Mycological Methods for Routine Air Sampling and Interpretation of Results in Operating Theaters.

Many infectious diseases are transmitted via the air and are, therefore, particularly difficult to combat. These infections include various invasive mycoses caused by molds. The usual route of infection is the inhalation of conidia. In hospitals, infection can also occur through the deposition of conidia in otherwise sterile anatomical sites during surgical and other invasive procedures. Therefore, knowledge of airborne mold concentrations can lead to measures to protect patients from fungal infections. The literature on this topic contains insufficient and sometimes ambiguous information. This is evidenced by the fact that there are no international recommendations or guidelines defining the methodology of air sampling and the interpretation of the results obtained. Surgical departments, intensive care units and medical mycology laboratories are, therefore, left to their own devices, leading to significant differences in the implementation of mycological surveillance in hospitals. The aim of this mini-review is to provide an overview of the current methods of air sampling and interpretation of results used in medical mycology laboratories.

Effects of Anthropogenic Disturbance and Seasonal Variation on Aerobiota in Highly Visited Show Caves in Slovenia.

Aerosols in caves are natural tracers and, together with climatic parameters, provide a detailed insight into atmospheric conditions, responses to climatic changes and anthropogenic influences in caves. Microbiological air monitoring in show caves is becoming increasingly useful to understand changes in cave ecosystems and to implement and review measures for sustainable cave use and tourism development. In 2017 and 2018, air along tourist trails in caves Postojnska jama and Skocjanske jame (Slovenia) was sampled before and after tourist visits. Samples were analysed using culture-dependent methods, flow cytometry, detection of ß-D-glucan and lipopolysaccharide and compared with CO2 and temperature data to measure anthropogenic influences and seasonality on aerobiota. While the presence of tourists significantly increased concentrations of airborne microorganisms (p < 0.05), ß-D-glucan and CO2 did not show such a trend and were more dependent on seasonal changes. Locally, concentrations of cultivable microorganisms above 1000 CFU/m3 were detected, which could have negative effects on the autochthonous microbiota and possibly on human health. A mixture of bacteria typically associated with humans was found in the air and identified with MALDI-TOF MS. Using MALDI-TOF MS, we achieved a 69.6% success rate in identification. Micrococcus luteus, Streptococcus mitis, Staphylococcus epidermidis and Moraxella spp. were recognized as good indicators of cave anthropisation.

Predictors for Prolonged Hospital Stay Solely to Complete Intravenous Antifungal Treatment in Patients with Candidemia: Results from the ECMM Candida III Multinational European Observational Cohort Study.

BACKGROUND: To date, azoles represent the only viable option for oral treatment of invasive Candida infections, while rates of azole resistance among non-albicans Candida spp. continue to increase. The objective of this sub-analysis of the European multicenter observational cohort study Candida III was to describe demographical and clinical characteristics of the cohort requiring prolonged hospitalization solely to complete intravenous (iv) antifungal treatment (AF Tx). METHODS: Each participating hospital (number of eligible hospitals per country determined by population size) included the first ~ 10 blood culture proven adult candidemia cases occurring consecutively after July 1st, 2018, and treating physicians answered the question on whether hospital stay was prolonged only for completion of intravenous antifungal therapy. Descriptive analyses as well as binary logistic regression was used to assess for predictors of prolonged hospitalization solely to complete iv AF Tx. FINDINGS: Hospital stay was prolonged solely for the completion of iv AF Tx in 16% (100/621) of candidemia cases by a median of 16 days (IQR 8 - 28). In the multivariable model, initial echinocandin treatment was a positive predictor for prolonged hospitalization to complete iv AF Tx (aOR 2.87, 95% CI 1.55 - 5.32, p < 0.001), while (i) neutropenia, (ii) intensive care unit admission, (iii) catheter related candidemia, (iv) total parenteral nutrition, and (v) C. parapsilosis as causative pathogen were found to be negative predictors (aOR 0.22 - 0.45; p < 0.03). INTERPRETATION: Hospital stays were prolonged due to need of iv AF Tx in 16% of patients with candidemia. Those patients were more likely to receive echinocandins as initial treatment and were less severely ill and less likely infected with C. parapsilosis.

Different BD BACTEC™ Blood Culture Bottle Types for the Detection of Fungi in Simulated Sterile Body Fluid Samples.

Blood culture systems are a potential alternative to classical cultivation of fungi on mycological media, but there are limited data on the suitability of these systems for culturing other sample types (e.g., sterile body fluids). We conducted a prospective study to evaluate different types of blood culture (BC) bottles for the detection of different fungal species in non-blood samples. A total of 43 fungal isolates were tested for their ability to grow in BD BACTEC Mycosis-IC/F (Mycosis bottles), BD BACTEC Plus Aerobic/F (Aerobic bottles) and BD BACTEC Plus Anaerobic/F (Anaerobic bottles) (Becton Dickinson, East Rutherford, NJ, USA) BC bottles inoculated with spiked samples without the addition of blood or fastidious organism supplement. Time to detection (TTD) was determined for all BC types tested and compared between groups. In general, Mycosis and Aerobic bottles were similar (p > 0.05). The Anaerobic bottles failed to support growth in >86% of cases. The Mycosis bottles were superior in detecting Candida glabrata, Cryptococcus spp. and Aspergillus spp. (p < 0.05). The performance of Mycosis and Aerobic bottles was similar, but if cryptococcosis or aspergillosis is suspected, the use of Mycosis bottles is recommended. Anaerobic bottles are not recommended for fungal detection.

Long-Term Monitoring of Bioaerosols in an Environment without UV and Desiccation Stress, an Example from the Cave Postojnska Jama, Slovenia.

A natural cave environment subject to regular human visitation was selected for aerobiological study to minimize the effects of severe temperature fluctuations, UV radiation, and desiccation stress on the aerobiome. The longer sampling period of bioaerosols, up to 22 months, was generally not associated with a proportionally incremental and cumulative increase of microbial biomass. The culture-independent biomass indicator ATP enabled quick and reliable determination of the total microbial biomass. Total airborne microbial biomass was influenced by human visitation to the cave, as confirmed by significantly higher concentrations being observed along tourist footpaths (p < 0.05). Airborne beta-glucans (BG) and lipopolysaccharide (LPS) are present in cave air, but their impact on the cave remains to be evaluated. Staphylococcus spp., as an indicator of human presence, was detected at all sites studied. Their long-term survival decrease is likely due to high relative humidity, low temperature, the material to which they adhere, and potentially natural elevated radon concentration. The most commonly recorded species were: S. saprophyticus, which was identified in 52% of the studied sites, S. equorum in 29%, and S. warneri in 24% of the studied sites. Only a few isolates were assigned to Risk group 2: S. aureus, S. epidermidis, S. haemolyticus, S. pasteuri, and S. saprophyticus.

Interaction between Microalgae P. tricornutum and Bacteria Thalassospira sp. for Removal of Bisphenols from Conditioned Media.

We studied the efficiency of three culture series of the microalgae Phaeodactylum tricornutum (P. tricornutum) and bacteria Thalassospira sp. (axenic microalgae, bacterial culture and co-culture of the two) in removing bisphenols (BPs) from their growth medium. Bacteria were identified by 16S ribosomal RNA polymerase chain reaction (16S rRNA PCR). The microorganism growth rate was determined by flow cytometry. Cultures and isolates of their small cellular particles (SCPs) were imaged by scanning electron microscopy (SEM) and cryogenic transmission electron microscopy (Cryo-TEM). BPs were analyzed by gas chromatography coupled with tandem mass spectrometry (GC-MS/MS). Our results indicate that some organisms may have the ability to remove a specific pollutant with high efficiency. P. tricornutum in axenic culture and in mixed culture removed almost all (more than 99%) of BPC2. Notable differences in the removal of 8 out of 18 BPs between the axenic, mixed and bacterial cultures were found. The overall removals of BPs in axenic P. tricornutum, mixed and bacterial cultures were 11%, 18% and 10%, respectively. Finding the respective organisms and creating microbe societies seems to be key for the improvement of wastewater treatment. As a possible mediating factor, numerous small cellular particles from all three cultures were detected by electron microscopy. Further research on the mechanisms of interspecies communication is needed to advance the understanding of microbial communities at the nano-level.

Candidaemia in Central Slovenia: A 12-year retrospective survey.

BACKGROUND: Candida bloodstream infections (BSI) became an important invasive disease in the late 20th century, in particular among immunocompromised patients. Although considerable progress has been made in the management of patients with invasive mycoses, Candida BSI are still widespread among hospitalised patients and are associated with relatively high mortality. OBJECTIVES: We conducted a retrospective study to evaluate patient characteristics, incidence, species distribution and antifungal susceptibility of BSI isolates of Candida spp. as well as outcomes of Candida BSI from 2001 to 2012, before the widespread use of echinocandins. This is the first epidemiological study of Candida BSI in Slovenia so far. METHODS: All documented candidaemia cases from 2001 to 2012 in two major hospitals-University Medical Centre and Institute of Oncology in Ljubljana, Slovenia-were taken into consideration. Candida BSI were identified in 422 patients (250 male, 172 female). Laboratory and clinical data of these patients were retrospectively analysed. Mann-Whitney U test was used to compare continuous variables and Fisher's exact test or chi-squared test for categorical variables. RESULTS AND CONCLUSIONS: The average incidence of Candida BSI was 0.524/10.000 patient-days (0,317/1000 admissions); 16/422 were younger than 1 year and 251/422 patients were over 60 years old. The most commonly isolated species were Candida albicans and Candida glabrata, followed by Candida parapsilosis. Majority of the patients had a single episode of Candida BSI, multiple episodes of Candida BSI occurred in 18/434 patients (4.1%); in 25/434 patients (5.8%) mixed Candida BSI were observed. Crude 30-day case-fatality rate was 55.4%.

Phenotypes Associated with Pathogenicity: Their Expression in Arctic Fungal Isolates.

Around 85% of the environments on Earth are permanently or seasonally colder than 5 °C. Among those, the poles constitute unique biomes, which harbor a broad variety of microbial life, including an abundance of fungi. Many fungi have an outstanding ability to withstand extreme conditions and play vital ecosystem roles of decomposers as well as obligate or facultative symbionts of many other organisms. Due to their dispersal capabilities, microorganisms from cryosphere samples can be distributed around the world. Such dispersal involves both species with undefined pathogenicity and potentially pathogenic strains. Here we describe the isolation of fungal species from pristine Arctic locations in Greenland and Svalbard and the testing of the expression of characteristics usually associated with pathogenic species, such as growth at 37 °C, hemolytic ability, and susceptibility to antifungal agents. A total of 320 fungal isolates were obtained, and 24 of the most abundant and representative species were further analyzed. Species known as emerging pathogens, like Aureobasidium melanogenum, Naganishia albida, and Rhodotorula mucilaginosa, were able to grow at 37 °C, showed beta-hemolytic activity, and were intrinsically resistant to commonly used antifungals such as azoles and echinocandins. Antifungal resistance screening revealed a low susceptibility to voriconazole in N. albida and Penicillium spp. and to fluconazole in Glaciozyma watsonii and Glaciozyma-related taxon.

Molecular Characterization and Antifungal Susceptibility Testing of Sequentially Obtained Clinical Cryptococcus deneoformans and Cryptococcus neoformans Isolates from Ljubljana, Slovenia.

AIM: To retrospectively investigate the epidemiology of cryptococcosis in Ljubljana, Slovenia. METHODOLOGY: Forty-six sequentially obtained isolates from 19 patients were subjected to amplified fragment length polymorphism (AFLP) genotyping, microsatellite typing, mating- and serotype PCRs and antifungal susceptibility testing. RESULTS: Majority of the isolates were Cryptococcus deneoformans (n = 29/46; 63%) followed by Cryptococcus neoformans (n = 16/46; 34.8%) and their interspecies hybrid (n = 1/46; 2.2%). Mating-type α was predominant, two mating-type a C. deneoformans isolates and one mating-type a/α isolate were observed. Several mixed infections were found by microsatellite typing; one patient had a persisting C. deneoformans infection for > 2.5 years. For C. deneoformans, the in vitro antifungal MIC90 and susceptibility ranges were for amphotericin B 0.25 µg/ml (0.031-0.25 µg/ml), 5-fluorocytosine 0.25 µg/ml (0.063-4 µg/ml), fluconazole 8 µg/ml (0.5-16 µg/ml), voriconazole 0.063 µg/ml (0.008-0.125 µg/ml), posaconazole 0.063 µg/ml (0.008-0.063 µg/ml) and itraconazole 0.063 µg/ml (0.031-0.125 µg/ml). For C. neoformans, these values were for amphotericin B 0.25 µg/ml (0.063-0.5 µg/ml), 5-fluorocytosine 1 µg/ml (0.063-1 µg/ml), fluconazole 16 µg/ml (0.5-64 µg/ml), voriconazole 0.125 µg/ml (0.008-0.25 µg/ml), posaconazole 0.063 µg/ml (0.008-0.063 µg/ml) and itraconazole 0.063 µg/ml (0.031-0.125 µg/ml). CONCLUSIONS: Majority of the cases were caused by C. deneoformans; mating-type α was predominant. Several mixed infections were identified by AFLP genotyping and microsatellite typing. Despite antifungal therapy, a cryptococcal isolate could persist for years. Voriconazole, itraconazole and posaconazole were the most potent antifungal drugs.

First report of otitis externa caused by Schizophyllum commune and review of the literature.

Basidiomycete Schizophyllum commune is a widely distributed cellulolytic fungus that is a well-known pathogen. It can cause a wide range of different infections, and here we describe the first case of otitis externa and a molecularly based identification process.

Risk factors for colonization with ampicillin and high-level aminoglycoside-resistant enterococci during hospitalization in the ICU and the impact of prior antimicrobial exposure definition: a prospective cohort study.

The aim of our prospective cohort study was to determine the incidence, genetic relatedness and risk factors for colonization with ampicillin and high-level aminoglycoside-resistant enterococci (ARHLARE) among patients hospitalized in the intensive care unit. During 15-month period, we included 105 patients. The only independent risk factor for ARHLARE colonization was days of cefotaxime/ceftriaxone therapy [odds ratio (OR): 1.13; 95% confidence interval (CI) 1.10-1.27; P  =  0.045]. Patients with higher total use of antibiotics, patients on prolonged mechanical ventilation, and patients with urinary tract infection (UTI), were also found to be at increased risk to become colonized with ARHLARE. Pulsed-field gel electrophoresis suggested multifocal origin of the majority of the colonizing strains. Our results show that an increase in total antibiotic consumption for 10 defined daily doses (DDD)/patient increased the odds of colonization with ARHLARE for 36%. Further efforts to optimize antimicrobial use in high risk patients are proposed.