We evaluated the role of prescribing antibiotics for AOM.

We performed a meta-analysis of randomized controlled trials (RCTs) that were retrieved from searches performed in the PubMed and Cochrane databases, and compared antibiotic treatment with placebo or watchful waiting (delayed antibiotic treatment if clinically indicated) for patients with AOM.

We identified seven trials comparing antibiotic treatment with placebo (all double-blinded) and four trials comparing antibiotic treatment with watchful waiting (two investigator-blinded and two open-label) trials, all of which involved children (6 months to 12 years). Clinical success was more likely with antibiotics than comparator treatment in: placebo-controlled trials [seven RCTs, 1405 patients, risk ratio (RR) = 1.11, 95% confidence interval (CI) = 1.05–1.18]; watchful waiting trials (four RCTs, 915 patients, RR = 1.18, 95% CI = 1.07–1.32); and all trials combined (11 RCTs, 2320 patients, RR = 1.13, 95% CI = 1.08–1.19). Similarly, persistence of symptoms 2–4 days after treatment initiation was less likely with antibiotics in: placebo-controlled trials (four RCTs, 1014 patients, RR = 0.75, 95% CI = 0.64–0.88) and all trials combined (five RCTs, 1299 patients, RR = 0.68, 95% CI = 0.54–0.85). Diarrhoea was more likely with antibiotics (seven RCTs, 1807 patients, RR = 1.50, 95% CI = 1.16–1.95). No differences between the compared treatments were found regarding other effectiveness and safety outcomes.

Antibiotic treatment is associated with a more favourable clinical course in children with AOM, compared with placebo, and also compared with watchful waiting. However, safety issues and the rather small treatment effect difference render the consideration of additional factors necessary in relevant clinical decision making.

At pre-treatment screening, the integrase gene from plasma samples from patients infected with subtype B and non-B viruses was analysed. Raltegravir resistance evolution was further evaluated in 10 heavily pre-treated patients.

Two hundred and nine plasma samples from 94 subtype B and 115 non-B patients were sequenced. No signature/primary raltegravir resistance mutations were detected at baseline. The secondary mutations L74M, T97A, V151I and G163R were observed with a frequency of <4%. The primary mutations N155H, Q148R/H or Q143R were observed during raltegravir therapy. The Q148R/H was detected only in subtype B. A switch of the primary mutation during raltegravir treatment was not restricted to the subtype B viruses. The prevalence of each primary mutation varied depending on the length of the raltegravir therapy. The Q148R/H was mostly detected after short exposure to raltegravir, while the Y143R was observed only after prolonged raltegravir exposure. We detected an association between the presence of the T206S in the baseline genotype and the absence of the primary Q148R/H mutation or any secondary mutation accompanying the N155H following raltegravir failure.

A number of secondary and additional mutations were found in baseline genotypes. During therapy, when the virus was not optimally suppressed, resistance mutations developed, which were dependent on subtype and time on raltegravir.

A panel of six matched DPS/DBS was generated using blood collected from HIV-infected donors. Replicate cards were prepared in 903 filter paper using 50 µL of blood and stored at either –20°C or at 37°C/100% humidity. Nucleic acids were extracted at baseline and after 1, 2, 8 and 16 weeks of storage and were amplified and sequenced using an in-house RT-nested PCR method or the ViroSeq assay.

HIV-1 pol was successfully amplified in all DBS/DPS at baseline and in those stored for up to 16 weeks at –20°C by the in-house assay. In contrast, amplification was rapidly lost during storage at 37°C/100% humidity with only 6/6 and 4/6 DBS specimens amplifiable by the in-house assay at weeks 1 and 2, respectively. Similarly, only two DPS stored at 37°C/100% humidity were amplified by the in-house assay at week 1.

We show that resistance testing from DBS and DPS is severely compromised after 2 and 1 weeks of storage at 37°C/100% humidity with desiccant, respectively. These findings underscore the importance of temperature and humidity for the efficient genotyping of HIV-1 from DBS and DPS, and reiterate the need to rapidly transport specimens from collection sites to locations that have appropriate storage conditions such as –20°C.

Using established procedures, SA13011 HeR to HoR selection was performed by using subinhibitory concentrations of oxacillin and examined for mutability. Real-time RT-PCR and transcriptional profiling by DNA microarray were used to compare gene expression between both populations and related genetically modified SA13011 strain.

We found that HeR/HoR selection by oxacillin was associated with increased mutation rate and oxacillin-mediated SOS response. We determined increased expression of both mecA and SOS response lexA/recA regulators. Mutational inactivation of lexA repressor resulted in a significant decrease in both mutation rate and oxacillin resistance in the HoR cells. Complementation of the lexA mutant strain restored oxacillin resistance to the high levels observed in the corresponding HoR wild-type strain.

The present results support the notion that SOS response is mechanistically involved in generating mutations that, in addition to mecA induction, allow the selection of a highly oxacillin-resistant population.

During July to December from 2004 to 2006, a surveillance of eight major hospitals in the Hiroshima region identified 387 non-duplicate isolates resistant to imipenem (MIC ≥ 16 mg/L). They were screened for resistance to amikacin (MIC ≥ 64 mg/L) and ciprofloxacin (MIC ≥ 4 mg/L) and MBL-encoding genes. The structure of the variable regions of the integrons was determined using PCR mapping. Clonality was assessed using PFGE and multilocus sequence typing (MLST).

The frequency of MBL-positive isolates in MDR P. aeruginosa isolates significantly increased from 42.3% in 2004 to 81.4% in 2006. Most of the MBL-positive isolates produced IMP-1 followed by VIM-2. The bla_IMP-1 and bla_VIM-2 genes were present in class 1 integrons. Characterization of the variable regions of the integron showed the presence of six different gene cassette arrays in bla_IMP-1 cassettes and a single array in bla_VIM-2 cassettes. The IMP-1 producers belonged to two clonal lineages using PFGE and MLST analyses and the integron variations correlated well with the clonal complexes. Among them, strains positive for a newly identified In113-derived bla_IMP-1 gene cassette array were most widely distributed in Hiroshima.

This study shows a dramatic increase in MBL genes, primarily bla_IMP-1, in MDR P. aeruginosa isolates in Hiroshima during these 3 years. In addition, MDR P. aeruginosa with the newly discovered In113-derived bla_IMP-1 gene cassette array appears to be clonally expanding.

We developed a duplex real-time PCR for the detection of the six 23S rRNA mutations associated with macrolide resistance in M. pneumoniae and a simplex real-time PCR for the identification of the A2058G mutation, the most common one. Both methods rely on fluorescence resonance energy transfer coupled to melting curve analysis and are directly applicable to clinical samples. The duplex real-time PCR assay, first validated on 40 genetically characterized M. pneumoniae strains, was then applied directly on 248 French respiratory tract clinical samples.

Among M. pneumoniae-positive specimens collected before 2005, no macrolide-resistant M. pneumoniae isolate was detected. In contrast, among 51 samples collected between 2005 and 2007, five (9.8%) yielded a resistant genotype, suggesting a recent increase in macrolide-resistant M. pneumoniae isolates in France.

The epidemiological monitoring of macrolide resistance in this species has become necessary in France and Europe, and will be made easier by using these PCR assays.

Tin (IV) chlorin e6 (SnCe6) was conjugated to bacteriophage 11, a transducing phage that can infect Staphylococcus aureus NCTC 8325-4, but not epidemic methicillin-resistant S. aureus (EMRSA)-16. The conjugate and appropriate controls were incubated with these bacteria and either exposed to laser light at 632.8 nm or kept in the dark.

The SnCe6/11 conjugate achieved a statistically significant reduction in the number of viable bacteria of both 8325-4 and EMRSA-16 strains by 2.31 log₁₀ and 2.63 log₁₀, respectively. The conjugate could not however instigate lethal photosensitization of Escherichia coli. None of the other combinations of controls, such as an equivalent concentration of SnCe6 only, an equivalent titre of bacteriophage only or experiments conducted without laser light, yielded significant reductions in the number of viable bacteria recovered.

The inability of a bacteriophage to infect S. aureus does not prevent it from specifically delivering a photosensitizer to a bacterium enabling its lethal photosensitization.

Staphylococcus aureus and human reporter cells continuously generating a fluorescence signal were competitively co-cultivated. The fluorescence signals were determined in the presence and absence of the specific antibiotic streptomycin and the toxic compound sodium azide. The results were compared with a standard cfu assay.

In the absence of an effective antibiotic, S. aureus outgrew the human reporter cells and thus abolished the fluorescence signal. Conversely, the addition of streptomycin resulted in the growth of the reporter cells and a strong fluorescence signal. When sodium azide was added instead of streptomycin, only a very low background signal was obtained indicating toxicity and damage to the human reporter cells. The assay proved to be highly reliable (Z-factor >0.9) and high fluorescence signals correctly correlated with the efficient inhibition of S. aureus, as determined in comparative cfu assays.

In contrast to conventional cfu assays, the co-cultivation system allows the effects of a drug candidate on pathogens and human cells to be monitored simultaneously. Cytotoxic compounds can, therefore, be quickly ruled out during a primary screen. The nature of the screen also enables effective antibiotics to be identified without engineering the target pathogen to yield a fluorescence signal.

Acinetobacter spp. isolates with tigecycline MICs of ≥0.5 mg/L determined by commercially developed Etests strips (January 2006 to July 2007) in five Spanish hospitals were considered. Values were rounded to the nearest upper double-dilution. Susceptibility by broth microdilution following CLSI (formerly NCCLS) recommendations, as the reference method, was determined in a central laboratory. BSAC breakpoints were used: susceptible ≤1 mg/L; intermediate = 2 mg/L; and resistant >2 mg/L.

One hundred and forty-eight isolates were collected: 12 isolates with a tigecycline Etest MIC of 0.5 mg/L, 14 with 1 mg/L, 86 with 2 mg/L, 31 with 4 mg/L and 5 with 8 mg/L. Isolates with Etest MICs of 0.5–1 mg/L showed the same values by broth microdilution. Among isolates with Etest MICs of 2 mg/L, only 5.8% of strains showed the same value by both methods (88.4% showed values that were one or two dilutions lower by microdilution). None of the 36 isolates with Etest MICs of 4–8 mg/L showed the same value by both methods, with values at least two dilutions lower by microdilution. Weak correlation (R = 0.238; P ≤ 0.001) was found between both methods. All 26 Etest susceptible isolates, 80/86 (93.0%) Etest intermediate and 32/36 (88.9%) Etest resistant strains were susceptible by microdilution.

Caution should be taken in interpreting Etest MICs of ≥2 mg/L for Acinetobacter spp. since strains with Etest MICs of 2–4 mg/L are susceptible when tested by microdilution. False non-susceptibility by Etest may exclude tigecycline as a therapeutic option in a field where multiresistance is the rule.

MICs of 19 antimicrobial agents for 125 clinical isolates of the Nocardia species were determined by the broth microdilution method.

Nocardia brasiliensis (n = 61), Nocardia asteroides (n = 45), Nocardia flavorosea (n = 5), Nocardia otitidiscaviarum (n = 4), Nocardia farcinica (n = 3), Nocardia beijingensis (n = 2), Nocardia puris (n = 2) and one each of Nocardia nova, Nocardia jinanensis and Nocardia takedensis were identified based on a 16S rRNA gene sequencing analysis. For N. brasiliensis isolates, the MIC₉₀s of the tested quinolones were in the order nemonoxacin < gemifloxacin = moxifloxacin < levofloxacin = ciprofloxacin, and the MIC₉₀s of the tested carbapenems were in the order doripenem = meropenem < ertapenem < imipenem. Tigecycline had a lower MIC₉₀ (1 mg/L) than linezolid (8 mg/L). For N. asteroides isolates, the MIC₉₀s of the tested quinolones were in the order nemonoxacin < gemifloxacin = moxifloxacin < levofloxacin < ciprofloxacin, and the MIC₉₀s of the tested carbapenems were in the order doripenem = meropenem = imipenem < ertapenem. For the other 19 Nocardia species isolates, nemonoxacin showed good activity with the lowest MIC₉₀ of the tested quinolones. Among the four tested carbapenems, doripenem and meropenem had comparatively lower MIC₉₀s.

The results of this in vitro study suggest that nemonoxacin, linezolid and tigecycline show promise as treatment options for nocardiosis. Further investigation of their clinical role is warranted.

MICs of isavuconazole, voriconazole, posaconazole, fluconazole, amphotericin B and flucytosine were determined for 54 Trichosporon, 7 Geotrichum capitatum, 18 Saccharomyces cerevisiae, 11 Pichia and 14 Rhodotorula species in accordance with the M27-A2 reference method. Minimum fungicidal concentrations were also measured for each agent.

Isavuconazole demonstrated excellent in vitro activity against each species tested. MIC₉₀ values ranged between 0.125 and 0.25 mg/L against Trichosporon isolates, and between 0.03 and 0.5 mg/L against the other yeast tested. The geometric mean MICs of isavuconazole were similar to those of voriconazole and similar or less than those of posaconazole, fluconazole, amphotericin B and flucytosine for all species tested. This activity was also maintained against one Trichosporon asahii and nine Rhodotorula isolates that were resistant to fluconazole.

Isavuconazole is a welcome addition to the growing antifungal armamentarium with potent in vitro activity against emerging yeast pathogens. Although this agent may be useful in the treatment of the rare yeasts, clinical data are needed to verify these results.

The antileishmanial activity of physalins B, D and F was tested in Leishmania-infected macrophage cultures. For the in vivo studies, BALB/c mice were infected with Leishmania amazonensis subcutaneously in the ear pinna and treated with physalin F by topical administration.

Physalins B and F were able to reduce the percentage of Leishmania-infected macrophages and the intracellular parasite number in vitro at concentrations non-cytotoxic to macrophages. More importantly, topical treatment with physalin F significantly reduced the lesion size, the parasite load and histopathological alterations in BALB/c mice infected with L. amazonensis.

Our results demonstrate the potent antileishmanial activity of physalins, especially physalin F, and suggest these molecules as the basis for the development of new therapeutic options for cutaneous leishmaniasis.

We investigated the potential synergistic antimicrobial and antibiofilm activity of triclosan and DspB by biofilm assays. The in vitro antimicrobial efficacy of triclosan + DspB-coated catheters was determined by microbial colonization assays. Antimicrobial durability of the coated catheters was tested by soaking segments in bovine serum for 7 days and determining antimicrobial activity, and by a serial plate transfer method. The in vivo efficacy of triclosan + DspB-coated catheters compared with CH-SS-coated and uncoated catheters was assessed by subcutaneous implantation of segments in a rabbit model of S. aureus infection.

The combination of triclosan and DspB showed synergistic antimicrobial and antibiofilm activity against S. aureus, Staphylococcus epidermidis and Escherichia coli, significantly reduced bacterial colonization (P < 0.05) and generally demonstrated a prolonged superior antimicrobial activity against clinical pathogens compared with CH-SS-coated catheters. Triclosan + DspB-coated and CH-SS-coated catheters exhibited equal in vivo efficacy (P ≤ 0.05) in reducing colonization by S. aureus compared with uncoated catheters.

Catheters coated with the triclosan + DspB combination showed synergistic, broad-spectrum and durable antimicrobial activity. Furthermore, the in vivo efficacy of catheters coated with this unique antimicrobial/antibiofilm composition prompts clinical evaluation of such an innovative approach.

Jugular vein catheterized mice (four to six per group) challenged with S. aureus developed multiorgan infection and biofilm infections on the catheters. The infected mice with established biofilms received various doses of recombinant lysostaphin through the catheters, administered up to three times daily for up to 4 days. Some mice also received lysostaphin combined with nafcillin. Following treatment, mice were sacrificed and cfu on the catheter and in the liver and heart were determined. In another set of experiments, implanted jugular vein catheters in mice were pre-instilled with lysostaphin to determine whether this pre-treatment would protect the mice from biofilm infection.

Lysostaphin administered at 15 mg/kg in combination with 50 mg/kg nafcillin three times per day for 4 days eradicated established S. aureus, including methicillin-resistant S. aureus, biofilms from implanted catheters and sterilized heart and liver infections of S. aureus-infected mice. Furthermore, a single pre-instillation of 10 mg/kg lysostaphin in catheters completely protected catheterized mice from a subsequent biofilm infection. These results demonstrate that lysostaphin is an effective treatment as well as prophylaxis for S. aureus biofilms on indwelling catheters.

Rats were allocated into the following treatment groups: oral gavage of AmB dispersed in mono/diglyceride–phospholipid formulation at doses of 4.5 and 10 mg/kg; iv bolus administration of 0.8 mg/kg Fungizone^®; iv bolus of 5 mg/kg Abelcet^® and iv bolus of 5 mg/kg AmBisome^®. Blood was sampled from jugular vein cannula at certain time points. The animals were sacrificed 72 h following administration of AmB and multiple tissues were harvested. The concentration of AmB in plasma and tissues was determined by means of HPLC. The plasma creatinine concentrations were determined using an enzymatic kit.

The pharmacokinetics and tissue distribution of AmB following iv administrations of the commercial formulations were found to be highly formulation dependent. The terminal half-life and biodistribution of orally administered AmB in a mono/diglyceride–phospholipid formulation resembled those of Fungizone^®. The larger volume of the co-administered lipid-based formulation in the case of the higher dose of orally administered AmB resulted in flip-flop kinetics and in preferential distribution into the kidneys. No nephrotoxicity was detected for any formulation and route of administration.

Oral administration of AmB in a mono/diglyceride–phospholipid formulation to rats resulted in significant intestinal absorption into the systemic circulation with pharmacokinetic and biodistribution properties similar to a micellar iv preparation.

We retrospectively enrolled HIV-infected patients undergoing therapeutic drug monitoring (TDM) of NNRTIs and PIs during routine outpatient visits. Plasma drug concentrations were measured by HPLC-UV and were considered therapeutic if above the proposed minimum efficacy trough concentration. Inter-individual and intra-individual variabilities were evaluated through the coefficient of variation (CV).

A total of 457 PI and 172 NNRTI plasma concentrations were measured from 363 patients (HIV-RNA <50 copies/mL in 70.8%, median CD4 count 434 cells/mm³). NNRTIs showed less inter-individual (CV_inter 54.8% versus 84.3%) and intra-individual (CV_intra 19.0% versus 38.1%) pharmacokinetic variabilities than PIs. Intra-individual variability was constantly lower than inter-individual variability for each drug. Subtherapeutic drug concentrations were observed in 106 samples (16.9%). Older age (P = 0.020) and higher viral load (P = 0.013) were associated with subtherapeutic levels. Patients with therapeutic levels had a viral load of <50 copies/mL more frequently than those with subtherapeutic levels (74.8% versus 63.2%, P = 0.020). The estimated proportion with virological failure at 24 weeks was 0.21 in patients with suboptimal baseline drug levels and 0.08 in those with optimal levels (P < 0.001). In the multivariate analysis, therapeutic drug levels showed an independent negative association with virological failure (P = 0.004).

A wide inter-individual and limited intra-individual pharmacokinetic variabilities, together with the demonstration of a concentration–response relationship, suggest that TDM is a useful tool for the clinical management of patients treated with NNRTIs or PIs.

Thirty patients received fosamprenavir/atazanavir/ritonavir (Group 1) and 31 patients received saquinavir/atazanavir/ritonavir (Group 2). The primary endpoint for efficacy was the rate of early virological success, defined as plasma viral load <50 copies/mL at week 16. The study is registered with ClinicalTrials.gov (NCT00122603).

At baseline, median (range) viral load was 4.8 log₁₀ copies/mL (4.0–5.7) and the median CD4 cell count was 271/mm³ (197–740). Viral load was <50 copies/mL in 12/30 patients [40%, 95% confidence interval (CI) 23%–58%] and 13/31 patients (42%, 95% CI 25%–59%) at week 16 in Groups 1 and 2, respectively. Patients with failing regimens (viral load ≥400 copies/mL at week 16 or ≥50 copies/mL at week 24) were switched to a standard antiretroviral regimen. At week 48, by an intention-to-treat analysis, 23/30 patients (77%) and 26/31 patients (84%) had plasma HIV-1 RNA <50 copies/mL in Groups 1 and 2, respectively. Four patients discontinued treatment for adverse events, all before week 4. No major changes in the protease gene were detected at treatment failure relative to baseline. Baseline viral load <50 000 copies/mL was the only predictor of virological success at week 16.

Ritonavir-boosted dual protease inhibitor regimens targeting only one step of viral replication were insufficient to rapidly suppress plasma HIV RNA to <50 copies/mL in antiretroviral-naive patients with high viral load at baseline.

A non-inferiority, randomized, multicentre, open-label trial was performed in 24 AIDS clinical centres in France randomizing 143 patients [treated for ≥6 months, plasma viral load (pVL) <50 copies/mL, no prior history of treatment failure] to receive a two-drug regimen [tenofovir disoproxil fumarate (tenofovir DF) and efavirenz] or to maintain a three-drug treatment (tenofovir DF, lamivudine and efavirenz). The main outcome measure was the success rate (percentage of patients with pVL <50 copies/mL without treatment modifications) at week 48.

Success rates for the intention-to-treat analysis were 97.2% (70/72) versus 81.7% (58/71) in the three-drug versus two-drug maintenance regimen groups, respectively [difference, 15.5%; upper limit of one-sided 95% confidence interval (CI), 23.7%], and 100% (70/70) versus 90% (54/60) for the per protocol analysis, respectively (difference, 10%; upper limit of one-sided 95% CI, 16.4%), with a non-inferiority margin set at 14%. Three patients from the two-drug group experienced virological failure with selection of efavirenz-associated mutations. Overall, CD4 counts were significantly increased from baseline (median, +24 cells/mm³; P = 0.007). Four patients discontinued study treatment due to adverse events in the two-drug group and none in the three-drug group. No significant changes in creatinine clearance or phosphataemia were reported. Overall, levels of triglycerides, total and high-density lipoprotein cholesterol were improved; low-density lipoprotein cholesterol was improved only in the three-drug group.

The non-inferiority of the two-drug versus the three-drug regimen was not demonstrated. Lipid parameters improved after switching from twice-daily highly active antiretroviral therapy (HAART) to once-daily tenofovir-based HAART.

V3 loop sequences were amplified from HIV-1-DNA and analysed with a combination of five genotypic rules to predict tropism: (i) the ‘11/25 rule’; (ii) the net charge rule; (iii) the PSSM_X4/DM algorithm; (iv) the PSSM_SI/NSI algorithm; and (v) the SVM_Geno2pheno algorithm.

A high proportion (62/390, 15.9%) of patients harboured X4/DM-tropic viruses. This prevalence was stable over time: 18.1% before 2003 versus 14.8% since 2003. No difference according to HIV tropism was noted in HIV-RNA levels, CD4 cell count, time between infection and enrolment, and HIV infection risk factor. The frequency of X4/DM-tropic virus was similar among patients infected with a resistant virus (12/62, 19.4%) compared with patients harbouring wild-type strains (50/328, 15.2%).

This large French epidemiological study evidenced a high proportion of patients (15.9%) harbouring X4/DM-tropic viruses in PBMCs at the time of PHI, suggesting the existence of a cellular X4/DM viral reservoir that could persist for lengthy period of time. Several reports identified that HIV-1 CXCR4 usage was more frequent among patients who developed AIDS and was a powerful predictor of the response to antiretrovirals. Further studies are needed to evaluate the impact of such strains on the outcome of HIV disease, when they are detected at the time of primary infection.

We randomized 10 patients with sepsis to receive meropenem by intermittent bolus administration (n = 5; 1 g 8 hourly) or an equal dose administered by continuous infusion (n = 5). Serial subcutaneous tissue concentrations were determined using microdialysis and compared with plasma data for first-dose and steady-state pharmacokinetics. Population pharmacokinetic modelling of plasma data and Monte Carlo simulations were then undertaken with NONMEM^®.

It was found that continuous infusion maintains higher median trough concentrations, in both plasma (intermittent bolus 0 versus infusion 7 mg/L) and subcutaneous tissue (0 versus 4 mg/L). All simulated intermittent bolus, extended and continuous infusion dosing achieved 100% of pharmacodynamic targets against most Gram-negative pathogens. Superior obtainment of pharmacodynamic targets was achieved using administration by extended or continuous infusion against less susceptible Pseudomonas aeruginosa and Acinetobacter species.

This is the first study to compare the relative concentration–time data of bolus and continuous administration of meropenem at the subcutaneous tissue and plasma levels. We found that the administration of meropenem by continuous infusion maintains higher concentrations in subcutaneous tissue and plasma than by intermittent bolus dosing. Administration by extended or continuous infusion will achieve superior CFR against less-susceptible organisms in patients without renal dysfunction.

Study 1 was an open-label, single-dose, two-period, crossover study in which each subject received 6 mg/kg daptomycin administered as a 30 min iv infusion (n = 15) and as a 2 min iv injection (n = 16). In Study 2, a single-blind, multiple-dose, parallel-group study, subjects received a once-daily 2 min iv injection of 6 mg/kg daptomycin (n = 12), 4 mg/kg daptomycin (n = 8) or placebo (n = 4) for 7 days. Single-dose pharmacokinetics were assessed at various timepoints up to 36 and 24 h post-dose in Study 1 and Study 2, respectively, and multiple-dose pharmacokinetics were assessed in Study 2 at day 7 for 48 h post-dose.

In Study 1, pharmacokinetic comparability between the two administration regimens was demonstrated by meeting the bioequivalence criteria for the exposure parameters, AUC_0–t, AUC_0– and C_max. In Study 2, time-invariant pharmacokinetic properties as well as dose-proportional pharmacokinetics were demonstrated for the daptomycin 2 min iv injection regimen. In both studies, daptomycin was well tolerated and the majority of treatment-emergent adverse events were of mild intensity and considered to be unrelated to daptomycin.

The similar pharmacokinetic and safety profiles of the two administration regimens suggest that the 2 min iv injection may be a convenient treatment option for both patients and healthcare professionals.

We enrolled 370 hospitalized patients (66 ± 17 years; 42% females) with proven CAP. Venous blood samples were collected at the time of inclusion into the study and as soon as possible after the diagnosis of CAP. Copeptin (B.R.A.H.M.S. AG, Henningsdorf, Germany) levels were determined in venous blood on admission.

Eighty-five patients had antibiotic pre-treatment and 285 patients did not. Copeptin levels increased with increasing severity of CAP in patients without antibiotic pre-treatment but not in patients with antibiotic pre-treatment. Patients with prior antibiotic treatment showed significantly lower levels of copeptin [median (interquartile range): 12.8 (5.3–22.6) versus 20.8 (11.1–37.8) pmol/L, P < 0.0001] and procalcitonin [0.15 (0.07–0.38) versus 0.27 (0.10–1.52) ng/mL, P = 0.0003], but not C-reactive protein [113 (46–229) versus 122 (49–231) mg/mL, not significant] and leucocytes [12.2x10³ (8.1x10³–15.4x10³) versus 12.5x10³ (9.4x10³–16.3x10³) cells/mm³, not significant] compared with those without antibiotic pre-treatment.

Copeptin serum levels are higher in patients without antibiotic pre-treatment compared with those with antibiotic pre-treatment. Copeptin serum levels increase with an increasing severity of CAP in patients without, but not in patients with, antibiotic pre-treatment. Thus, antibiotic pre-treatment has to be taken into account for the correct interpretation of copeptin levels in CAP.

The population-based cohort study included 1533 unique isolates of E. coli from Danish patients with CAB during a 10 year period. Triplex PCR was used to classify the phylogenetic groups, and susceptibility testing was performed by disc diffusion. Data were analysed using contingency tables and logistic regression.

Overall, 65.9% of the 1533 E. coli isolates belonged to phylogroup B2, 16.6% to D, 13.1% to A and 4.4% to B1. B2 was the most prevalent group for all sites of infection, ranging from 69.9% in cases with a urinary tract site of infection to 54.8% in cases with a hepatobiliary tract site of infection. Antibiotic resistance to one and more than three antibiotics, respectively, was most frequent in group D (11.4%/33.9%), followed by A (5.5%/26.9%), B1 (5.9%/19.1%) and B2 (6.7%/7.5%). Regression analysis, with group B2 as reference, confirmed that groups A and B1 were associated with a site of infection other than the urinary tract and that groups A and D were associated with resistance to antibiotics including ampicillin, sulphonamide, trimethoprim, gentamicin and quinolones.

Phylogenetic group B2 was predominant in E. coli CAB. This was the least resistant of the four groups. Phylogroups A and B1 were associated with sites of infection other than the urinary tract, and resistance to multiple antibiotics was most prevalent for groups A and D.

In this retrospective population-based incidence study, we identified 461 unique patients with first episodes of E. coli bloodstream infection (BSI) from 1 January 1998 to 31 December 2007 through microbiology records at the two laboratories in Olmsted County, Minnesota. Logistic regression was used to examine temporal changes in antimicrobial resistance and Poisson regression for changes in incidence rates.

The median age of patients with E. coli BSI was 69 years; 306 (66.4%) were female. The age-adjusted incidence rate of E. coli BSI per 100 000 person-years was 48.0 (95% CI: 42.5–53.4) in females and 34.0 (95% CI: 28.6–39.6) in males. The urinary tract was the most common primary source of infection (79.8%). During the study period, resistance rates of E. coli bloodstream isolates increased from 32% to 53% for ampicillin, from 23% to 45% for ampicillin/sulbactam, from 9% to 28% for trimethoprim/sulfamethoxazole and from 0% to 12% for ciprofloxacin. Resistance rates to carbapenems, cephalosporins and piperacillin/tazobactam remained low and stable.

To our knowledge, this is the first population-based study on antimicrobial resistance trends of E. coli bloodstream isolates in the USA. We demonstrated linear trends of increasing resistance among these isolates to three different classes of antimicrobial over the past decade.

A retrospective study was performed on 98 adult patients with VRE bacteraemia admitted to two hospitals between September 2003 and December 2007 to compare the efficacy of daptomycin with that of linezolid. Multivariable analyses were performed to compare the microbiological and clinical outcomes of both groups.

Out of 98 patients with VRE bacteraemia, 68 were treated with linezolid and 30 with daptomycin. Univariate analyses showed no significant differences between the groups regarding baseline demographic and clinical characteristics, severity of illness and co-morbidity. Daptomycin was associated with a trend towards a higher mortality rate (26.7% versus 20.6%), longer median duration of bacteraemia (3 days versus 2 days) and higher relapse rate (6.7% versus 2.9%), but these differences did not reach statistical significance (P > 0.2). Microbiological cure rates were 90% for the daptomycin group and 88.2% for the linezolid group (P = 0.92).

Despite a trend towards worse outcomes, daptomycin was as effective as linezolid in treating VRE bacteraemia. A randomized clinical trial is needed to confirm these results.

Patients received 15–25 mg/kg/day for 3 days, then 15–25 mg/kg thrice weekly. Trough concentrations were measured weekly and doses adjusted to maintain 20–30 or 10–20 mg/L according to clinical condition. Concentration–time data were analysed using the pharmacokinetic package NONMEM and the final model was used to develop new dosage guidelines.

Data from 94 and 36 patients were used for model development and validation, respectively. Patient ages ranged from 15 to 94 years, weights from 43 to 146 kg and estimated CL_CR from 9 to 195 mL/min. Teicoplanin concentrations (n = 670) ranged from 6.7 to 66.9 mg/L and a one-compartment model adequately described the data. The typical estimate of CL was 0.542 L/h and changed by 10.6% for every 10 mL/min difference from a CL_CR of 66 mL/min. V was 1.62 L/kg. Dosage guidelines based on body weight and CL_CR can be expected to lead to a significant improvement in the proportion of concentrations in the range 20–30 mg/L. Alternative doses aimed at lower target concentrations have also been developed.

New dosage guidelines have been developed to support thrice-weekly administration of teicoplanin in an OPAT setting.

During a 12 month intervention study, a printed checklist of criteria for switching on the third day of iv treatment was placed in the medical charts. The decision to switch was left to the discretion of the attending physician. Outcome parameters of a 4 month control phase before intervention were compared with the equivalent 4 month period during the intervention phase to control for seasonal confounding (before–after study; April to July of 2006 and 2007, respectively): 250 episodes (215 patients) during the intervention period were compared with the control group of 176 episodes (162 patients). The main outcome measure was the duration of iv therapy. Additionally, safety, adherence to the checklist, reasons against switching patients and antibiotic cost were analysed during the whole year of the intervention (n = 698 episodes).

In 38% (246/646) of episodes of continued iv antibiotic therapy, patients met all criteria for switching to oral antibiotics on the third day, and 151/246 (61.4%) were switched. The number of days of iv antibiotic treatment were reduced by 19% (95% confidence interval 9%–29%, P = 0.001; 6.0–5.0 days in median) with no increase in complications. The main reasons against switching were persisting fever (41%, n = 187) and absence of clinical improvement (41%, n = 185).

On general medical wards, a checklist with bedside criteria for switching to oral antibiotics can shorten the duration of iv therapy without any negative effect on treatment outcome. The criteria were successfully applied to all patients on the wards, independently of the indication (empirical or directed treatment), the type of (presumed) infection, the underlying disease or the group of antibiotics being used.

Within the European Surveillance of Antimicrobial Consumption (ESAC; www.esac.ua.ac.be), using the anatomic therapeutic chemical (ATC) and defined daily dose (DDD) classification, data on outpatient use of antibacterials for systemic use (ATC J01), aggregated at the level of the active substance and expressed in DDD per 1000 inhabitants per day (DID; WHO version 2007), were extracted for 2006 by route of administration and by country. Parenteral use was expressed as a percentage of the total outpatient use in DID.

In 20 European countries, the total outpatient antibiotic use ranged from 27.91 DID in France to 9.58 DID in Russia. The proportion of outpatient parenteral antibiotic treatment ranged from 6.75% in Russia to 0.001% in Iceland. The three most commonly used antibiotic groups for parenteral treatment in Europe were the cephalosporins (J01D; 44.58%), the aminoglycosides (J01G; 25.27%) and the penicillins (J01C; 17.78%). Four antibiotics [gentamicin (J01GB03) 18.53%; ceftriaxone (J01DD04) 17.85%; cefazolin (J01DB04) 13.16%; and lincomycin (J01FF02) 5.47%] represented more than half of the use.

In all 20 European countries studied together, 2.04% of outpatient antibiotics were used for parenteral treatment. However, as for the total outpatient antibiotic use and the use of different antibiotic groups and antibiotics, there is a striking variation in the proportions of parenteral antibiotic use in Europe. More in-depth data on outpatient antibiotic use are needed to explain this variation.

Forty E. faecalis isolates (22 vancomycin-susceptible and 18 vancomycin-resistant) representing disseminated and/or multiresistant strains from different sources (humans, animals and the environment) were characterized by PFGE and multilocus sequence typing. Genes encoding putative virulence markers and the backbone of Tn1546 were investigated by PCR. Plasmid analysis included determination of size, content (S1 hybridization) and comparison of restriction fragment length polymorphism patterns.

The 40 E. faecalis isolates (22 PFGE types) mostly clustered within the worldwide-spread clonal complexes (CCs) CC2 (13 ST6 mostly corresponding to an epidemic strain, where ST stands for sequence type), CC21 (3 ST21, 1 ST22 and 1 ST224) and ST16 (n = 7), but also comprised ST159, ST35, ST19, ST26, ST30, ST41, ST55, ST59, ST117, ST160 and ST200. CC2 and CC21 were isolated from both hospital and community settings. Similar Tn1546-like elements encoding VanA were found on related plasmids within strains belonging to different clonal lineages and recovered in distinct hospitals over several years.

The predominance of E. faecalis CC2 is mainly due to the dissemination of a particular clone persistently recovered for 11 years. The presence in the community of specific strains belonging to major clonal lineages highlights the role of community-associated hosts as possible reservoirs of putative human pathogenic enterococci. Both clonal expansion and dissemination of epidemic conjugative VanA plasmids seem to join forces in the establishment of pathogenic E. faecalis strains.

Spontaneous trimethoprim-resistant mutants of S. aureus SH1000 were recovered in vitro, resistance genotypes characterized by DNA sequencing of the gene encoding the drug target (dfrA) and the fitness of mutants determined by pair-wise growth competition assays with SH1000. Novel resistance genotypes were confirmed by ectopic expression of dfrA alleles in a trimethoprim-sensitive S. aureus strain. Molecular models of S. aureus dihydrofolate reductase (DHFR) were constructed to explore the structural basis of trimethoprim resistance, and to rationalize the observed in vitro fitness of trimethoprim-resistant mutants.

In addition to known amino acid substitutions in DHFR mediating trimethoprim resistance (F₉₉Y and H₁₅₀R), two novel resistance polymorphisms (L₄₁F and F₉₉S) were identified among the trimethoprim-resistant mutants selected in vitro. Molecular modelling of mutated DHFR enzymes provided insight into the structural basis of trimethoprim resistance. Calculated binding energies of the substrate (dihydrofolate) for the mutant and wild-type enzymes were similar, consistent with apparent lack of fitness costs for the resistance mutations in vitro.

Reduced susceptibility to trimethoprim of DHFR enzymes carrying substitutions L₄₁F, F₉₉S, F₉₉Y and H₁₅₀R appears to result from structural changes that reduce trimethoprim binding to the enzyme. However, the mutations conferring trimethoprim resistance are not associated with fitness costs in vitro, suggesting that the survival of trimethoprim-resistant strains emerging in the clinic may not be subject to a fitness disadvantage.

We determined the proportion of rpoB mutants in the chemostat culture and characterized the sequence of mutations found in the rifampicin resistance-determining region of rpoB in a steady-state chemostat at pH 7.0 and 6.2.

The overall proportion of rpoB mutants of strain H37Rv remained constant for 37 days at pH 7.0, ranging between 3.6 x 10^–8 and 8.9 x 10^–8; however, the spectrum of mutations varied. The most commonly detected mutation, serine to leucine mutation at codon 531 (S531L), increased from 40% to 89%, while other mutations (S531W, H526Y, H526D, H526R, S522L and D516V) decreased over the 37 day sampling period. Changing the pH from 7.0 to 6.2 did not significantly alter the overall proportion of mutants, but resulted in a decrease in the percentage of strains harbouring S531L (from 89% to 50%) accompanied by an increase in the range of different mutations from 4 to 12.

The data confirm that the fitness of strains with the S531L mutation is greater than that of strains containing other mutations. We also conclude that at low pH the environment is permissive for a wider spectrum of mutations, which may provide opportunities for a successful mutant to survive.

A total of 49 MDR-TB and 19 fully susceptible non-MDR-TB isolates, as determined by conventional drug susceptibility testing using the BACTEC-MGIT960 system, were used to evaluate the suitability of HRM curve analysis as a rapid and accurate screening system for rifampicin resistance.

HRM analysis of the rpoB cluster I site allowed the correct allocation of 44 of the 49 MDR-TB isolates and all non-MDR-TB isolates. Three of the five MDR-TB isolates (60%) falsely identified as non-MDR-TB harboured the V176F mutation that could be specifically detected by an additional HRM assay. The combined HRM analysis of all strains and isolates exhibited 95.9% sensitivity and 100% specificity.

With a positive predictive value of 100% and a negative predictive value of at least 99.9%, this combined HRM curve analysis is an ideal screening method for the TB laboratory, with minimal requirements of cost and time. The method is a closed-tube assay that can be performed in an interchangeable 96- or 384-well microplate format enabling a rapid, reliable, simple and cost-effective handling of even large sample numbers.

A random mutagenesis technique was used to predict the evolutionary potential of these PRPs against nalidixic acid and fluoroquinolones. After comparing the amino acid sequences of these and other PRPs protecting bacteria from quinolone activity, several conserved positions were found. The role of these residues in their effect against quinolones was evaluated by site-directed mutagenesis.

Three different phenotypes (similar resistance, higher resistance or lower resistance to quinolones) were obtained in the random mutagenesis assays when compared with wild-type phenotypes. Only one mutant increased quinolone resistance: QnrS1 containing D185Y substitution (4-fold for ciprofloxacin). Using site-directed mutagenesis, residues G56, C72, C92, G96, F114, C115, S116, A117 and L159, according to the sequence of QnrA1, were analysed and despite the wide amino acid variability of the PRPs, most conserved residues analysed were critical to QnrA1, QnrB1 and QnrS1.

Amino acid sequences of PRPs QnrA1, QnrB1 and QnrS1 could be already optimized for quinolone resistance. One or several changes appear to be insufficient to obtain variants producing fluoroquinolone clinical resistance (MIC > 1 mg/L). Critical residues for quinolone resistance in PRPs were described. Interestingly, different effects were observed for QnrA1, QnrB1 and QnrS1 with the same substitution in several positions.

In the first part of this study, 105 ESBL-producing E. coli isolates (February 2006 to March 2007) were characterized with regard to ESBL enzymes, serotypes, virulence genes, phylogenetic groups, multilocus sequence typing (MLST) and PFGE. In the second part of this study, 249 ESBL-producing E. coli isolates (April 2007 to May 2008) were investigated only for the detection of clone O25b:H4-ST131 producing CTX-M-15 using a triplex PCR developed in this study and based on the detection of the new operon afa FM955459 and the targets rfbO25b and 3' end of the bla_CTX-M-15 gene.

Of the 105 ESBL-producing E. coli isolates, 60 (57.1%) were positive for CTX-M-14, 23 (21.9%) for CTX-M-15, 10 (9.5%) for SHV-12 and 7 (6.7%) for CTX-M-32. Serotypes, virulence genes, phylogenetic groups and molecular typing by PFGE demonstrated high homogeneity within those producing CTX-M-15 and high diversity within E. coli producing CTX-M-14 and other ESBLs. By PFGE, CTX-M-15-producing E. coli isolates O25b:H4 belonging to the phylogenetic group B2 and MLST profile ST131 were grouped in the same cluster. The epidemic strain of clone O25b:H4-ST131 represented 23.1%, 22.5% and 20.0% of all ESBL-producing E. coli isolated in 2006, 2007 and 2008, respectively.

CTX-M-type ESBLs, primarily CTX-M-14 and CTX-M-15, have emerged as the predominant types of ESBL produced by E. coli isolates in Lugo. In view of the reported findings, long-term care facilities for elderly people may represent a significant reservoir for E. coli clone O25b:H4-ST131 producing CTX-M-15. The triplex PCR developed in this work will be useful for rapid and simple detection of this clone.