Johann Hausdorfer

Activities of Antifungal Agents against Yeasts and Filamentous Fungi: Assessment according to the Methodology of the European Committee on Antimicrobial Susceptibility Testing

ABSTRACT 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 μg/ml; liposomal amphotericin B (l-AMB); 0.35 μg/ml; itraconazole (ITC), 0.56 μg/ml; voriconazole (VRC), 0.45 μg/ml; posaconazole (POS), 0.44 μg/ml; and caspofungin (CPF), 0.45 μg/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 μg/ml, 1.44 μg/ml, 0.65 μg/ml, 0.34 μg/ml, 0.25 μg/ml, and 0.32 μg/ml, respectively. For the various zygomycetes, the average GM MICs of AMB, l-AMB, ITC, and POS were 1.36 μg/ml, 1.42 μg/ml, 4.37 μg/ml, and 1.65 μg/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.

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.

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.

Plasma protein binding of fluoroquinolones affects antimicrobial activity

OBJECTIVES In contrast to most antimicrobial classes, there is a doubt about the impact of protein binding (PB) on the antimicrobial activity of fluoroquinolones. We set out to evaluate the suitability of previously used models for investigating the influence of PB on bacterial killing by fluoroquinolones. METHODS PB of moxifloxacin and trovafloxacin was determined in Mueller-Hinton broth (MHB) containing different concentrations of human serum or albumin. Bacterial growth curves of Staphylococcus aureus and Pseudomonas aeruginosa were determined in pure serum, pure MHB and MHB containing different amounts of serum or albumin. Killing of both strains at concentrations equal to the MIC was investigated for moxifloxacin and trovafloxacin in MHB and also in medium that showed PB values identical to those of pure serum. RESULTS Frequently used media for investigating the impact of PB, i.e. MHB containing 20% to 70% serum or 4% albumin, did not reach the level of PB achieved in pure serum or significantly hampered bacterial growth compared with pure MHB. PB in MHB containing 12% albumin was identical to that in pure serum but did not impair bacterial growth. Addition of 12% albumin significantly reduced bacterial killing by both fluoroquinolones compared with that found in pure MHB. CONCLUSIONS For fluoroquinolones, standard media might be insufficient to investigate the impact of PB on bacterial killing. MHB containing 12% albumin seems to be a promising medium in this context. For moxifloxacin and trovafloxacin, PB leads to significant reduction of antimicrobial activity.

Staphylococcus aureus biofilm formation and antibiotic susceptibility tests on polystyrene and metal surfaces

Aim:  We compared the MBEC™‐HTP assay plates made of polystyrene with metal discs composed of TMZF® and CrCo as substrates for biofilm formation.

Diagnosis of Bacteria In Vitro by Mass Spectrometric Fingerprinting:A Pilot Study

The identification of bacteria by using conventional microbiological techniques can be very time-consuming and circumstantial. In contrast, the headspace screening of bacterial cultures by analyzing their emitted volatile compounds using mass spectrometry might provide a novel approach in diagnostic microbiology. In the present study different strains of Escherichia coli, Klebsiella, Citrobacter, Pseudomonas aeruginosa, Staphylococcus aureus, and Helicobacter pylori were investigated. The volatile compounds emitted by these bacteria in vitro were analyzed using proton-transfer-reaction mass spectrometry, which allows rapid and sensitive measurement. The detected patterns of volatile compounds produced by the investigated bacteria were compared and substantial differences regarding both quantity and quality were observed. In conclusion, the present study is the first to describe headspace screening of bacterial cultures as a potential diagnostic approach in medical microbiology.

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.

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.

Bactericidal Activity of N-Chlorotaurine against Biofilm-Forming Bacteria Grown on Metal Disks

ABSTRACT 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.

Tigecycline has no effect on cytokine release in an ex vivo endotoxin model of human whole blood.

In previous studies, tetracyclines have been shown to decrease the release of cytokines in experimental settings of endotoxaemia. Tigecycline is the first member of the closely related glycylglycines and, due to its broad antimicrobial spectrum, it is considered useful in the treatment of sepsis. We therefore tested its ability to influence the concentrations of the proinflammatory cytokines interleukin (IL)-1beta, tumour necrosis factor-alpha (TNFalpha) and IL-6 in an established ex vivo model of human endotoxaemia. Whole blood from ten healthy volunteers was incubated with either saline (negative control), tigecycline (1 microg/mL [therapeutic concentration] or 100 microg/mL [supratherapeutic concentration]), lipopolysaccharide (LPS; 50 pg/mL, control) or a combination of tigecycline plus LPS (test group). Concentrations of IL-1beta, TNFalpha and IL-6 in the supernatant were measured using commercially available enzyme-linked immunosorbent assay (ELISA) kits. As expected, incubation with LPS significantly increased the cytokine concentrations in whole blood compared with baseline (P<0.05). The combination of tigecycline plus LPS did not exert any significant effect on the concentrations of IL-1beta, IL-6 and TNFalpha after 2h and 4h of incubation compared with LPS alone. These results indicate that proinflammatory cytokines remained unaffected by tigecycline in an established ex vivo model of systemic inflammatory response.