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Herein, we report the hit-to-lead identification of a drug-like pleuromutilin conjugate 16 , based on a triaromatic hit reported in 2020. The lead arose as the clear candidate from a hit-optimization campaign in which Gram-positive antibacterial activity, solubility, and P-gp affinity were optimized. Conjugate 16 was extensively evaluated for its in vitro ADMET performance which, apart from solubility, was overall on par with lefamulin. This evaluation included Caco-2 cell permeability, plasma protein binding, hERG inhibition, cytotoxicity, metabolism in microsomes and CYP3A4, resistance induction, and time-kill kinetics. Intravenous pharmacokinetics of 16 proved satisfactory in both mice and pigs; however, oral bioavailability was limited likely due to insufficient solubility. The in vivo efficacy was evaluated in mice, systemically infected with Staphylococcus aureus , where 16 showed rapid reduction in blood bacteriaemia. Through our comprehensive studies, lead 16 has emerged as a highly promising and safe antibiotic candidate for the treatment of Gram-positive bacterial infections.

Background: The recommended duration of antimicrobial treatment for Staphylococcus aureus bacteremia (SAB) is a minimum of 14 days. We compared the clinical outcomes of patients receiving short-course (SC; 6-10 days), or prolonged-course (PC; 11-16 days) antibiotic therapy for low-risk methicillin-susceptible SAB (MS-SAB)., Methods: Adults with MS-SAB in 1995-2018 were included from 3 independent retrospective cohorts. Logistic regression models fitted with inverse probability of treatment weighting were used to assess the association between the primary outcome of 90-day mortality and treatment duration for the individual cohorts as well as a pooled cohort analysis., Results: A total of 645, 219, and 141 patients with low-risk MS-SAB were included from cohorts I, II, and III. Median treatment duration in the 3 SC groups was 8 days (interquartile range [IQR], 7-10), 9 days (IQR, 8-10), and 8 days (IQR, 7-10). In the PC groups, patients received a median therapy of 14 days (IQR, 13-15), 14 days (IQR, 13-15), and 13 days (IQR, 12-15). No significant differences in 90-day mortality were observed between the SC and PC group in cohort I (odds ratio [OR], 0.85 [95% confidence interval {CI}, .49-1.41]), cohort II (OR, 1.24 [95% CI, .60-2.62]), or cohort III (OR, 1.15 [95% CI, .24-4.01]). This result was consistent in the pooled cohort analysis (OR, 1.05 [95% CI, .71-1.51]). Furthermore, duration of therapy was not associated with the risk of relapse., Conclusions: In patients with low-risk MS-SAB, shorter courses of antimicrobial therapy yielded similar clinical outcomes as longer courses of therapy., (© The Author(s) 2021. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Introduction. Cefuroxime is an important antibiotic to treat several serious infections. Rapid elimination through the kidneys and the variation in MICs of various susceptible pathogens such as Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus and Streptococcus pneumoniae give rise to dosing issues, especially in otherwise healthy patients. Aim. To investigate the probability of target attainment (PTA) for obtaining the optimal dosage regimens for cefuroxime in healthy young people. Methodology. Two weeks apart 750 and 1500 mg cefuroxime were administered as an intravenous bolus to 20 healthy volunteers (mean age: 27 years). Population modelling and simulation studies were done based on the obtained data for cefuroxime plasma concentration. Results. With a target value of time above MIC ( T >MIC ) greater than 50 % the simulations revealed that a PTA of >99 % is obtained for S. pneumoniae with a dosage regimen of 750 mg q12h. For E. coli and K. pneumoniae the PTA was <90 % even with the highest, simulated dosage of 1500 mg q6h. For S. aureus a dosage of 1500 mg q8h gave a PTA above 97 %. Conclusions. S. pneumoniae is most likely treatable with a two-daily dose of 750 mg cefuroxime. Not treatable are K. pneumoniae and E. coli . For S. aureus 1500 mg q8h constitutes an optimal dosing schedule.

Background: Staphylococcus aureus bacteremia (SAB) is frequently encountered in the hospital setting, and current guidelines recommend at least 14 days of antibiotic treatment for SAB in order to minimize risks of secondary deep infections and relapse. However, evidence to support these treatment recommendations remains scarce. Patients with uncomplicated SAB are known to have a low of risk of recurrence and death. Reducing treatment length in uncomplicated SAB would reduce the total consumption of antibiotics, duration of hospital admission, and potentially the risk of adverse events. With SAB7 we seek to determine if 7 days of antibiotic treatment in patients with uncomplicated SAB is non-inferior to 14 days of treatment., Methods/design: The study is designed as a randomized, non-blinded, non-inferiority, multicenter interventional study. Primary measure of outcome will be 90-day survival without clinical or microbiological failure to treatment or relapse. Secondary outcomes include the prevalence of severe adverse effects, in particular secondary infection with Clostridium difficile, all-cause mortality, as well as public health related costs. Patients identified with uncomplicated SAB who have received 7 days of protocol-approved antibiotics will be eligible for inclusion and randomized 1:1 in two parallel arms to either (i) discontinue antibiotic treatment at day 7 or (ii) to continue antibiotic treatment for a total of 14 days. Main exclusion criteria include signs of complicated SAB, such as the presence of secondary deep infections, persistent bacteremia, and implantable devices. Patients are followed for 6 months with clinical examinations, consecutive blood tests, and registration of adverse events. A total of 284 patients are to be included at ten centers across Denmark. The primary endpoint will be tested with a statistical non-inferiority margin of 10 percentage points., Discussion: SAB 7 will determine if 7 days of antibiotic treatment in patients with uncomplicated SAB is sufficient and safe. Results of the study will provide important knowledge on optimized SAB management and could potentially modify the current treatment recommendations., Trial Registration: ClinicalTrails.gov, H-17027414 . Registered on May 2, 2018. The Danish Medicines Agency (EudraCT), 2017-003529-13. Registered on October 30, 2017.

Development of antimicrobial resistance has been assigned to excess and misuse of antimicrobial agents. Staphylococci are part of the normal flora but are also potential pathogens that have become essentially resistant to many known antibiotics. Resistances in coagulase negative staphylococci (CoNS) are suggested to evolve due to positive selective pressure following antibiotic treatment. This study investigated the presence of the nine most commonly used antimicrobial agents in human urine from outpatients in two hospitals in Ghana in relation to CoNS resistance. Urine and CoNS were sampled (n = 246 and n = 96 respectively) from patients in two hospitals in Ghana. CoNS were identified using Gram staining, coagulase test, and MALDI-TOF/MS, and the antimicrobial susceptibility to 12 commonly used antimicrobials was determined by disk diffusion. Moreover an analytical method was developed for the determination of the nine most commonly used antimicrobial agents in Ghana by using solid-phase extraction in combination with HPLC-MS/MS using electron spray ionization. The highest frequency of resistance to CoNS was observed for penicillin V (98%), trimethoprim (67%), and tetracycline (63%). S. haemolyticus was the most common isolate (75%), followed by S. epidermidis (13%) and S. hominis (6%). S. haemolyticus was also the species displaying the highest resistance prevalence (82%). 69% of the isolated CoNS were multiple drug resistant (≧ 4 antibiotics) and 45% of the CoNS were methicillin resistant. Antimicrobial agents were detected in 64% of the analysed urine samples (n = 121) where the most frequently detected antimicrobials were ciprofloxacin (30%), trimethoprim (27%), and metronidazole (17%). The major findings of this study was that the prevalence of detected antimicrobials in urine was more frequent than the use reported by the patients and the prevalence of resistant S. haemolyticus was more frequent than other resistant CoNS species when antimicrobial agents were detected in the urine.

Objectives: Penicillin-susceptible Staphylococcus aureus isolates account for a fifth of cases of S. aureus bacteraemia (SAB) in Denmark, but little is known about treatment outcomes with penicillins or other antimicrobials. Here we compare penicillin, dicloxacillin and cefuroxime as definitive treatments in relation to 30 day mortality., Methods: A retrospective chart review of 588 penicillin-susceptible S. aureus cases at five centres from January 1995 to December 2010. Data on demographics, antimicrobial treatment, clinical signs and symptoms, and mortality at day 30 were collected. Hazard ratios (HRs) with 95% CIs associated with mortality were modelled using propensity-score-adjusted Cox proportional hazards regression analysis. Propensity-score-matched case-control studies were carried out., Results: Definitive therapy with cefuroxime was associated with an increased risk of 30 day mortality compared with penicillin (adjusted HR 2.54, 95% CI 1.49-4.32). Other variables that were statistically significantly associated with 30 day mortality included increasing age, disease severity and a primary respiratory focus. Osteomyelitis/arthritis was associated with a lower risk of death than were other secondary manifestations. Propensity-score-matched case-control studies confirmed an increased risk of 30 day mortality: cefuroxime treatment (39%) versus penicillin treatment (20%), P = 0.037; and cefuroxime treatment (38%) versus dicloxacillin treatment (10%), P = 0.004., Conclusions: Definitive therapy for penicillin-susceptible SAB with cefuroxime was associated with a significantly higher mortality than was seen with therapy with penicillin or dicloxacillin.

Objectives: Denmark and several other countries experienced the first epidemic of methicillin-resistant Staphylococcus aureus (MRSA) during the period 1965-75, which was caused by multiresistant isolates of phage complex 83A. In Denmark these MRSA isolates disappeared almost completely, being replaced by other phage types, predominantly only penicillin resistant. We investigated whether isolates of this epidemic were associated with a fitness cost, and we employed a mathematical model to ask whether these fitness costs could have led to the observed reduction in frequency., Methods: Bacteraemia isolates of S. aureus from Denmark have been stored since 1957. We chose 40 S. aureus isolates belonging to phage complex 83A, clonal complex 8 based on spa type, ranging in time of isolation from 1957 to 1980 and with various antibiograms, including both methicillin-resistant and -susceptible isolates. The relative fitness of each isolate was determined in a growth competition assay with a reference isolate., Results: Significant fitness costs of 2%-15% were determined for the MRSA isolates studied. There was a significant negative correlation between number of antibiotic resistances and relative fitness. Multiple regression analysis found significantly independent negative correlations between fitness and the presence of mecA or streptomycin resistance. Mathematical modelling confirmed that fitness costs of the magnitude carried by these isolates could result in the disappearance of MRSA prevalence during a time span similar to that seen in Denmark., Conclusions: We propose a significant fitness cost of resistance as the main bacteriological explanation for the disappearance of the multiresistant complex 83A MRSA in Denmark following a reduction in antibiotic usage.

Antibiotic treatment of Staphylococcus aureus infections is often problematic due to the slow response and recurrences. The intracellular persistence of the staphylococci offers a plausible explanation for the treatment difficulties because of the impaired intracellular efficacies of the antibiotics. The intra- and extracellular time- and concentration-kill relationships were examined in vitro with THP-1 cells and in vivo by use of a mouse peritonitis model. The in vivo model was further used to estimate the most predictive pharmacokinetic/pharmacodynamic (PK/PD) indices (the ratio of the maximum concentration of drug in plasma/MIC, the ratio of the area under the concentration-time curve/MIC, or the cumulative percentage of a 24-h period that the free [f] drug concentration exceeded the MIC under steady-state pharmacokinetic conditions [fT(MIC)]) for dicloxacillin (DCX) intra- and extracellularly. In general, DCX was found to have similar intracellular activities, regardless of the model used. Both models showed (i) the relative maximal efficacy (1-log-unit reduction in the numbers of CFU) of DCX intracellularly and (ii) the equal relative potency of DCX intra- and extracellularly, with the MIC being a good indicator of the overall response in both situations. Discordant results, based on data obtained different times after dosing, were obtained from the two models when the extracellular activity of DCX was measured, in which the in vitro model showed a considerable reduction in the number of CFU from that in the original inoculum (3-log-unit decrease in the number of CFU after 24 h), whereas the extracellular CFU reduction achieved in vivo after 4 h did not exceed 1 log unit. Multiple dosing of DCX in vivo revealed increased extra- and intracellular efficacies (2.5 log and 2 log units of reduction in the numbers of CFU after 24 h, respectively), confirming that DCX is a highly active antistaphylococcal antibiotic. PK/PD analysis revealed that fT(MIC) is the index that is the most predictive of the outcome of infection both intra- and extracellularly.

Antibiotic treatment of Staphylococcus aureus infections is often problematic due to the slow response to therapy and the high frequency of infection recurrence. The intracellular persistence of staphylococci has been recognized and could offer a good explanation for these treatment difficulties. Knowledge of the interplay between intracellular antibiotic activity and the overall outcome of infection is therefore important. Several intracellular in vitro models have been developed, but few experimental animal models have been published. The mouse peritonitis/sepsis model was used as the basic in vivo model exploring a quantitative ex vivo extra- and intracellular differentiation assay. The intracellular presence of S. aureus was documented by electron microscopy. Five antibiotics, dicloxacillin, cefuroxime, gentamicin, azithromycin, and rifampin (rifampicin), were tested in the new in vivo model; and the model was able to distinguish between their extra- and intracellular effects. The intracellular effects of the five antibiotics could be ranked as follows as the mean change in the log(10) number of CFU/ml (Delta log(10) CFU/ml) between treated and untreated mice after 4 h of treatment: dicloxacillin (3.70 Delta log(10) CFU/ml) > cefuroxime (3.56 Delta log(10) CFU/ml) > rifampin (1.86 Delta log(10) CFU/ml) > gentamicin (0.61 Delta log(10) CFU/ml) > azithromycin (0.21 Delta log(10) CFU/ml). We could also show that the important factors during testing of intracellular activity in vivo are the size, number, and frequency of doses; the time of exposure; and the timing between the start of infection and treatment. A poor correlation between the intracellular accumulation of the antibiotics and the actual intracellular effect was found. This stresses the importance of performing experimental studies, like those with the new in vivo model described here, to measure actual intracellular activity instead of making predictions based on cellular pharmacokinetic and MICs.

Background: Staphylococcus aureus is known to be a leading cause of bacteremia in childhood, and is associated with severe morbidity and increased mortality. To determine developments in incidence and mortality rates, as well as risk factors associated with outcome, we analyzed data from 1971 through 2000., Methods: Nationwide registration of S. aureus bacteremia (SAB) among children and adolescents from birth to 20 years of age was performed. Data on age, sex, source of bacteremia, comorbidity and outcome were extracted from discharge records. Rates were population adjusted and risk factors for death were assessed by multivariate logistic regression analysis., Results: During the 30-year study period, 2648 cases of SAB were reported. Incidence increased from 4.6 to 8.4 cases per 100,000 population and case-mortality rates decreased from 19.6% to 2.5% (P = 0.0001). Incidence in the infant age group (<1 year) were 10- to 17-fold greater compared with that in the other age strata and mortality rate was twice as high. Hospital-acquired infections dominated the infant group, accounting for 73.9%-91.0% versus 39.2%-50.5% in the other age groups. By multivariate analysis, pulmonary infection and endocarditis for all age groups, comorbidity for the older than 1 year, and hospital-acquired infections for the oldest group were independently associated with an increased risk of death., Conclusions: Mortality rates associated with SAB decreased significantly in the past 3 decades, possibly because of new and improved treatment modalities. However, incidence rates have increased significantly in the same period, underscoring that S. aureus remains an important invasive pathogen.

Staphylococcus aureus is a leading cause of bacteraemia. This study analysed temporal trends from 18,702 adult cases of S. aureus bacteraemia in Denmark between 1981 and 2000. After stratification for mode of acquisition, 57% of cases were hospital-acquired (HA), 28% were community-acquired (CA) and 15% were of undetermined acquisition (UA). Incidence rates increased from 18.2 to 30.5 cases/100,000 population. Annual rates increased by 6.4% for CA, by 2.2% for HA and by 3.6% for UA cases, respectively. Case-mortality associated with HA bacteraemia decreased from 36.2% to 20.7% (43% rate reduction, p 0.0001), compared with a decrease from 34.5% to 26.5% (23% rate reduction, p 0.0001) for CA bacteraemia. Following multivariate analysis, age, pneumonia, endocarditis and chronic illness were associated with increased mortality, regardless of the mode of acquisition. Overall, mortality associated with S. aureus bacteraemia declined significantly between 1981 and 2000, but incidence rates doubled, so that the total number of deaths increased. These data emphasise the public health importance of S. aureus bacteraemia and the need for further preventive measures and improved care in order to reduce incidence rates and improve outcomes.

In order to find a disk diffusion method with both high sensitivity and specificity for determination of methicillin resistance primarily for S. aureus but also for coagulase-negative staphylococci we screened several methodological variants using a material of 66 S. aureus comprising of 11 methicillin-susceptible, 18 borderline-resistant, and 37 methicillin-resistant strains. Only four of the combinations studied performed with both high sensitivity and specificity. Two of these, the Columbia agar +4.5% NaCl and Mueller Hinton agar +2% NaCl combined with a 5 microg oxacillin disk, confluent inoculum and 24 h incubation at 35 degrees C were further evaluated using 105 MRSA and 91 mecA-negative S. aureus and 193 clinical isolates of coagulase-negative staphylococci. The Columbia agar +4.5% NaCl performed excellently for both S. aureus and coagulase-negative staphylococci. For Columbia agar +4.5% NaCl using a 5 microg oxacillin disk we suggest an interpretive zone diameter of R < or =15 mm and S > or =16 mm for S. aureus and R < or =24 mm and S >or =26 mm for coagulase-negative staphylococci. The Mueller Hinton agar +2% NaCl performed well for coagulase-negative staphylococci but for S. aureus at least three (3%) very major errors were found, making this method less attractive.

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.

Infections caused by Staphylococcus aureus comprise relatively benign local skin infections, as well as serious generalised conditions. In Denmark, more than 85% of all S. aureus isolates are found resistant to penicillin, whereas resistance to methicillin is rare (< 1%) and therefore one of the penicillinase-stable penicillins is still the drug of choice. Dicloxacillin is usually chosen, because of its superior penicillinase stability. Infections caused by methicillin resistant strains may be treated with different combinations of macrolides, fusidic acid, aminoglycosides, fluoroquinolones, and rifampicin, but vancomycin is generally used. Newer drugs like the oxazolidinones and streptogramins are effective against methicillin-resistant strains and will be available in Denmark within a short time. Antibiotic treatment, however, is most often only a supplement to surgical intervention. This paper deals with the clinical picture and treatment of some common S. aureus infections.

Background: Staphylococcus aureus bacteremia is still a serious problem, and the optimal treatment is under debate. Only a few studies concerning treatment are available., Methods: The study population was all patients with a positive blood culture result for S aureus in Copenhagen County, Denmark, from May 1994 through April 1996. Of 278 patients with S aureus bacteremia, 186 were evaluated according to outcome in a prospective, observational follow-up study. The time above the minimum inhibitory concentration was estimated for dicloxacillin sodium for each treatment regimen and evaluated by logistic regression along with other potential risk factors., Results: The following variables were statistically associated with death: the presence of an uneradicated focus (odds ratio [OR], 6.7; 95% confidence interval [CI], 2.1-21.0); the presence of septic shock (OR, 3.7; 95% CI, 1.5-9.1); the total daily dose of penicillinase-stable penicillin less than 4 g (OR, 3.7; 95% CI, 1.3-11.1); and age 60 years or older (OR, 2.4; 95% CI, 1.1-5.3). The following variables were significantly associated with recurrence: the total daily dose of penicillinase-stable penicillin less than 3 g (OR, 3.9; 95% CI, 1.6-10.0) and the presence of a secondary focus (OR, 3.2; 95% CI, 1.3-7.7). Among 155 patients with observation time longer than duration of treatment, this factor (duration of treatment, <14 days) was significantly related to mortality (OR, 0.84; 95% CI, 0.76-0.94)., Conclusions: Focus eradication and the dosing of penicillinase-stable penicillin are important to the outcome of S aureus bacteremia. We recommend treatment with at least 1 g of penicillinase-stable penicillins 4 times daily for longer than 14 days.

The central S. aureus surveillance in Denmark made it possible to analyze the clinical features of S. aureus endocarditis in a nation-wide population of non-drug addicts. Almost all cases of bacteraemia with S. aureus are reported to the Staphylococcus laboratory, Copenhagen. The medical records were reviewed in cases from 1982 to 1991 in which the diagnosis of endocarditis was reported or suspected. Two hundred and sixty patients fulfilled the diagnostic criteria. In 83 patients the diagnosis of endocarditis was not suspected clinically. The overall mortality rate among those patients whose disease was diagnosed clinically was 46% and significantly associated with late congestive heart failure, age and involvement of the central nervous system. A more frequent use of echocardiography as a screening method seems essential to improve the prognosis of patients with S. aureus endocarditis. Involvement of the CNS constitutes a relative indication for early valve replacement.

Background: Staphylococcus aureus bacteremia (SAB) acquired in hospitals continues to be a frequent and serious complication to hospitalization, and no previous case-control studies dealing with risk factors of this severe disease are available., Methods: Based on a 1-year prospective analysis, the data from all patients with hospital-acquired SAB admitted to 4 hospitals in Copenhagen County, Denmark, from May 1, 1994, through April 30, 1995, were evaluated. Eighty-five patients with hospital-acquired SAB were matched to 85 control patients with a similar primary diagnosis at admission (matched controls). Of these, 62 patients with hospital-acquired SAB were compared with 118 other patients with a similar time of admission, who were randomly selected with no clinical evidence of SAB (unmatched controls)., Results: The incidence of hospital-acquired SAB was 0.71 per 1000 hospital admissions. The presence of a central venous catheter (odds ratio, 6.9; 95% confidence interval [CI], 2.8-17.0), anemia (odds ratio, 3.3; 95% CI, 1.4-7.6), and hyponatremia (odds ratio, 3.3; 95% CI, 1.5-7.0) was significantly associated with hospital-acquired SAB in a conditional and a usual logistic regression analysis. Nasal carriage was not an independent risk factor, but nasal carriers among patients in surgery (odds ratio, 4.0; 95% CI, 1.3-13.0) had a significantly higher risk for hospital-acquired SAB compared with matched and unmatched controls. The presence of hospital-acquired SAB increased the mortality rate 2.4-fold (95% CI, 1.1-5.2)., Conclusions: The presence of a central venous catheter is an important risk factor, and hyponatremia and anemia are associated with the development of hospital-acquired SAB. Furthermore, hospital-acquired SAB in itself increases mortality.

Background: Both morbidity and mortality resulting from Staphylococcus aureus endocarditis are known to be high, and the incidence of this disease seems to increase. The Statens Serum Institut, Copenhagen, Denmark, made it possible for us to analyze the clinical features of S aureus endocarditis in a nation-wide population of non-drug addicts., Methods: Almost all Danish cases of bacteremia due to S aureus are reported to the Staphylococcus laboratory, Statens Serum Institut. The medical records were reviewed in cases reported from 1982 to 1991 in which the diagnosis of endocarditis was reported or suspected., Results: A total of 260 patients, 145 males and 115 females, fulfilled the diagnostic criteria. The median age was 67.5 years. In 83 patients, the diagnosis of endocarditis was not suspected clinically. The overall mortality rate among those patients whose disease was diagnosed clinically was 46%. Among the subset of patients who received medical therapy only and appropriate antistaphylococcal treatment, mortality was significantly associated with late congestive heart failure, age, and involvement of the central nervous system., Conclusions: A raised awareness of the paucity of clinical findings and a more frequent use of echocardiography as a screening method seem essential to improve the prognosis of patients with S aureus endocarditis. Involvement of the central nervous system constitutes a relative indication of early valve replacement.

Background: The incidence of hematogenous Staphylococcus aureus osteomyelitis of the vertebral column is rapidly increasing and few studies dealing with the diagnosis, treatment, and outcome of this severe disease are available., Methods: Based on a nationwide registration, the clinical and bacteriological data were reviewed from 133 cases with a positive blood culture for S aureus and symptoms of vertebral osteomyelitis in Denmark for the period 1980 to 1990., Results: The 133 cases of vertebral S aureus osteomyelitis reviewed were mainly community-acquired infections (82%) in older patients (median age, 65 years) and often occurred with underlying diseases. Both symptoms and laboratory values were relatively unspecific. Bone scan methods proved to be more optimal for diagnosis of vertebral S aureus osteomyelitis in the early stages compared with conventional radiography that proved a lack of consistency in the formative stages. The infection was mostly (70%) localized in the lower part of the column. The recurrence rate and rate of therapeutic failure depended on the duration and dosage of penicillinase-stable penicillins, respectively. Patients treated with fusidic acid in addition to penicillinase-stable penicillins had a significantly lower recurrence rate. Based on these findings, we recommend treatment with penicillinase-stable penicillins and fusidic acid for a total of 8 weeks, with a daily dosage of penicillinase-stable penicillins higher than 4 g., Conclusions: The diagnosis of vertebral S aureus osteomyelitis based on clinical findings is difficult to ascertain. Bone scans are necessary because radiographic methods do not detect disease as early. Treatment with penicillinase-stable penicillins, at least 4 g/d for at least 8 weeks, is recommended.

Purpose: To investigate the neurologic manifestations of infective endocarditis caused by Staphylococcus aureus in a population of nondrug addicts with special emphasis on the clinical presentation, epidemiology, and mortality., Patients and Methods: During the period from 1982 to 1991 a total of 8,514 cases of bacteremia with S aureus were reported to the Staphylococcus Laboratory, Copenhagen, Denmark. The medical records of cases of suspected infective endocarditis were retrospectively reviewed and classified according to the new diagnostic criteria for endocarditis proposed by Durack., Results: A total of 260 cases from 63 hospitals fulfilled the diagnostic criteria. Overall, 91 patients (35%) experienced neurologic manifestations. Sixty-one presented with neurologic symptoms, whereas 30 patients developed neurologic complications at various intervals (median: 10 days) after the debut of the disease. The most frequent neurologic manifestation was unilateral hemiparesis, which occurred in 41 patients (45%). Forty-two percent of the females had neurologic manifestations compared to only 30% of the males (P = 0.06). Cases with native mitral valve infection had a significantly higher frequency of neurologic manifestations compared with all other valvular involvement (44% versus 29%, P = 0.02) but the frequency of neurologic complications was only nonsignificantly higher in those patients with native mitral valve infection than in those patients with native aortic valve infection (44% versus 31%, P = 0.10). Only two of the patients with tricuspid valve infection and none of those with congenital heart disorder experienced neurologic manifestations. A neurologic manifestation occurred in 22 (35%) of the 63 episodes in which vegetations were detected on the echocardiograms, compared with 17 (26%) of the 65 episodes without vegetations (P = 0.38). The mortality was 74% in patients with major neurologic manifestations and 56% in patients without neurologic manifestations (P = 0.008). In patients with neurologic complications the mortality was significantly higher among those treated with antibiotics alone as compared with those treated surgically (65 of 81, 80% versus 2 of 10, 20%; P = 0.0003)., Conclusions: In a population of nondrug addicts with infective endocarditis caused by S aureus the following main conclusions can be drawn: neurologic manifestations occur with a higher frequency in patients with native mitral valve infection. The presence of vegetations on echocardiograms is not a risk factor for developing neurologic complications but this conclusion is based on the results of transthoracic echocardiograms performed in only one half of the patients. The majority of the neurologic manifestations occur on presentation or shortly thereafter and the risk of recurrent embolism is low. Mortality is increased in patients with neurologic manifestations. A neurologic event per se may constitute an indication for surgical treatment.

From 1980 to 1990, 309 cases of haematogenous osteomyelitis were identified in Denmark. Haematogenous osteomyelitis of the vertebral column increased significantly (P < 0.01) from the first to the second half of the period due to an increased number of patients > 50 years of age with community-acquired infection. Vertebral osteomyelitis differed significantly from osteomyelitis of other bones in accordance to age distribution (median 66 vs. 16 years), male/female ratio (75/71 vs. 105/ 58) and patients with diabetes (13% vs. 6%). We found a higher risk of haematogenous osteomyelitis in patients > 50 years of age and among patients with community-acquired infection. The highest incidence (5%) of vertebral osteomyelitis in Staphylococcus aureus bacteraemia in this age group was found in cases without an identified portal of entry. The highest incidence (34%) of osteomyelitis of other bones was found in community-acquired cases in the age group 1-20 years and without an identified portal of entry. The present study discusses reasons for the continued increase of vertebral osteomyelitis among adults and describes incidence rates and major risk factors for developing haematogenous osteomyelitis among patients with S. aureus bacteraemia. We suggest that the localization of haematogenous S. aureus osteomyelitis is connected with the presence of red bone marrow.

A retrospective review of medical records from the Staphylococcus Laboratory, Copenhagen, 1982-1991, was carried out at the Department of Clinical Microbiology, Statens Serum Institut, 1994-1995, to investigate the clinical features and outcome of two subgroups of bacteremic Staphylococcus aureus endocarditis cases in non-drug addicts: patients with prosthetic valve endocarditis (PVE) and patients with native valve endocarditis treated surgically. Twenty-four cases of PVE were included. Six cases were early (within 60 days of valve implantation) and 18 were late. The overall in-hospital mortality was 42%. Surgical treatment resulted in a non-significantly lower mortality as compared with medical treatment alone (0% vs 50%, p = 0.19). Medical treatment of aortic and mitral valve endocarditis resulted in similar mortality rates (44% and 50%, respectively). Twenty-three cases of native valve infective endocarditis had the valve replaced surgically. The in-hospital mortality was 22%, which was significantly lower as compared with medical therapy (69%, p < 0.0001). The treatment changed significantly during the study period: 6 of 112 patients (5%) were treated surgically in the first half of the period (1982-1986) compared to 17 of 124 patients (14%) in the second half (1987-1991, p = 0.049). Severe congestive heart failure was the main indication for cardiac surgery in 21 patients. In conclusion, a shift towards a more aggressive surgical approach has taken place in the 10-year period. This development should be strengthened in the future as surgical intervention may improve survival in patients with infective endocarditis caused by S. aureus whether the infected valve is prosthetic or native.

In 34 rabbits, both tibiae were inoculated with Staphylococcus aureus. 14 legs received no treatment and served as controls. In 12 legs, the wound was treated with pure collagen and in 18 legs, collagen with gentamicin (Gentacoll) in a dose of 10 mg/kg body weight was applied to the wound before closure. Postoperatively 12 received 10 mg/kg body weight gentamicin intravenously and no local treatment. The animals were killed 7 days after inoculation and evaluated macroscopically and microbiologically for infection. 6 rabbits (12 legs) were used for pharmacokinetic studies only and they were killed after 2, 4, and 18 hours, respectively. 11/14 untreated legs developed a macroscopically acute osteomyelitis. No infection was found in the 18 legs treated with Gentacoll and 1/12 treated with gentamicin systemically had growth of the inoculated bacteria in tissue biopsies. The concentrations of gentamicin in the serum as well as locally reached peak values were well above the MIC value in all groups, with a maximum after 1-2 hours. No gentamicin could be detected after 18 hours, independently of the mode of administration.

In a retrospective study covering the years 1982-1989 episodes of Staphylococcus aureus endocarditis in 51 intravenous drug users were studied. Tricuspid involvement dominated (34/51), but the frequency of left-sided involvement (33.3%) was greater than in earlier reports. Involvement of both sides of the heart was not detected, but 27.8% of the left-sided endocarditis cases had multiple pulmonary infiltrates, indicating that some of them might have had a concomitant right-sided endocarditis. The 2 groups were compared: patients with left-sided endocarditis were significantly older and with a longer time of intravenous drug use. The complication rate was the same (44.1%) as was the duration of antibiotic treatment (median 42 days). In total, five patients underwent surgery, two (5.8%) due to right-sided failure and three (29.4%) because of left-sided endocarditis. The mortality of tricuspid endocarditis was low (2.9%), whereas 5 patients (29.4%) with left-sided involvement died. The patients who died were significantly older and had a shorter duration of symptoms before hospitalization.

This retrospective nationwide study of 756 cases of S. aureus bacteremia in hematological patients, between 1977-1990, confirms that S. aureus bacteremia is disproportionately prevalent among, and a major cause of morbidity and mortality in, hematological patients. The case mortality rate attributable to S. aureus bacteremia (44%) was significantly higher than the rate (29%) found in patients without hematological disease. A decreasing mortality was, however, observed from the first to the last part of the study period (53 and 37% respectively). A logistic regression analysis showed that mortality was further dependent on age, the type of hematological disease and origin of infection/primary focus of infection. Analysis of clinical features confirmed that in comparison with other patients, S. aureus bacteremia in hematological patients more often was hospital-acquired, acquired without known portal of infection/primary focus of infection and much less frequently resulted in osteomyelitis or endocarditis. From phage typing and antibiotic susceptibility patterns no specific types of S. aureus were associated with hematological patients.

A new mouse model of foreign body infection has been developed. Intraperitoneal placement of a silicone catheter followed by injection of 10(8) Staphylococcus aureus organisms resulted in a reproducible, localized foreign body infection. The infection persisted as an intra-abdominal abscess surrounding the catheter for at least 30 days. Treatment with up to nine doses of methicillin or gentamicin or both was started 3 days after infection. The treatment showed a significant effect (P < 0.05), measured as reduction of bacteria on the foreign body, for all three regimens with a reduction of up to 2 log units, but no synergism was observed. The result of the treatment was poor, despite the facts that the local concentrations of methicillin were greater than the MIC for at least 72 h and that nine peak concentrations of gentamicin of > 13 micrograms/ml were obtained. The poor result of the treatment was not caused by development of antibiotic resistance or influenced by protein concentration, pH, or local presence in the pus of inhibitors of antibiotics. Both antibiotics showed good effects in time-kill studies in vitro on bacteria on catheters taken out of infected mice and catheters infected in vitro. During treatment, the proportion of intracellular bacteria increased in all treated mice to 60 to 75% compared with 20 to 30% in nontreated mice (P < 0.05). This indicates that intracellular survival of staphylococci may influence the outcome of the treatment in foreign body infections.

Methods: Based on a nationwide registration, the clinical and bacteriologic data from 61 postoperative and 43 hematogenous cases of Staphylococcus aureus meningitis in Denmark from 1986 through 1989 were reviewed., Results: Postoperative meningitis was a foreign body infection in 89% of the cases and had a lower mortality (18% [11/61]) compared with hematogenous meningitis (56% [24/43]). Hematogenous S aureus meningitis seems to be part of an overwhelming, disseminated infection as indicated by the following: 81% of the patients had bacteremia, 21% had endocarditis, and 12% had osteomyelitis. Most patients were older, often with underlying diseases, community-acquired infections, and a clinical picture of severe meningitis. The major findings were mental status changes and a high rate (34%) of focal neurological changes. The initial leukocyte count in the cerebrospinal fluid sample was low, and the bacteria were seen in Gram's stain smears in 40% of cases only. The prognosis was related to the age of the patients and the initial antibiotic treatment. Patients treated with penicillinase-stable penicillins in combination with fusidic acid may have a better prognosis. Three (12%) of 25 surviving patients had severe sequelae., Conclusions: Hematogenous S aureus meningitis is a severe disease with a high mortality related to age, presence of shock, and infection with strains of phage type 95.

Among 3394 patients with Staphylococcus aureus bacteraemia from the years 1986-89, 88 patients were found whose intravenous catheter and blood grew organisms of the same phage type. Strains of phage type 95 were more frequent among the patients with confirmed catheter-related bacteraemia than among other bacteraemia cases. Strains with particular phage-type patterns occurring with increasing frequency in Denmark during recent years also occurred with significantly higher frequencies among the confirmed catheter-related bacteraemias. No major differences in antibiotic resistance were observed. Patients with catheter-related bacteraemia had, in spite of a higher frequency of underlying diseases, a lower mortality compared with other bacteraemia patients, and endocarditis occurred less frequently (2% vs. 6%). Among 201 S. aureus isolates from catheters in 1988 only strains of group I occurred with increased frequency. The possible role of catheters as selection pressure on the S. aureus population is discussed.

Of the 15,170 cases of bacteremia due to Staphylococcus aureus that occurred in Denmark between 1959 and 1988, we review 525 cases of acute hematogenous osteomyelitis and 185 cases of septic arthritis that developed subsequent to the bacteremia and 134 cases of contiguous osteomyelitis in which the bacteremia developed secondarily. The pattern of acute infections of bones and joints has changed over the three decades studied. The frequency of secondary bone or joint infections due to S. aureus bacteremia has changed, as have the phage-type pattern and antibiotic resistance of the infective strains. The prevalence of hospital-acquired cases has increased and the age distribution of patients has changed, as is reflected in an increasing number of older patients. The localization of hematogenous osteomyelitis has shifted, and the vertebral column is now the most common site of infection. The rate of chronic cases of osteomyelitis that occur following acute hematogenous osteomyelitis has been reduced from 34% to 6%. The mortality associated with S. aureus bacteremic infections of bones or joints is low compared to that associated with other cases of S. aureus bacteremia.

Human serum and plasma have been demonstrated to enhance mortality in Staphylococcus aureus intraperitoneal infection in mice. Two different mechanisms seem to be involved. The effect of serum could be removed by adsorption to S. aureus protein A coupled to Sepharose 4 B and could be reconstituted by the addition of human IgG to IgG-depleted serum. Plasma diluted 1/10 in combination with purified fibrinogen also enhanced mouse mortality. Both effects could be demonstrated, when a coagulase-free variant of S. aureus was used. The results of viable counts of S. aureus in blood and peritoneum of the mice indicate that the enhancing effect of IgG alone and the enhancing effect of fibrinogen in combination with diluted plasma have different mechanisms.

Staphylococcus aureus endocarditis cases in Denmark from 1976 to 1981 were reviewed. A total of 119 patients--61 female and 58 male, with a median age of 63 years (range, 1 month to 85 years)--fulfilled the diagnostic criteria. Community-acquired infections were most common (62%), but the frequency of hospital-acquired cases (38%) was greater than in earlier reports. The clinical picture was relatively nonspecific, and 32% of the patients had no heart murmurs initially. In 65 cases (55%), endocarditis was not suspected clinically, and the diagnosis was first obtained at autopsy. The mortality was 71% and correlated with age, hospital-acquired infection, and the presence of heart failure and arterial embolism.

Clinical and bacteriological information of Staphylococcus aureus endocarditis was reviewed in 119 cases from all over Denmark. Overall mortality was 71%. Survival correlated with antistaphylococcal treatment, short duration from onset of infection to start of treatment, and long duration of treatment. In spite of relevant treatment, mortality was significantly lower in cases infected with penicillin-susceptible strains than when penicillin-resistant strains were isolated. There were no differences in the effect of various anti-staphylococcal treatment regimens; in particular, there were no differences in mortality with regard to beta-lactam antibiotics alone as compared to beta-lactam antibiotics in combination with aminoglycosides. However, embolic manifestations occurred more often after start of treatment with combination therapy than with beta-lactam antibiotics alone.

The bacteriology, epidemiology, infection types and mortality were reviewed in 407 cases of Staphylococcus aureus bacteraemia in the period 1967-1984 in children less than one year old. The number of bacteraemia cases in this age group increased in the seventies, but only 4% of the bacteraemias seemed to be due to epidemic spreading. Neonates might be at higher risk for S. aureus bacteraemia than older children less than one year old, and nearly all infections in the neonatal period were hospital-acquired. The mortality was 24%, and did not correlate with type of infection, but in neonates it correlated with low birthweight.

A total of 6,253 cases of Staphylococcus aureus bacteremia, including 274 (4.4%) endocarditis cases, were registered in Denmark in the period 1975-1984. Patients with hematological malignancies and/or agranulocytosis accounted for 479 of the bacteremia cases. The incidence of endocarditis in this group of patients was only 0.4% as compared to 4.7% in other patients with staphylococcal bacteremia (p less than 0.01). The lower incidence of endocarditis complicating bacteremia in these patients may justify a shorter course of therapy than usually recommended for suspected endocarditis. Patients with hematological malignancies and other patients with agranulocytosis had a higher mortality (49 and 46%, respectively) than other patients with S. aureus bacteremia (33%). The highest mortality was found in patients with multiple myeloma (71%, p less than 0.01), the lowest in patients with acute lymphocytic leukemia (28%, p less than 0.01). The higher mortality in these patients may indicate that empiric antibiotic regimens in granulocytopenic patients should include a specific anti-staphylococcal agent.

Clinical and bacteriological information on Staphylococcus aureus endocarditis from hospitals all over Denmark in the period 1976-1981 was reviewed in 119 cases, 61 females and 58 males. Patient ages ranged from 16 days to 85 years, with a median age of 63 years. The overall mortality was 71%. The mortality correlated significantly with such factors as age, hospital-acquired infections and resistance to penicillin in infecting strains. Hospital-acquired infections occurred in 38% of the patients. The distribution of phage types among strains isolated from blood cultures from patients with endocarditis corresponded to that of strains from other septicaemia cases. Group I and group III strains and strains of the 94, 96 complex comprised 74% of the phage types of the present material. Infections of the skin were the most common portal of entry for the infecting strains. Apart from drug addicts, of which 11 cases were included, mortality did not correlate with the presence of any underlying diseases.

We investigated the diffusion of penicillin-G, cloxacillin, clindamycin, and netilmicin into synovial fluid and membrane in rabbits. Purulent arthritis was induced in the right knee of each rabbit by inoculation of Staphylococcus aureus phage type 3C, whereas sterile saline was injected into the left knee to serve as a control. Two days later, concentrations of antibiotics were determined in serum, synovial fluid, and membrane after an intramuscular single dose. All four drugs diffused readily into infected joints, whereas the corresponding concentrations in the normal joints were 2-3 times lower. Clindamycin showed the highest intraarticular penetration, cloxacillin the lowest. The lower penetration of cloxacillin corresponded to its higher protein binding in rabbit serum. Considering the sufficient local concentrations achieved, parenteral treatment obviates the need for local instillation of these antibiotics.

We treated septic arthritis of the knee in 38 rabbits with cloxacillin i.m. once and twice daily combined with probenecid for 7 or 21 days, respectively, or with only cloxacillin i.m. thrice daily for 7 days. The animals were killed weekly in groups up to 5 weeks after inoculation. Aspirated cultures obtained after 4 days of treatment were always negative. Histologic specimens revealed progressive joint destruction, but at a slower rate after frequent treatment independent of the period. We concluded that antibiotic therapy alone could not prevent destruction of articular cartilage once bacterial arthritis was established.