The pharmacodynamics of minocycline alone and in combination with rifampicin against Staphylococcus aureus studied in an in vitro pharmacokinetic model of infection.

BACKGROUND: Tetracyclines are widely used as oral therapy of MRSA infection, however, the pharmacodynamic underpinning is absent. OBJECTIVES: We employed an in vitro pharmacokinetic model to study the pharmacodynamics of minocycline alone and in combination with rifampicin. METHODS: An exposure-ranging design was used to establish fAUC/MIC targets for static, -1 log drop and -2 log drop effects against Staphylococcus aureus for minocycline and in combination with rifampicin. We then simulated 7-10 day human dosing of minocycline and the combination. RESULTS: The minocycline fAUC/MIC for 24 h static effect and -1 log drop in bacterial load were 12.5 ±â€Š7.1 and 23.3 ±â€Š12.4. fAUC/MIC targets for static and -1 log drop were greater at 48 and 72 h. The addition of simulated free rifampicin associated with dosing 300 mg q12h reduced the 24 h minocycline fAUC/MICs. Simulations performed over 7-10 days exposure indicated that for minocycline standard human doses there was a 1-3 log reduction in viable count and no changes in population profiles. Addition of rifampicin resulted in larger reductions in staphylococcal load but emergence of resistance to rifampicin. There was no resistance to minocycline. CONCLUSIONS: An fAUC/MIC minocycline target of 12-36 is appropriate for S. aureus. Addition of rifampicin decreases bacterial load but results in emergence of resistance to rifampicin. Unusually, there was no emergence of resistance to minocycline.

In vitro pharmacodynamics of omadacycline against Escherichia coli and Acinetobacter baumannii.

BACKGROUND: The pharmacodynamics of omadacycline have been extensively studied against Gram-positive pathogens but less information is available for Gram-negative pathogens. We describe the pre-clinical pharmacodynamics of omadacycline against Escherichia coli and Acinetobacter baumannii. METHODS: An in vitro dilutional pharmacokinetic model was used. Exposure experiments with fAUC/MIC ratios ranging from 0 to 1200 were performed using five strains of E. coli and five strains of A. baumannii. Reduction in bacterial load and changes in population profiles were measured. RESULTS: The fAUC/MIC targets against E. coli for 24 h static and -1 log reduction in load were 25.3 ±â€Š17.2 and 42.7 ±â€Š32.5, respectively. For A. baumannii the fAUC/MIC for 24 h static effect was 108.1 ±â€Š38.6. Changes in population profiles were observed for E. coli at fAUC/MIC ratios of ≤200 and for A. baumannii up to 1200. MICs were increased 2-32 fold. CONCLUSIONS: fAUC/MIC targets for A. baumannii are greater than for E.coli and changes in population profiles more likely. E. coli fAUC/MIC targets align with in vivo data and will be useful in determining omadacycline dosing for this pathogen.

An in vitro biofilm model of Staphylococcus aureus infection of bone.

Chronic osteomyelitis is difficult to treat, with biofilm growth and the diffusion barrier to antibiotics presented by bone contributory factors. The aim of this study was to develop and evaluate an in vitro model of osteomyelitis. A bioluminescent strain of Staphylococcus aureus was grown in bone blocks made from bovine femur. Light output was insufficient for detection of bacterial cells within bone by 24 h and viable counting of crushed bone blocks was used to determine bacterial survival. Challenge of 72 h biofilms with gentamicin and daptomycin for 24 h demonstrated that only concentrations of 10 times the clinical peak serum target levels (100 mg l-1 gentamicin and 1000 mg l-1 daptomycin) resulted in significant reductions in cell viability compared to controls. Once daily dosing over 7 days resulted in ≥3 log reductions in cell numbers by 48 h. Thereafter no significant reduction was achieved, although emergence of resistance was suppressed. Determination of antibiotic concentration in bone blocks over 7 days indicated that neither agent was able to consistently reach levels in bone of >10% of the original dose. The model was, therefore, able to demonstrate the challenges posed by biofilm growth on and within bone. SIGNIFICANCE AND IMPACT OF THE STUDY: The majority of studies of antibiotic efficacy in the treatment of chronic osteomyelitis are carried out in animals. We developed an in vitro model of Staphylococcus aureus infection of bone to evaluate the ability of antibiotics to eradicate mature biofilms on surfaces analogous to necrotic bone. The results demonstrated the difficulties which occur in osteomyelitis treatment, with only very high concentrations of antibiotic able to penetrate the bone sufficiently to reduce bacterial survival whilst still failing to eradicate biofilms. This model could be of use in initial screening of novel compounds intended for use in the treatment of osteomyelitis.

Pharmacodynamics of taniborbactam in combination with cefepime studied in an in vitro model of infection.

OBJECTIVES: To define the in vitro pharmacodynamics of taniborbactam against Enterobacterales with CTXM-15, KPC, AmpC, and OXA-48 ß-lactamases. METHODS: An in vitro pharmacokinetic model was used to simulate serum concentrations associated with cefepime 2G by 1 h infusion 8 h. Taniborbactam was given in exposure ranging and fractionation simulations. Reduction in viable count at 24 h (Δ 24) was the primary end point and four strains were used: Escherichia coli expressing CTXM-15 or AmpC and Klebsiella pneumoniae expressing KPC or OXA-48 enzymes. RESULTS: Taniborbactam was administered as continuous infusions; ≥4 log kill was attained with taniborbactam concentrations of ≥0.01 mg/L against CTXM-15 E. coli, ≥0.5 mg/L against KPC- and OXA-48 K. pneumoniae, and ≥4 mg/L against AmpC E. coli. Analyses were conducted to determine the pharmacokinetic/dynamic driver for each strain. For E. coli (CTXM-15) and E. coli (AmpC), area under the concentration-time curve (AUC) was best related to change in viable count (R20.74 and 0.72, respectively). For K. pneumoniae (KPC) AUC and T > 0.25 mg/L were equally related to bacterial clearance (R20.72 for both), and for K. pneumoniae (OXA-48) T > 0.25 mg/L was the best predictor (R20.94). The taniborbactam AUC range to produce a 1-log10 reduction in viable count was 4.4-11.2 mg·h/L. Analysis of data from all strains indicated T > MIC divided by 4 was best related to change in viable count; however, curve fit was poor R2 < 0.49. CONCLUSIONS: Taniborbactam was effective in combination with cefepime in producing bacterial clearance for B lactam resistant Enterobacterales. The primary pharmacodynamic driver was AUC or time > threshold, both being closely related to antibacterial effect.

Comparative antibacterial effects of moxifloxacin and levofloxacin on Streptococcus pneumoniae strains with defined mechanisms of resistance: impact of bacterial inoculum.

OBJECTIVES: We aim to further define the impact of the mechanism of fluoroquinolone resistance and inoculum load on the pharmacodynamic effects of levofloxacin and moxifloxacin on Streptococcus pneumoniae. METHODS: The antibacterial effects of and emergence of resistance (EoR) to moxifloxacin (400 mg once daily) or levofloxacin (750 mg once daily or 500 mg twice daily) were compared using five S. pneumoniae strains containing no known resistance mechanisms, efflux resistance mechanisms, a parC mutation or parC and gyrA mutations, at high (10(8) cfu/mL) and low (10(6) cfu/mL) inocula. An in vitro pharmacokinetic model was used and simulations were performed over 96 h. After drug exposure, isolates were tested for the presence of efflux pumps and mutations in the quinolone resistance-determining regions. RESULTS: A high inoculum diminished the antibacterial effect of moxifloxacin and levofloxacin. Levofloxacin at both dosages produced EoR with all strains. Levofloxacin regimens with AUC/MIC ratios <100 produced EoR. Moxifloxacin produced EoR with the parC strain only. CONCLUSIONS: Levofloxacin dosing regimens with low AUC/MIC ratios select for efflux pump overexpression, leading to fluoroquinolone resistance. Levofloxacin dosing may select for gyrA mutations, inducing moxifloxacin resistance. These data confirm that a fluoroquinolone AUC/MIC ratio of >100 is required for prevention of EoR.

Lack of upward creep of glycopeptide MICs for methicillin-resistant Staphylococcus aureus (MRSA) isolated in the UK and Ireland 2001-07.

OBJECTIVES: There have been several reports of upward creep in vancomycin MICs for Staphylococcus aureus [predominantly methicillin-resistant S. aureus (MRSA)] over recent years, but only in single centres or using contemporaneous results. We aimed to test the hypothesis of MIC creep in a multicentre study, testing all the isolates concurrently. METHODS: Nineteen laboratories in the UK and Ireland contributed isolates from blood to the BSAC Bacteraemia Resistance Surveillance Programme every year between 2001 and 2007. MICs for 271 MRSA from these sites were re-measured at a single central laboratory during a single week by the BSAC agar dilution method, but with √2-fold instead of conventional 2-fold dilutions. Re-test results were compared with the original results obtained each year at the same central laboratory. RESULTS: The re-test results were much less variable than the original results and avoided the confounding of experimental variation with year of collection. They demonstrated statistically significant but very slow downward trends in MICs of vancomycin and teicoplanin, at 0.027 and 0.055 doubling dilutions/year, respectively. The original results had suggested more rapid trends in MICs, upward for vancomycin and downward for teicoplanin. The proportion of EMRSA-16 fell from 21% to 9% over the study period, while EMRSA-15 rose from 76% to 85%. CONCLUSIONS: Historical data can give a misleading impression of trends in MIC values because of experimental variation between tests conducted at different times. There was no upward creep in glycopeptide MICs for MRSA in the UK and Ireland between 2001 and 2007.

Role of early intravenous to oral antibiotic switch therapy in the management of prosthetic hip infection treated with one- or two-stage replacement.

OBJECTIVES: To determine outcomes for an antibiotic regimen using early switch to oral antibiotics for treatment of infected total hip replacement (THR) treated by either a one-stage or two-stage procedure. METHODS: Cases of infected THR were identified from the microbiology records on all orthopaedic infections in a 24 month period. Diagnosis was made by microbiological culture of theatre specimens and findings at the time of surgery. A standard approach of 10-14 days intravenous (iv) antibiotic followed by a switch to oral antibiotics either for 6-8 weeks until second-stage re-implantation or for up to 3 months following a one-stage procedure was used. The exact date of oral switch and antibiotic duration was determined by clinical resolution and C-reactive protein (CRP). Outcome was recorded as no microbiological or clinical evidence of relapse of infection or relapse after completing the antibiotic course. Follow-up duration for all cases at the time of study was 24-36 months after completion of antibiotic treatment. RESULTS: In 24 months, 19 patients underwent two-stage THR for infection, of which 17 were treated with oral antibiotics after a median of 14 days initial iv antibiotics. None relapsed. Four patients underwent one-stage THR and had 12-20 days iv then 6-26 weeks oral antibiotics with no relapse. CONCLUSIONS: Early oral antibiotic switch therapy was effective in patients treated at the Avon Orthopaedic Centre with infected THR and plays an important role in enabling patients to return to independence after revision surgery and avoid complications of prolonged iv access.

Analyses of teicoplanin concentrations from 1994 to 2006 from a UK assay service.

Objectives An analysis of the trough serum concentrations sent to the UK Antimicrobial Reference Laboratory for teicoplanin therapeutic drug monitoring (TDM). Methods All trough concentrations over a 13 year period were analysed and the percentages were calculated for the following: <10 mg/L (a sub-optimal concentration for all); ≥10-<20 mg/L (the target used for ordinary Gram-positive infections); ≥20-<60 mg/L (the target for all severe staphylococcal infections including endocarditis); and ≥60 mg/L (the concentration associated with toxicity). Results The percentage of patients with concentrations of <10 mg/L decreased each year to 13% in 2006. Almost 40% of the samples each year were in the ≥10-<20 mg/L range. In 1996, the percentage of samples in the ≥20-<60 mg/L range reached a study high of ∼70%. That percentage then fell to 30% and increased slowly to 50% at the end of the study. Fewer than 5% of the samples were ≥60 mg/L. Conclusions Our study shows that there is a need to increase the initial dose or extend the number of days that the loading dose is used in a significant number of patients. With such a wide optimal range and a low potential for toxicity, it is unclear why optimal therapy is not achieved in a higher percentage of patients.

Development, evaluation and application of an isocratic high-performance liquid chromatography (HPLC) assay for the simultaneous determination of aciclovir and its metabolite 9-carboxymethoxymethylguanine in human serum and cerebrospinal fluid.

9-Carboxymethoxymethylguanine (CMMG), the main metabolite of aciclovir (ACV), is a putative neurotoxin. Measurement of CMMG in body fluids may aid patient management. We describe the development, validation and application of a high-performance liquid chromatography (HPLC) method for the simultaneous determination of ACV and CMMG in human serum and cerebrospinal fluid (CSF). Recovery was between 94% and 100% at all concentrations both from serum (range 0-20 mg/L) and CSF (0-5 mg/L). The intra-assay precision (coefficient of variation (CV)) was <2% and the inter-assay precision (CV) was <5%. The limits of detection and quantification were 0.1 and 0.25 mg/L, respectively, in both body fluids. Significant interference from endogenous material or from drugs in clinical samples was not seen. CMMG was detected in most of the 55 clinical samples containing ACV, but little correlation was found between the levels of the drug and its metabolite.

Expression of tcaA and mprF and glycopeptide resistance in clinical glycopeptide-intermediate Staphylococcus aureus (GISA) and heteroGISA strains.

Two genes recently associated with glycopeptide intermediate resistance in Staphylococcus aureus (GISA) are mprF and tcaA, with inactivation causing shifts in vancomycin resistance. This study reveals that expression levels of both genes are similar in groups of clinical GISA, heteroGISA and glycopeptide susceptible strains, suggesting no association with clinical isolates.

European Committee on Antimicrobial Susceptibility Testing (EUCAST) Technical Notes on antimicrobial susceptibility testing.

The main objectives of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) are to harmonise breakpoints for antimicrobial agents in Europe, and to act as the breakpoint committee for the European Medicines Agency (EMEA) during the registration of new antimicrobial agents. Detailed EUCAST procedures for harmonising and setting breakpoints for antimicrobial agents are available on the EUCAST website. Beginning with the current issue, a series of EUCAST Technical Notes will be published in CMI, based on the rationale documents produced by EUCAST for each of the antimicrobial agents studied, with the aim of highlighting important background information underlying decisions on breakpoints made by EUCAST.

Sequence analysis of the L1 metallo-beta-lactamase from Xanthomonas maltophilia.

The amino acid sequence deduced from the L1 beta-lactamase gene of Xanthomonas maltophilia shows a significant variation from that of the CphA and Blm metallo-beta-lactamases of Aeromonas hydrophila and Bacillus cereus, respectively. Whilst the N-terminus of the L1 protein shows some similarity, particularly at the histidine residues previously suggested as a zinc-binding motif, the C-terminus of the protein demonstrates very little similarity. Such differences amongst this group of enzymes would argue for at least three subclasses within the Group 3 beta-lactamases. However, in order to predict their phylogenetic ancestry more sequence data are required from other possible metallo-beta-lactamases.

Development of a novel assay method for colistin sulphomethate.

Increased use of colistin therapy for infections caused by Pseudomonas aeruginosa has indicated a need for a more robust microbiological assay technique. This report describes a quick and simple microbiological assay for quantifying levels of colistin sulphomethate in serum and urine samples from cystic fibrosis patients. The technique uses no specialised or costly equipment and is suitable for use in all routine diagnostic microbiology laboratories.

Continuous infusion of beta-lactam antibiotics.

There are considerable laboratory data and information from animal and continuous culture in vitro models to support continuous infusion therapy for penicillins and cephalosporins, but, as yet, the only existing clinical data relate to cephalosporins. Penicillins do not exert concentration-dependent killing in the therapeutic range but have a post-antibiotic effect (PAE) against Gram-positive cocci but not Gram-negative rods. Animal models indicate the time (T) during which the serum concentrations exceed the minimum inhibitory concentration (MIC) of the pathogen [T > MIC] determines outcomes. Pharmacokinetic studies in humans indicate that continuous infusion with penicillins is possible but there are no clinical data on efficacy. Cephalosporins have similar pharmacodynamic properties to penicillins; T > MIC determines outcome. Data related to ceftazidime indicate that the drug concentration at steady-state (Css) should exceed the pathogen MIC by > 1-fold and perhaps by 4- to 5-fold or more. Human pharmacokinetics of ceftazidime administered by continuous infusion to a wide variety of patient groups indicates that Css of > 20 mg/L can easily be achieved using conventional daily doses. Clinical data indicate increased effectiveness of a continuous regimen in neutropenic patients with Gram-negative infection. Furthermore cefuroxime administration by continuous infusion has resulted in lower doses and shorter course durations. Little is known of the pharmacodynamics of monobactams and there are few clinical data on continuous infusion therapy. Carbapenems have different pharmacodynamics to other beta-lactams as they have concentration-dependent killing and a PAE with both Gram-positive and Gram-negative bacteria. While T > MIC has a role in determining outcomes, the proportion of the dosing interval for which serum drug concentrations should exceed the pathogen MIC is less than for other beta-lactams. In vitro models have shown that continuous infusion is effective, as is less frequent dosing. There are few data on continuous infusion of carbapenems but some patients have been treated with once-daily dosing. Clinically, continuous infusion therapy with penicillins and cephalosporins should be considered in patients infected with susceptible Gram-negative rods not responding to conventional therapy. As an approximation, the same total daily dose should be given but a bolus intravenous injection should be give at the start of continuous infusion to ensure Css is reached rapidly. The Css may be difficult to predict and determination of serum drug concentrations may be indicated. Ideally, the Css should be calculated based on the MIC of the potential pathogen and may be higher or lower than the Css achieved by a conventional daily dose.

A preliminary report on the pharmacokinetics of ofloxacin, desmethyl ofloxacin and ofloxacin N-oxide in patients with chronic renal failure.

The in vitro activities of ofloxacin, desmethyl ofloxacin and ofloxacin N-oxide were determined and a specific high performance liquid chromatography (HPLC) assay for these 3 compounds was devised as part of a study of the pharmacokinetics of ofloxacin and its metabolites in patients with impaired renal function. Desmethyl ofloxacin had significant antimicrobial activity but less than that of the parent drug. In 2 patients with chronic renal failure, specific HPLC assay indicated an extended half-life for ofloxacin (approximately 13 hours) and the appearance in serum of low concentrations of both metabolites after 10 hours, persisting until the last blood sample was taken (32 hours). Further studies using specific assays are needed, particularly in patients undergoing haemodialysis and after administration of multiple doses.

Streptococcus milleri and second trimester abortion.

Review of 214 fetal necropsies performed in the department of pathology, University of Aberdeen, showed 40 cases of chorioamnionitis or intrauterine pneumonia, five of which were associated with Streptococcus milleri. In two cases there was good evidence to implicate S milleri as the cause of infected abortion while in the other cases its pathogenic role was less clear.

PIP: A review of 214 fetal necropsies submitted to the University of Aberdeen's Department of Pathology in 1983-86 revealed 40 cases of chorioamnionitis or intrauterine pneumonia, 5 of which were associated with Streptococcus milleri. Abortion had occurred between 18-23 weeks' gestation. There was no systemic evidence of infection in any of the 5 mothers. 3 of the women had prolonged (longer than 24 hours) rupture of membranes and 1 had an IUD in place. 1 woman had a cervical suture inserted in this pregnancy after a cone biopsy in 1975; another had had a previous septic abortion and 2 terminations. There was good evidence to implicate S milleri as a cause of septic abortion in 2 cases. In both these women, a profuse growth of S milleri was obtained from a maternal high vaginal swab collected before abortion and from a swab taken from the fetal airways at necropsy. the pathogenic role of S milleri was less clear in the remaining 3 cases. It is also unknown whether intrauterine infection follows spontaneous rupture of membranes or whether heavy vaginal colonization with a particular organism predisposes to rupture and preterm labor.

Measurement of urinary lipopolysaccharide antibodies by ELISA as a screen for urinary tract infection.

Five hundred and twenty two clinical urine specimens submitted for routine microbiological examination were tested in parallel by conventional microscopy and culture and for lipopolysaccharide antibodies by an enzyme linked immunoabsorbent assay (ELISA) to assess the ELISA as a screen for urinary tract infection. When the ELISA alone was compared with routine methods the specificity sensitivity, and predictive value of positive and negative tests was 73.2%, 75.7%, 51.1% and 38.5%. For ELISA with microscopy the same variables were 71.1%, 82.2%, and 92.4% and 94.7%, respectively. The ELISA absorbency increased with increasing bacterial numbers, but results varied widely. Only 65.4% of urines which contained greater than or equal to 10(5) bacteria/ml were positive by ELISA; 36.8% of urines with less than 10(3) bacteria/ml were positive by ELISA; 100% of greater than or equal to 10(5) bacteria/ml cultures of Pseudomonas sp (n = 4), Staphylococcus aureus (n = 3), and Streptococcus faecalis (n = 2) were positive by ELISA but only 71.4% of Proteus sp (n = 7), 61.4% coliforms (n = 70), and 25% of coagulase negative staphylococci (n = 4). It is concluded that further development is required before the ELISA can be used for routine screening for urinary tract infection.