Identification and Morphological Characterization of Biofilms Formed by Strains Causing Infection in Orthopedic Implants.

Objectives: For a better understanding of the mechanisms involved in biofilm formation, we performed a broad identification and characterization of the strains affecting implants by evaluating the morphology of biofilms formed in vitro in correlation with tests of the strains' antibiotic susceptibility in planktonic form. The ability of the strains to form biofilms in vitro was evaluated by means of colony forming units counting, metabolic activity tests of biofilm cells, and scanning electron microscopy. Methods: A total of 140 strains were isolated from patients with orthopedic implant-related infections during the period of 2015 to 2018. The identification of the isolates was carried out through microbiological cultures and confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Antibiotic susceptibility rates of the isolates were accessed according to EUCAST (European Committee on Antimicrobial Susceptibility Testing). The ability of all isolates to form biofilms in vitro was evaluated by counting the colony forming units, by measuring the metabolic activity of biofilm cells, and by analyzing the morphology of the formed biofilms using scanning electron microscopy. Results: From all the isolates, 41.84% (62 strains) were Staphylococcus epidermidis and 15.60% (22 strains) were Staphylococcus aureus. A significant difference in the capacity of biofilm formation was observed among the isolates. When correlating the biofilm forming capacity of the isolates to their antibiotic susceptibility rates, we observed that not all strains that were classified as resistant were biofilm producers in vitro. In other words, bacteria that are not good biofilm formers can show increased tolerance to multiple antibiotic substances. Conclusion: From 2015 until 2018, Staphylococcus epidermidis was the strain that caused most of the orthopedic implant-related infections in our hospital. Not all strains causing infection in orthopedic implants are able to form biofilms under in vitro conditions. Differences were observed in the number of cells and morphology of the biofilms. In addition, antibiotic resistance is not directly related to the capacity of the strains to form biofilms in vitro. Further studies should consider the use of in vitro culture conditions that better reproduce the joint environment and the growth of biofilms in humans.

Antibiotic resistance of blood cultures in regional and tertiary hospital settings of Tyrol, Austria (2006-2015): Impacts & trends.

Blood stream infections rank among the top seven causes of death of the general population. The aim of our study was to better understand the epidemiology of BSI in order to improve diagnostics and patient outcome. We used retrospective aggregated laboratory data of blood samples received from all public hospitals in Tyrol, Austria between 2006 and 2015. Microorganisms were categorized into obligatory, facultative, unusual pathogens and contaminants. The distribution, the cumulative incidence and antimicrobial susceptibility patterns were compared between the tertiary (TH) and regional peripheral hospitals (PH). Among 256,364 blood samples, 76.1% were from the TH The incidence of obligatory pathogens was 1.7 fold, and up to 3 times higher for facultative, unusual pathogens and contaminants in the TH and increased mainly due to an increase of E.coli, which was the most common isolated pathogen (n = 2,869), followed by Staphylococcus aureus (n = 1,439), Enterococcus sp. (n = 953) and Klebsiella sp. (n = 816). The distribution of obligatory pathogens differed between the hospital settings: In the TH Enterococcus sp. accounted for 40.8% and E.coli for 70.4%, respectively, whereas in the PH for 25.4% (p<0.0001) and 57.8%, respectively (p<0.0001) Antibiotic resistance of Gram negative bacteria and Staphylococcus aureus did not change during the observation period. Carbapenem resistance of Klebsiella sp. and vancomycin and linezolid resistance of Enterococcus faecium showed a non-significant increase since 2010 in the TH setting. We concluded that the incidence of BSI in TH was higher compared to PH. We observed higher contamination rates in the TH. We could not interpret the data of coagulase negative staphylococci due to lack of clinical data. We strongly recommend enhancement of training on blood culture sampling to decrease the rate of contamination. Due to differences in pathogen distribution and antimicrobial resistance between different hospital settings we recommend separate treatment guidelines for BSI by hospital setting.

The impact of bacteremia on lipoprotein concentrations and patient's outcome: a retrospective analysis.

Bacteremia is a major clinical challenge requiring early treatment. Metabolic alterations occur during bacteremia, and accordingly plasma concentrations of lipoproteins LDL-C and HDL-C are substantially changed. We questioned whether bacteremia with Gram-negative versus Gram-positive bacteria causes contrasting changes of lipoprotein levels in order to differentiate between the 2-g stain types and if there is a relation with outcome parameters namely ICU-admission, 30-day mortality, duration of hospitalization. This is a retrospective dual-center cross-sectional study, including 258 patients with bacteremia. Plasma lipid levels were analyzed within 48 h to positive blood culture. Upon admission, HDL-C, LDL-C, and total cholesterol (p = 0.99) in plasma did not significantly differ between patients with Gram-negative and Gram-positive bacteremia, while significantly higher triglyceride concentrations were found in Gram-negative bacteremia (p < 0.05). 30-day mortality and ICU admission were associated with lower LDL-C and HDL-C concentrations as compared to survivors and non-ICU patients, and patients with HDL-C < 20 mg dl-1 and LDL-C < 55 mg dl-1 had a relative risk (RR) of 2.85 for ICU therapy requirement and RR = 2 of death within 30 days. Reduced HDL-C and LDL-C concentrations were associated with adverse patient's outcome in bacteremia. Discrimination between Gram-negative and Gram-positive pathogens upon lipoprotein patterns is unlikely.

Lyophilized allogeneic bone tissue as an antibiotic carrier.

The rising number of primary joint replacements worldwide causes an increase of revision surgery of endoprostheses due bacterial infection. Revision surgery using non-cemented implants seems beneficial for the long-term outcome and the use of antibiotic-impregnated bone grafts might control the infection and give a good support for the implant. In this study we evaluated the release of antibiotics from fresh-frozen and lyophilized allogeneic bone grafts. Lyophilized bone chips and fresh frozen bone chips were mixed with gentamicin sulphate, gentamicin palmitate, vancomycin, calcium carbonate/calcium sulphate impregnated with gentamicin sulphate, and calcium carbonate/calcium sulphate bone substitute material impregnated with vancomycin. The efficacy of each preparation was measured by drug release tests and bacterial susceptibility using B. subtilis, S. aureus and methicillin-resistant Staphylococcus aureus. The release of gentamicin from lyophilized bone was similar to the release rate from fresh frozen bone during all the experimental time. That fact might be related to the similar porosity and microstructure of the bone chips. The release of gentamicin from lyophilized and fresh frozen bone was high in the first and second day, decreasing and keeping a low rate until the end of the second week. Depending on the surgical strategy either polymethylmethacrylate or allogeneic bone are able to deliver sufficient concentrations of gentamicin to achieve bacterial inhibition within two weeks after surgery. In case of uncemented revision of joint replacements allogeneic bone is able to deliver therapeutic doses of gentamicin and peak levels immediately after implantation during a fortnight. The use of lyophilized and fresh frozen bone allografts as antibiotic carriers is recommended for prophylaxis of bone infection.

Antibiotic-loaded calcium carbonate/calcium sulfate granules as co-adjuvant for bone grafting.

In this study HERAFILL(®) granules containing gentamicin was evaluated as a bone void filling material once mixed with allograft bone grafts. The efficacy of the bone grafts mixed with HERAFILL(®) was measured by drug release tests and bacterial susceptibility using Bacillus subtilis, Staphylococcus epidermidis and Staphylococcus aureus. The effect of storage at -80 °C on the delivery and efficacy of gentamicin from bone grafts mixed with HERAFILL(®) was also investigated. Higher elution of gentamicin was detected in all stored groups (1 and 6 months) in comparison with non-stored samples. The gentamicin elution released from all groups was efficient on reducing S. aureus and S. epidermidis CFU. The susceptibility tests using S. aureus showed less resistance of the strain after 1 month of the elution storage. That resistance was not observed after 6 months of storage. The capacity of bone grafts to act as gentamicin carriers has been confirmed in this study. The different granules sizes did not interfere in the delivery rate of the antibiotics or in the activity against the bacteria. Storage at -80 °C does not interfere on the antibiotic activity.

Influence of poly-N-acetylglucosamine in the extracellular matrix on N-chlorotaurine mediated killing of Staphylococcus epidermidis.

N-chlorotaurine (NCT) has recently been shown to have bactericidal activity against bacterial biofilm on metal discs (Coraca-Huber et al., 2014). In a biofilm, Staphylococcus epidermidis polymerizes poly-N-acetylglucosamine (PNAG) to form an extracellular matrix (ECM). Pseudomonas aeruginosa does not express this PNAG and has been shown to be highly susceptible to NCT. We compared the action of NCT on S. epidermidis 1457, a PNAG positive strain (SE1457) and S. epidermidis 1457- M10 an isogenic PNAG negative mutant (SE1457 M10). NCT-mediated killing was more effective and quicker on the PNAG negative strain SE1457 M10. Bacteria hidden in biofilms for prolonged periods of time were generally more susceptible than freshly formed biofilms. The differences in NCT-mediated killing might not be direct effects since NCT did not react with the monomeric N-acetylglucosamine, but might be explained by denser growth in the PNAG-containing biofilm produced by the wild type strain, which results in delayed penetration of NCT. The higher susceptibility of older biofilms to NCTmediated killing could be explained by more pronounced 3D architecture and subsequent larger surface area for interactions with NCT.

Calcium carbonate powder containing gentamicin for mixing with bone grafts.

Bone grafts are used for reconstructing bone defects caused by implant-associated complications, trauma, and tumors. Surgery with bone allografts is complex and time consuming; therefore, it is prone to a higher infection rate (2.0%-2.5%). In the case of site infection, systemically administered antibiotics cannot reach the infected bone graft. This study evaluated the use of resorbable bone graft substitute powder (HERAFILL; Heraeus Medical GmbH, Wehrheim, Germany) as a bone void-filling material as well as an antibiotic carrier for mixing with bone grafts. The antibiotic activity of the bone chips mixed with HERAFILL powder was measured by drug release tests and bacterial susceptibility with Bacillus subtilis, Staphylococcus epidermidis, and Staphylococcus aureus. HERAFILL powder was added to the bone chips (bone chips/HERAFILL; w/w = 1:1), mixed with a spatula, and vortexed for 1 minute. Gentamicin base release was evaluated in phosphate-buffered saline for up to 7 days using B subtilis bioassay. Antimicrobial efficacy was tested with S aureus and S epidermidis. The average amount of gentamicin base released from bone chips mixed with HERAFILL at 0 to 12 hours was 99.66 mg/mL. On day 7, the gentamicin base released 0.42 mg/mL. The elution released from bone chips mixed with HERAFILL promoted the formation of a zone of inhibition on S epidermidis and S aureus plates. This study confirmed the capacity of bone grafts to act as antibiotic carriers once mixed with HERAFILL powder. Bone chips mixed with HERAFILL showed efficacy against S aureus and S epidermidis.

Effect of freezing on the release rate of gentamicin palmitate and gentamicin sulfate from bone tissue.

In this study we evaluated gentamicin palmitate salt and gentamicin sulfate salt mixed with bone chips after storage at -80°C. Different concentration rates of gentamicin sulfate and gentamicin palmitate were mixed with human bone chips and stored for 1-6 months at -80°C. Nonstored samples were used as control. The release of the antibiotics from the bone was carried out in phosphate-buffered saline. Antibiotic concentrations in the elutions were determined with microbiological agar diffusion assay using Bacillus subtilis. Susceptibility tests were carried out using Staphylococci strains. The rate of gentamicin base (GB) released from bone was similar for all gentamicin salts and all storage conditions. The elutions released were efficient on reducing S. aureus and S. epidermidis CFU during all storage time. In resume, the capacity of bone grafts to act as gentamicin carriers has been confirmed in this study. GS + GP showed equivalent efficacy against S. aureus and S. epidermidis compared with GS pure. The lower delivery rate of GS + GP, related to its affinity with fat tissue can be an advantage for longer release times, increasing the local protection against infections. Storage at -80°C does not interfere on the gentamicin salts activity used.

Bactericidal activity of N-chlorotaurine against biofilm-forming bacteria grown on metal disks.

Many orthopedic surgeons consider surgical irrigation and debridement with prosthesis retention as a treatment option for postoperative infections. Usually, saline solution with no added antimicrobial agent is used for irrigation. We investigated the activity of N-chlorotaurine (NCT) against various biofilm-forming bacteria in vitro and thereby gained significant information on its usability as a soluble and well-tolerated active chlorine compound in orthopedic surgery. Biofilms of Staphylococcus aureus were grown on metal alloy disks and in polystyrene dishes for 48 h. Subsequently, they were incubated for 15 min to 7 h in buffered solutions containing therapeutically applicable concentrations of NCT (1%, 0.5%, and 0.1%; 5.5 to 55 mM) at 37°C. NCT inactivated the biofilm in a time- and dose-dependent manner. Scanning electron microscopy revealed disturbance of the biofilm architecture by rupture of the extracellular matrix. Assays with reduction of carboxanilide (XTT) showed inhibition of the metabolism of the bacteria in biofilms. Quantitative cultures confirmed killing of S. aureus, Staphylococcus epidermidis, and Pseudomonas aeruginosa biofilms on metal alloy disks by NCT. Clinical isolates were slightly more resistant than ATCC type strains, but counts of CFU were reduced at least 10-fold by 1% NCT within 15 min in all cases. NCT showed microbicidal activity against various bacterial strains in biofilms. Whether this can be transferred to the clinical situation should be the aim of future studies.

Gentamicin palmitate as a new antibiotic formulation for mixing with bone tissue and local release.

During surgery with bone grafting, the impaction of bone tissue creates an avascular area where local circulation is disrupted. If infections arise, they may prevent systemically administered antibiotics from reaching the infected bone. In this study we evaluated gentamicin palmitate (GP) mixed with gentamicin sulfate (GS) as a coating for bone chips (BCh). The efficacy of the coated BCh was measured by gentamicin base release tests using B. subtilis, S. epidermidis and S. aureus. Gentamicin base release was evaluated in phosphate-buffered saline for up to 7 days using B. subtilis bioassay. Antimicrobial efficacy was tested with S. aureus and S. epidermidis. A significant difference on the release of gentamicin base between GS and GS + GP was observed. S. epidermidis are significantly more susceptible to GS + GP and GS than S. aureus. BCh can act as gentamicin carriers and showed efficacy against S. aureus and S. epidermidis.

Efficacy of antibacterial bioactive glass S53P4 against S. aureus biofilms grown on titanium discs in vitro.

We evaluated the effectiveness of different sizes of bioactive glass S53P4 against Staphylococcus aureus biofilms grown on metal discs in vitro. S. aureus biofilms were cultivated on titanium discs. BAG-S53P4 (0.5-0.8 mm and <45 µm) were placed in contact with the discs containing biofilms. Glass beads (0.5 mm) were used as a control. After each interval, the pH from each sample was measured. Colony forming units were counted for the biofilm recovery verification. In parallel, we tested the activity of bioactive glass against S. aureus planktonic cells. We found that BAG-S53P4 can suppress S. aureus biofilm formation on titanium discs in vitro. The suppression rate of biofilm cells by BAG-S53P4 <45 µm was significantly higher than by BAG-S53P4 0.5-0.8 mm. BAG-S53P4 has a clear growth-inhibitory effect on S. aureus biofilms. BAG-S53P4 <45 µm is more efficient against biofilm growth in vitro comparing with BAG-S53P4 0.5-0.8 mm. Bioactive glass S53P4 has potential to be used as bone substitute for the resolution of infection complications in joint replacement surgeries and treatment of chronic osteomyelitis.

Sepsis otopathy: experimental sepsis leads to significant hearing impairment due to apoptosis and glutamate excitotoxicity in murine cochlea.

Hearing loss is frequent in intensive care patients and can be due to several causes. However, sepsis has not been examined as a possible cause. The aim of this study is to assess the influence of experimental sepsis on hearing thresholds and to evaluate pathological changes in the cochlea. The cecal ligation puncture technique was used to induce sepsis in 18 mice. Results were compared with those from 13 sham-operated and 13 untreated control mice. The hearing thresholds of the animals were evaluated with auditory evoked brainstem responses prior to the induction of sepsis and again at the peak of the disease. Immediately after the second measurement, the mice were sacrificed and the inner ears harvested and prepared for further evaluation. The cochleae were examined with light microscopy, electron microscopy and immunohistochemistry for Bax, cleaved caspase-3 and Bcl-2. The mice with sepsis showed a significant hearing loss but not the control groups. Induction of apoptosis could be shown in the supporting cells of the organ of Corti. Furthermore, excitotoxicity could be shown at the basal pole of the inner hair cells. In this murine model, sepsis leads to significant hearing impairment. The physiological alteration could be linked to apoptosis in the supporting cells of the organ of Corti and to a disturbance of the synapses of the inner hair cells.

Rapid detection of bloodstream pathogens by real-time PCR in patients with sepsis.

Rapid detection of bloodstream infections is an important issue for a better patient outcome. The aim of our study was thus to evaluate the LightCycler SeptiFast assay for diagnosis of bloodstream pathogens in a tertiary hospital in Western Austria. The 71 blood samples of 61 patients with presumed sepsis were investigated and compared with conventional blood culture system results. In both assays, 51 samples (71.8 %) were negative. In 20 positive samples (28.2 %), 10 different pathogens were detected by either blood culture system or SeptiFast assay or by both methods. Five samples were positive in both assays. The agreement rate of blood culture system and SeptiFast assay was 78.9 %, the negative predictive value of SeptiFast assay versus blood culture system was 0.94, sensitivity was 0.63, and specificity 0.81. In 12 samples where a positive SeptiFast assay and a negative blood culture system result were obtained, the same pathogens as identified by SeptiFast assay were detected in samples from other body sites suggesting a correct positive detection. In 11.3 % of cases, the SeptiFast assay resulted in an adjustment of the patients' therapy. In 3 samples, the blood culture assay was positive whereas the SeptiFast assay yielded negative results. In two of these cases, the pathogens involved were not included in the SeptiFast detection list, in the third case SeptiFast assay failed to detect Candida glabrata.Thus we recommend the SeptiFast assay as a valuable tool for rapid diagnosis of bloodstream infections in addition to, but not as replacement for, the blood culture test.

Evaluation of MBEC™-HTP biofilm model for studies of implant associated infections.

The bacteria in implant-related infections can evade host defenses by forming biofilms. The more we understand biofilm behavior, the better we can fight against then clinically. In vitro models for biofilms allow tests simulating in vivo conditions. In this study we evaluated the Minimum Biofilm Eradication Concentration-High Throughput Plates (MBEC™-HTP) as biofilm in vitro model for studies of implant associated infections. Staphylococcus aureus and Staphylococcus epidermidis biofilms were grown on MBEC™-HTP. To ensure the biofilm formation, antibiotic susceptibility tests and scanning electron microscopy (SEM) was carried out. Susceptibility tests were carried out using gentamicin, vancomycin, rifampicin, fosfomycin, clindamycin, and linezolid. Colony forming units counting were carried out. Minimal inhibitory concentration (MIC) and biofilm inhibitory concentration (BIC) were estimated. The CFU counting showed potency of rifampicin and daptomycin against S. epidermidis biofilms and rifampicin against S. aureus biofilms. SEM images showed proteic material in contact with cells. The differences between BIC and MIC demonstrated the biofilm formation as well as the SEM images. Rifampicin and daptomycin are good choices against biofilm related infections. Moreover, after suggested modifications, the model used in this study is eligible to further studies of implant associated infections.

Enhanced activity of linezolid against Staphylococcus aureus in cerebrospinal fluid.

Linezolid is considered for treatment of central nervous system (CNS) infections caused by multidrug-resistant Gram-positive bacteria. Therefore, the influence of cerebrospinal fluid (CSF) on the antimicrobial activity of linezolid was evaluated in vitro. Time-kill curves were conducted in CSF and Mueller-Hinton broth (MHB) using Staphylococcus aureus (ATCC 29213) and Staphylococcus epidermidis (ATCC 12228) strains. In CSF lower linezolid concentrations were needed against S. aureus (1× MIC) and S. epidermidis (0.5× MIC) to achieve bacteriostasis than in MHB (4× MIC for both strains). Good activity of linezolid in CSF supports performance of clinical trials evaluating its potential for treatment of CNS infections.

Cerebrospinal fluid impairs antimicrobial activity of fosfomycin in vitro.

OBJECTIVES: Fosfomycin penetrates well into cerebrospinal fluid (CSF) and is considered for treatment of infections of the central nervous system (CNS). This study evaluated the influence of human CSF on the antimicrobial activity of fosfomycin. METHODS: Time-kill curves were performed in Mueller-Hinton broth (MHB) and in pooled human CSF using fosfomycin concentrations ranging from 0.25x to 8x MIC for a clinical Staphylococcus aureus isolate. To estimate the activity of fosfomycin at the target site, the concentration-time curve measured in CSF of a patient at steady state was simulated in vitro in human CSF using two S. aureus isolates. RESULTS: In CSF a higher fosfomycin concentration (8x MIC) was required to achieve sustained bacterial killing than in MHB (1x MIC). In vitro simulation of the pharmacokinetic profile measured in CSF of the selected patient showed initial killing, but terminal re-growth of both test strains. CONCLUSIONS: The antibacterial activity of fosfomycin is lower in CSF than in MHB, and drug concentrations slightly exceeding the MIC may not be sufficient to achieve bactericidal effects in the CNS.

Streptococcus milleri group isolates from blood cultures: consider surgical sepsis.

BACKGROUND: Streptococcus milleri group bacteria (SMG) frequently are involved in purulent infections, often resulting in abscess formation with a high recurrence rate. Blood stream infections with these organisms are infrequent. PATIENTS AND METHODS: Review of epidemiologic and clinical characteristics of SMG-related bacteremia. RESULTS: During a four-year period, 637 SMG-positive samples from 475 patients, including 45 positive blood cultures from 25 patients (7.1%), were identified. In one pediatric patient, the SMG was a contaminant, so 24 patients (5.1%) with 44 positive blood cultures (6.9%) were available for further evaluation. The study group consisted of 17 male and seven female patients with a median age of 56 years and a median hospital stay of 23 days (range 8-83 days). In 21 patients, monomicrobial SMG blood stream infection was found, whereas in three patients, polymicrobial infection was diagnosed (coagulase-negative staphylococci in two, Escherichia coli and Bacteroides fragilis in one). The source of the organism in the blood was intra-abdominal infection in six cases, soft tissue infection in another six cases, and cardiovascular and nervous system infection in three cases each. In the remaining cases, miscellaneous sources were identified. In 14 infected patients (58.3%), surgical interventions were performed before SMG bacteremia was diagnosed. All patients received antibiotic treatment with a median duration of 11.5 d (range 6-25 d). Five patients died, but no death was related directly to SMG infection. CONCLUSIONS: Sepsis with SMG is in most cases associated with surgical infection. Source control together with directed antibiotic therapy leads to an acceptable outcome. Immunosuppression and significant co-morbidities are common in patients with this type of infection.

Multiple-dose pharmacokinetics of telithromycin in peripheral soft tissues.

Based on clinicians' expectations of high concentrations of telithromycin (TEL) in tissues, combined with its excellent in vitro antimicrobial characteristics, TEL is casually considered as a potential therapeutic option for the therapy of minor cases of soft tissue or bite-wound infections. To clarify this clinically important issue, the present investigation was carried out to measure the pharmacokinetic profile of TEL in the interstitial space fluid (ISF) of skeletal muscle and subcutaneous adipose tissue by means of the microdialysis technique in 10 healthy subjects following repetitive daily doses of 800 mg TEL. These data were compared with free concentrations of TEL determined in plasma. External controls for the present examination were the use of historic, single-dose data collected by our study group utilising identical methods and the same trial subjects. Despite an increase in the median half-life from ca. 3 h after a single dose to ca. 10h at steady-state conditions in all compartments, accumulation of TEL in ISF of soft tissues and plasma was clinically non-relevant. Median free peak concentrations in plasma, skeletal muscle and subcutis were 0.52, 0.13 and 0.19 mg/L, respectively. The median ratios of the tissue to plasma free areas under the concentration-time curves from 0 to 24 h (fAUC(0-24) tissue/fAUC(0-24) plasma) were 0.27 and 0.58 for muscle and subcutis, respectively (P>0.05). The present multiple-dose investigation of TEL is in line with a previous single-dose study confirming that TEL 800 mg/day may not be optimally effective in the therapy of soft tissue infections.

Activities of antifungal agents against yeasts and filamentous fungi: assessment according to the methodology of the European Committee on Antimicrobial Susceptibility Testing.

We compared the activities of antifungal agents against a wide range of yeasts and filamentous fungi. The methodology of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) for yeasts and spore-forming molds was applied; and a total of 349 clinical isolates of Candida spp., other yeast species, Aspergillus spp., and nondermatophyte non-Aspergillus spp. were investigated. The average geometric mean (GM) of the MICs of the various drugs for Candida spp. were as follows: amphotericin B (AMB), 0.55 microg/ml; liposomal amphotericin B (l-AMB); 0.35 microg/ml; itraconazole (ITC), 0.56 microg/ml; voriconazole (VRC), 0.45 microg/ml; posaconazole (POS), 0.44 microg/ml; and caspofungin (CPF), 0.45 microg/ml. The data indicated that the majority of Candida spp. were susceptible to the traditional and new antifungal drugs. For Aspergillus spp., the average GM MICs of AMB, l-AMB, ITC, VRC, POS, and CPF were 1.49 microg/ml, 1.44 microg/ml, 0.65 microg/ml, 0.34 microg/ml, 0.25 microg/ml, and 0.32 microg/ml, respectively. For the various zygomycetes, the average GM MICs of AMB, l-AMB, ITC, and POS were 1.36 microg/ml, 1.42 microg/ml, 4.37 microg/ml, and 1.65 microg/ml, respectively. Other yeastlike fungi and molds displayed various patterns of susceptibility. In general, the minimal fungicidal concentrations were 1 to 3 dilutions higher than the corresponding MICs. POS, AMB, and l-AMB showed activities against a broader range of fungi than ITC, VRC, and CPF did. Emerging pathogens such as Saccharomyces cerevisiae and Fusarium solani were not killed by any drug. In summary, the EUCAST data showed that the in vitro susceptibilities of yeasts and filamentous fungi are variable, that susceptibility occurs among and within various genera and species, and that susceptibility depends on the antifungal drug tested. AMB, l-AMB, and POS were active against the majority of pathogens, including species that cause rare and difficult-to-treat infections.