Detection of characteristic metabolites of Aspergillus fumigatus and Candida species using ion mobility spectrometry-metabolic profiling by volatile organic compounds.

Volatile metabolites of Aspergillus fumigatus and Candida species can be detected by gas chromatography/mass spectrometry (GC/MS). A multi-capillary column - ion mobility spectrometer (MCC-IMS) was used in this study to assess volatile organic compounds (VOCs) in the headspace above A. fumigatus and the four Candida species Candida albicans, Candida parapsilosis, Candida glabrata and Candida tropicalis in an innovative approach, validated for A. fumigatus and C. albicans by GC/MS analyses. For the detection of VOCs, a special stainless steel measurement chamber for the microbial cultures was used. The gas outlet was either attached to MCC-IMS or to adsorption tubes (Tenax GR) for GC/MS measurements. Isoamyl alcohol, cyclohexanone, 3-octanone and phenethylalcohol can be described as discriminating substances by means of GC/MS. With MCC-IMS, the results for 3-octanone and phenethylalcohol are concordant and additionally to GC/MS, ethanol and two further compounds (p_0642_1/p_683_1 and p_705_3) can be described. Isoamyl alcohol and cyclohexanone were not properly detectable with MCC-IMS. The major advantage of the MCC-IMS system is the feasibility of rapid analysis of complex gas mixtures without pre-concentration or preparation of samples and regardless of water vapour content in an online setup. Discrimination of fungi on genus level of the investigated germs by volatile metabolic profile and therefore detection of VOC is feasible. However, a further discrimination on species level for Candida species was not possible.

Difference in virulence between fluconazole-susceptible and fluconazole-resistant Candida albicans in a mouse model.

Two Candida albicans isolates were collected from a HIV-positive patient with recurrent oropharyngeal candidosis (OPC). One isolate was taken during the first episode of oral candidosis [fluconazole susceptible (FLU-S), minimal inhibitory concentration (MIC) = 0.25 mg l(-1) ] and the second after the patient developed refractory OPC and resistance to fluconazole (FLU-R, MIC = 64 mg l(-1)). Both isolates were clonally identical. Different in vitro studies were carried out to assess putative virulence factors of both isolates. Gene expressions of efflux pumps and CSH1 were determined as well as adherence to human epithelial cells, determination of proteinase secretion and biofilm formation activity. Virulence was studied using a disseminated mouse model. All mice challenged with the FLU-S isolate survived the experiment when FLU was given. However, when FLU was absent, the mortality of the FLU-S isolate was higher than that of the FLU-R isolate with no mice surviving the experiment. In vitro studies showed pronounced growth rates of the FLU-S isolate and a more intense biofilm-building activity compared with the FLU-R isolate. The FLU-R isolate highly up-regulated MDR1 and CSH1. This isolate also adhered stronger to the epithelial cell line. The results showed that FLU-S and FLU-R isolates exhibit different virulence factors, which enable the survival of both isolates in adapted environments.

Two serial check valves can prevent cross-contamination through intravenous tubing during total intravenous anesthesia.

BACKGROUND: Nonsterile handling of propofol for anesthesia has been linked with severe sepsis and death. Placing a single check valve in the IV tubing does not prevent retrograde ascension of pathogens into propofol-filled syringes, so we designed an IV tubing set with multiple check valves. To estimate the efficacy of this design, we measured the concentration of pathogens detected upstream in the IV tubing in relation to the pathogen concentration in a model of a contaminated patient. METHODS: A glass container with a rubber sealed port was filled with a suspension of either bacteria or phagocytes and kept at 37°C ("contaminated patient" model). A bag of normal saline was connected to an IV cannula, punctured through the rubber sealed port of the patient model. Two additional sidestream infusion lines were connected to syringes in 2 standard infusion pumps. One of the syringes contained propofol and the other contained normal saline as a substitute for an opioid preparation. After 5 hours of infusion, we obtained samples from different parts of the infusion lines and syringes. The samples were streaked out on blood agar plates and incubated at 37°C for 24 hours. We repeated this experiment with 6 different pathogens. RESULTS: We incubated 825 agar plates. Whereas the concentration of bacteria and phagocytes in the "patient" had significantly increased during the 5-hour experiments (positive control), no bacterial growth could be detected in any of the incubated plates. CONCLUSION: The data from this experimental setting suggest that the design with multiple check valves in paired configuration prevents retrograde contamination. Of note, this does not permit the reuse of propofol syringes because reusing is against the manufacturer's recommendations.

Decontamination efficacy of antiseptic agents on in vivo grown biofilms on rough titanium surfaces.

OBJECTIVE: Four common antimicrobial agents were evaluated for their efficacy in reduction of aerobic bacteria intraorally grown in biofilms on rough titanium samples. The solutions investigated contained chlorhexidine, essential oil, octenidine, or citric acid. METHOD AND MATERIALS: Twenty volunteers wore splints with titanium sleeves intraorally for 10 days. Following irrigation with the antiseptics, the sleeves were removed and biofilm samples were taken by swabbing. The bacteria were first examined microscopically by Gram staining. These samples from the surfaces were then cultured under aerobic conditions to identify and quantify the colonizing bacteria. RESULTS: Compared to untreated controls, significant (P < .05) differences in antimicrobial efficacy were observed for the different regimens depending on bacterial species or even the subtype. The reduction rates achieved varied from 30.0% after 2 minutes of rinsing with chlorhexidine to 99.8% after 8 minutes of rinsing with octenidine. CONCLUSION: The irrigation regimens studied in this investigation reduced bacterial colonization in a mature biofilm grown intraorally on rough titanium surfaces. The highest absolute reduction was achieved after 8 minutes, but only the 2-minute reduction rates are significant for clinical practice. Taking this into consideration, the distinct decontamination efficacy of octenidine and citric acid is evident.

Dental therapy before and after radiotherapy--an evaluation on patients with head and neck malignancies.

The present investigation evaluates the dental care situation of patients with head and neck cancer before and after radiotherapy. The situations of these patients in 1993 and 2005 were compared to detect similarities, differences and developments. In the years 1993 and 2005, 37 and 36 patients, respectively, with head and neck cancer treated by the local departments of otorhinolaryngology and of radiotherapy were examined consecutively according to their aftercare appointments. Time points of radiotherapy treatment of the patients evaluated in 1993 varied from 1984 to 1993. The patients evaluated in 2005 had received radiotherapy between 1998 and 2005. Therefore the applied radiotherapeutic regimen differed not only between the two groups of patients, but also within each group. The information for these investigations was provided anonymously. It was evaluated with descriptive statistics. The evaluation of the data shows distinct differences with respect to preventive and therapeutic dental care measures. In 2005, 35 out of 36 patients (97.2%) had a dental consultation before radiotherapy (1993, 65%). All 27 dentate patients (100%) obtained a splint for fluoride application (1993, none). 29% fewer edentulous patients were seen than in 1993. The number of teeth destroyed decreased from 19.2% (1993) to 7.8% in 2005. Mycoses due to Candida spp. and chronic failures in wound healing were rare (5.5%). In the course of the 12 years, prophylactic measures, such as the application of splints for fluoride treatment, were intensified. However, concepts for the dental care of patients undergoing radiotherapy, especially following the radiation, should be widened to avoid ruined teeth and long delayed wound healings.

Decontamination efficacy of erbium:yttrium-aluminium-garnet and diode laser light on oral Candida albicans isolates of a 5-day in vitro biofilm model.

The different forms of superficial and systemic candidiasis are often associated with biofilm formation on surfaces of host tissues or medical devices. The biofilm formation of Candida spp., in general, necessitates significantly increased amounts of antifungal agents for therapy. Often the therapeutic effect is doubtful. A 5-day biofilm model with oral Candida isolates was established according to Chandra et al. (J Dent Res 80:903-908, 2001) on glass and titanium surfaces and was modified by Sennhenn-Kirchner et al. (Z Zahnärztl Implantol 3:45-51, 2007) to investigate different aspects unanswered in the field of dentistry. In this model, the efficacy of erbium:yttrium-aluminium-garnet (Er:YAG) light (2940 nm, 100 mJ, 10 Hz, 300 micros pulsed mode applied for 80 s) and diode laser light (810 nm, 1 W, continuous wave mode applied for 20 s with four repetitions after 30 s pauses each) was evaluated and compared to untreated controls. The photometric evaluation of the samples was completed by observations on morphological changes of yeast cells grown in the biofilm. Compared to the untreated controls Candida cells grown in mature in vitro biofilms were significantly reduced by both wavelengths investigated. Comparison between the different methods of laser treatment additionally revealed a significantly greater effect of the Er:YAG over the diode laser. Scanning electron microscopy findings proved that the diode laser light was effective in direct contact mode. In contrast, in the areas without direct contact, the fungal cells were left almost unchanged. The Er:YAG laser damaged the fungal cells to a great extent wherever it was applied.

Preoperative sterilization and disinfection of drill guide templates.

The aim of the in vitro study was to evaluate the decontamination potential of common antiseptic solutions for heat-sensitive implantological drill guide templates. One hundred implantologists were evaluated on the basis of a questionnaire for their measures of disinfection. On the basis of these results, 80% alcohol, Octenidine 0.1%, and Chlorhexidine 0.12% were tested in an in vitro model for their decontamination efficacy for heat-sensitive plastic material infected with Pseudomonas aeruginosa, Acinetobacter baumannii, Enterococcus faecalis, Enterococcus faecium, Staphylococcus aureus, Enterobacter cloacae, Escherichia coli, and Candida albicans. The microorganisms were selected on the basis of results of environmental testing of dental laboratories. The results of the questionnaire revealed that Chlorhexidine was used by 30%, 80% alcohol by 23%, and Octenidine by 7% of the dentists. Using the in vitro model, with the exception of S. aureus, Chlorhexidine was not able to completely eliminate the microorganisms after 15 min of application. In contrast, the treatment with Octenidine revealed no further growth of the tested microorganisms after that time. The 80% alcohol was more efficient. No growth of microorganisms could be detected in any of the tests after 5 min of incubation. On the basis of our results and due to the fact that suitable installations for sterilization were hardly used by the dental practitioners, the disinfection of templates should be preferentially performed with 80% alcohol or Octenidine using an incubation time of 15 min with ultrasonication.

Epidemiology and antifungal susceptibilities of Candida spp. to six antifungal agents: results from a surveillance study on fungaemia in Germany from July 2004 to August 2005.

OBJECTIVES: Data on fungal infections occurring in Germany are rare to date. The aim of the present study was to survey the epidemiological situation in Germany, to provide data on the susceptibility of the fungal isolates to antifungals. METHODS: Five hundred and sixty-one Candida isolates were collected from primarily sterile sites of patients from July 2004 to August 2005 with the aid of a nationwide established laboratory network, MykolabNet-D. The MICs of amphotericin B, flucytosine, fluconazole, itraconazole, voriconazole and caspofungin were determined using the microdilution reference procedure M27-A2 of the CLSI. RESULTS: Candida albicans was the most frequently isolated species (58.5%), followed by Candida glabrata (19.1%), Candida parapsilosis (8.0%) and Candida tropicalis (7.5%). In contrast, the isolation rate of Candida krusei (1.4%) was low. Candida kefyr appeared as a new pathogen in this profile. Amphotericin B revealed excellent activity, with only three resistant isolates (0.5%). A total of 25 isolates (4.5%) showed resistance against flucytosine. All 25 isolates were identified as C. tropicalis indicating a peculiarity within German isolates. The resistance rate of all tested isolates to fluconazole and to itraconazole was 3.7% and 17.6%, respectively. According to the provisional breakpoints, two isolates (0.4%) were tested as resistant to voriconazole. Caspofungin was active against the majority of isolates where an intrinsic resistance is unknown. CONCLUSIONS: This latest German survey of isolates from patients with fungaemia demonstrates a favourable situation with respect to antifungal susceptibilities for the antifungal substances tested.

Decontamination of rough titanium surfaces with diode lasers: microbiological findings on in vivo grown biofilms.

OBJECTIVES: The bactericidal efficacy of diode lasers has already been demonstrated in vitro. We investigated the reduction of aerobe bacteria - colonizing rough titanium samples in biofilms intraorally grown - by diode lasers of different wave lengths. MATERIAL AND METHODS: Twenty-two volunteers participated in the trial. They were fitted for 10 days with custom-made intraoral plastic splints carrying titanium sleeves. A part of the sleeves was then irradiated with diode lasers in different modes. The other part remained non-irradiated and served as control. Directly after irradiation, the sleeves were swabbed and the gained bacteria were first examined microscopically and then were cultured under aerobic conditions. RESULTS: The bacteria in the controls and in the treated samples were quantified. A comparison with the controls revealed a marked overall reduction of bacterial colonization in all irradiated sleeves. Continuous irradiation for 20 s reduced bacteria counts by 99.67% at 810 nm and 99.58% at 980 nm. Repeating the 20 s exposure five times reduced counts by 99.98% at 810 nm and by 99.39% at 980 nm. A 98.86% reduction was seen after irradiation in pulsed mode. A further analysis in respect of different isolated bacteria revealed that the streptococci group was reduced by 99.29-99.99%, while the staphylococci group was reduced to a lesser extent in the range 94.67-99.99%. CONCLUSION: The results are of clinical relevance. In comparison with the mean bacterial counts of the untreated samples, all irradiation programs studied in this investigation reduced mean bacterial colonization in a biofilm on intraoral rough titanium surfaces by more than 98%. The actual extent of reduction was dependent on the bacteria species as well as on the irradiation mode.

Secreted aspartic proteases as virulence factors of Candida species.

Candida infections have emerged as a significant medical problem during the last few decades. Among the different virulence traits of C. albicans, secreted proteolytic activity has been intensively investigated. Pathogenesis of the various forms of candidiasis was shown to be associated with the differential and temporal regulation of the expression of genes coding for secreted aspartic proteases (Sap). These enzymes act as cytolysins in macrophages after phagocytosis of Candida, are present in tissue penetration and are also involved in adherence to epithelial cells. Since the introduction of new antiretroviral therapeutics such as HIV protease inhibitors, oropharyngeal candidiasis is less often observed in AIDS patients. Different HIV aspartic protease inhibitors were able to inhibit the C. albicans Saps involved in adherence. The lower rates of oropharyngeal candidiasis observed in individuals receiving antiretroviral combination therapy could reflect not only an improvement in the immune system but also direct inhibition of Candida Saps by HIV protease inhibitors. Therefore, the development of specific aspartic protease inhibitors might be of interest for the inhibition of candidiasis.

Effect of Micafungin (FK463) on Candida albicans adherence to epithelial cells.

BACKGROUND: Adherence is considered a major virulence trait of Candida albicans. FK463 is a new investigational intravenous antifungal of the 'candin family' with potent in vitro and in vivo activity against Candida spp. OBJECTIVE: The aim of the present study was to investigate the effect of Micafungin (FK463) on Candida adherence to epithelial cells of azole-sensitive and azole-resistant C. albicans isolates. METHODS: An in vitro assay using microtest plate technology and fluorescence measurement was developed to compare the adherence of C. albicans SC5314 and of paired C. albicans isolates to epithelial cells in the presence and in the absence of FK463. RESULTS: FK463 showed a marked inhibitory effect on the adherence of C. albicans SC5314. The addition of FK463 reduced the adherence of C. albicans SC5314 to 90% of the value of control without drug. A dose-dependent adherence inhibition was observed with FK463 in the range of 10-0.015 microg ml(-1). The comparison of paired C. albicans isolates, either a fluconazole-susceptible and a fluconazole-resistant isolate of one patient, revealed no significant difference in the adherence behavior between azole-susceptible and azole-resistant. CONCLUSION: Micafungin (FK463) has the capacity to reduce adherence of C. albicans azole-susceptible and azole-resistant strains to epithelial cells.

Adherence of different Candida dubliniensis isolates in the presence of fluconazole.

BACKGROUND: The recently described yeast species Candida dubliniensis is closely related to C. albicans and has been recovered predominantly from the oral cavities of HIV-infected individuals and AIDS patients who are often receiving fluconazole as prophylactic or therapeutic treatment for oropharyngeal candidiasis. Like C. albicans, C. dubliniensis secretes aspartic proteinases which in C. albicans have been shown to be involved in adherence. OBJECTIVE: To explain the increasing prevalence of C. dubliniensis in AIDS patients and to investigate the virulence factors of this yeast. METHODS: An in vitro assay was developed to compare the adherence to epithelial cells of C. dubliniensis from HIV-patients with that of C. albicans. RESULTS: All C. albicans isolates adhered better than the 22 C. dubliniensis isolates. In the presence of fluconazole, the C. dubliniensis isolates tested showed increased adherence as compared with controls without fluconazole. In contrast, all C. albicans isolates decreased in adherence to epithelial cells in the presence of fluconazole independently of their in vitro susceptibility to this drug. Proteinase antigens are present on the surface of C. dubliniensis cells adherent to epithelial target cells. In the presence of fluconazole this proteinase antigen was more strongly expressed. CONCLUSION: Increased adherence of C. dubliniensis strains in the presence of fluconazole could explain its high recovery rate from HIV-positive patients in recent years. The induction of proteinase secretion in the presence of fluconazole found for most of the C. dubliniensis isolates could be one of the factors involved in adherence.

Impact of N-chlorotaurine on viability and production of secreted aspartyl proteinases of Candida spp.

N-Chlorotaurine, an endogenous long-lived oxidant, demonstrated fungicidal activity against Candida spp. and a postantifungal effect. Secreted aspartyl proteinases, important fungal virulence factors, proved to be a first target of impact. These results provide support for the topical application of N-chlorotaurine as an antimicrobial agent in yeast infections.