabitant-days to 0.33 DDD/1000 inhabitant-days. During the same period, the frequency of ciprofloxacin resistance increased by 200%. A statistically significant correlation was found between the consumption of ciprofloxacin and the ciprofloxacin resistance rate in E. coli urine isolates, independent of the introduction of generic ciprofloxacin. CONCLUSIONS: After the introduction of generic ciprofloxacin, a significant increase in the total consumption of oral ciprofloxacin in PHC was observed in Denmark. The increase in consumption was significantly correlated with ciprofloxacin resistance in E. coli obtained from urine isolates.

Lipase inhibitory activity in alcohol extracts of worldwide occurring plants and propolis.

In the search for lipase inhibitory agents, 144 alcohol plant extracts and propolis were screened for pancreatic lipase activity in vitro using methods with methylresorufin and triolein as substrates. Ethanol extracts of Linum usitatissimum (oil flax) and Helianthus annuus (sunflower seeds) showed the strongest lipase inhibitory actions with ID(50) values of 1:370 and 1:166, respectively.

Affinity purification of recombinant protein-tyrosine phosphatase 1B using a highly selective inhibitor.

Our structure-based drug discovery program within the field of protein-tyrosine phosphatases (PTPs) demands delivery of significant amounts of protein with extraordinary purity specifications over prolonged time periods. Hence, replacement of classical, multi-step, low-yield protein purifications with efficient affinity techniques would be desirable. For this purpose, the highly selective PTP1B inhibitor 2-(oxalyl-amino)-4,5,6,7-tetrahydro-thieno[2,3-c]pyridine-3-carboxylic acid (OTP) was coupled to epoxy-activated Sepharose 6B (OTP Sepharose) and used for one-step affinity purification of tag-free PTP1B. The elution was performed with a combined pH and salt gradient. Importantly, since OTP Sepharose binds PTP1B with an intact active site only, the method ensures that the purified enzyme is fully active, a feature that might be particularly important in PTP research.

Selection of resistant Streptococcus pneumoniae during penicillin treatment in vitro and in three animal models.

Pharmacokinetic (PK) and pharmacodynamic (PD) properties for the selection of resistant pneumococci were studied by using three strains of the same serotype (6B) for mixed-culture infection in time-kill experiments in vitro and in three different animal models, the mouse peritonitis, the mouse thigh, and the rabbit tissue cage models. Treatment regimens with penicillin were designed to give a wide range of T(>MIC)s, the amounts of time for which the drug concentrations in serum were above the MIC. The mixed culture of the three pneumococcal strains, 10(7) CFU of strain A (MIC of penicillin, 0.016 micro g/ml; erythromycin resistant)/ml, 10(6) CFU of strain B (MIC of penicillin, 0.25 micro g/ml)/ml, and 10(5) CFU of strain C (MIC of penicillin, 4 micro g/ml)/ml, was used in the two mouse models, and a mixture of 10(5) CFU of strain A/ml, 10(4) CFU of strain B/ml, and 10(3) CFU of strain C/ml was used in the rabbit tissue cage model. During the different treatment regimens, the differences in numbers of CFU between treated and control animals were calculated to measure the efficacies of the regimens. Selective media with erythromycin or different penicillin concentrations were used to quantify the strains separately. The efficacies of penicillin in vitro were similar when individual strains or mixed cultures were studied. The eradication of the bacteria, independent of the susceptibility of the strain or strains or the presence of the strains in a mixture or on their own, followed the well-known PK and PD rules for treatment with beta-lactams: a maximum efficacy was seen when the T(>MIC) was >40 to 50% of the observation time and the ratio of the maximum concentration of the drug in serum to the MIC was >10. It was possible in all three models to select for the less-susceptible strains by using insufficient treatments. In the rabbit tissue cage model, a regrowth of pneumococci was observed; in the mouse thigh model, the ratio between the different strains changed in favor of the less-susceptible strains; and in the mouse peritonitis model, the susceptible strain disappeared and was overgrown by the less-susceptible strains. These findings with the experimental infection models confirm the importance of eradicating all the bacteria taking part in the infectious process in order to avoid selection of resistant clones.

Evaluation of fusidic acid in therapy of experimental Staphylococcus aureus meningitis.

OBJECTIVES: Combination therapy that includes fusidic acid, an antimicrobial agent highly active against staphylococci, has been recommended in the treatment of patients with Staphylococcus aureus meningitis. The aim of this study was to evaluate the pharmacokinetic, CSF bactericidal and anti-inflammatory properties of fusidic acid. METHODS: The pharmacokinetics, treatment efficacy and parameters of the meningeal inflammatory response were studied in rabbits, using an experimental meningitis model against S. aureus (MICs of fusidic acid and methicillin were 0.125 and 1 mg/L, respectively). RESULTS: Fusidic acid entered the CSF, with peak values within 0.5-1 h of the intravenous bolus injection/infusion and with a percentage penetration (AUCCSF/AUCserum) into uninfected and purulent CSF of 1.9% +/- 0.7 and 4.5% +/- 0.7, respectively. Rabbits treated with antibiotics [fusidic acid 80 mg/kg/6 h (n = 6), methicillin 80 mg/kg/3 h (n = 7) and the two combined (n = 6)] had significantly higher bacterial kill rates than untreated controls (n = 6, P < 0.05). Combination therapy was less effective, with significantly less killing after 6 h of treatment than methicillin alone (P < 0.05). CSF white blood cells and CSF levels of interleukin-8 (IL-8), glucose, lactate and protein were altered during staphylococcal meningitis, but with no significant difference between antibiotic-treated and untreated rabbits. CONCLUSIONS: Antagonism between methicillin and fusidic acid was observed in staphylococcal meningitis.