Search

The effective identification of bacterial and fungal isolates is essential for microbiological monitoring in environments like speleotherapeutic caves. This study compares MALDI-TOF MS and the OmniLog ID System, two high-throughput culture-based identification methods. MALDI-TOF MS identified 80.0% of bacterial isolates to the species level, while the OmniLog ID System identified 92.9%. However, species-level matches between the methods were only 48.8%, revealing considerable discrepancies. For discrepant results, MALDI-TOF MS matched molecular identification at the genus level in 90.5% of cases, while the OmniLog ID System matched only in 28.6%, demonstrating MALDI-TOF MS’s superiority. The OmniLog ID System had difficulties identifying genera from the order Micrococcales. Fungal identification success with MALDI-TOF MS was 30.6% at the species level, potentially improvable with a customised spectral library, compared to the OmniLog ID System’s 16.7%. Metagenomic approaches detected around 100 times more microbial taxa than culture-based methods, highlighting human-associated microorganisms, especially Staphylococcus spp. In addition to Staphylococcus spp. and Micrococcus spp. as indicators of cave anthropisation, metagenomics revealed another indicator, Cutibacterium acnes. This study advocates a multi-method approach combining MALDI-TOF MS, the OmniLog ID System, culture-based, and metagenomic analyses for comprehensive microbial identification. Metagenomic sampling on nitrocellulose filters provided superior read quality and microbial representation over liquid sampling, making it preferable for cave air sample collection.

Speleotherapy is one of the non-pharmacological methods for the treatment and rehabilitation of patients with chronic respiratory diseases, especially those with chronic obstructive pulmonary disease (COPD) and asthma. On the one hand, one of the alleged main advantages of speleotherapeutic caves is the low microbial load in the air and the absence of other aeroallergens, but on the other hand, due to the lack of comprehensive air monitoring, there is little information on the pristine and human-influenced aerobiota in such environments. The aim of this study was to assess the anthropogenic effects of speleotherapy on the air microbiota and to investigate its potential impact on human health in Sežana Hospital Cave (Slovenia). From May 2020 to January 2023, air samples were collected in the cave before and after speleotherapeutic activities using two different volumetric air sampling methods—impaction and impingement—to isolate airborne microbiota. Along with sampling, environmental data were measured (CO2, humidity, wind, and temperature) to explore the anthropogenic effects on the aerobiota. While the presence of patients increased microbial concentrations by at least 83.3%, other parameters exhibited a lower impact or were attributed to seasonal changes. The structure and dynamics of the airborne microbiota are similar to those in show caves, indicating anthropization of the cave. Locally, concentrations of culturable microorganisms above 1000 CFU/m3 were detected, which could have negative or unpredictable effects on the autochthonous microbiota and possibly on human health. A mixture of bacteria and fungi typically associated with human microbiota was found in the air and identified by MALDI-TOF MS with a 90.9% identification success rate. Micrococcus luteus, Kocuria rosea, Staphylococcus hominis, and Staphylococcus capitis were identified as reliable indicators of cave anthropization.

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.

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 Škocjanske jame (Slovenia) was sampled before and after tourist visits. Samples were analysed using culture-dependent methods, flow cytometry, detection of β-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), β-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.

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.

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.

Escherichia coli, one of the primary intestinal commensal bacteria in humans and endothermic animals, is commonly considered an indicator of faecal pollution. E. coli strains were isolated from karst rivers under different hydrological conditions, from footpaths in tourist caves and from bat guano. Isolates were tested for phenotypic resistance to ampicillin, chloramphenicol, ciprofloxacin, nalidixic acid, tetracycline and trimethoprim. The highest percentage of antimicrobial resistant E. coli was found in karst waters, followed by those from surface swabs and from bat guano. Several isolates from rivers and swabs exhibited multidrug-resistant phenotype. Environmental conditions impact the populations of E. coli; a positive correlation between dissolved oxygen and E. coli counts, and a negative correlation between conductivity and E. coli concentrations have been observed for karst rivers. Malenščica (Slovenia), a drinking water resource with an extensive catchment area, contained a relative high percentage of antimicrobial-resistant E. coli strains. None of the isolates from bat guano was resistant to ampicillin, chloramphenicol, and tetracycline. Future monitoring of bats should consider a regular follow-up of indicative microbial disease indicators in fresh guano. Regular cleansing of tourist footpaths in caves and disinfection barriers at the cave entrances reduce the concentration and transmission of E. coli significantly. A future, more detailed, study on characterization of additional E. coli isolates is needed to reveal their pathogeneicity, mechanisms of antibiotic resistance, mobile genetic elements, and gene transfer frequencies to other members of the karst microbiome.

Biofilms of different colours that colonize surfaces within karst caves represent a source of nutrients. They occur commonly and abundantly at sites with sediments, and close to seepages or underground rivers. Golden-yellow subaerial biofilms are particularly well observed because of their contrast with their surroundings, the characteristics of the pigment and recently, even more, due to the characteristics of light-emitting diode (LED) illumination. Yellow microbial biofilms were sampled from three caves in southwestern Slovenia, Dimnice, Križna jama and Sveta jama. The highest concentration of cultivable microbes (2.33×108 CFU/g) and the biggest number of identified bacteria (66.0%) were retrieved from a sample from Sveta jama. Using MALDI-TOF (Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight) for bacterial identification showed that different species of Pseudomonas prevailed in all samples. Yellow biofilms showed an absorption peak around 400 nm, and two emission peaks, a major in the blue (~460 nm) and a minor in the orange (~600 nm) parts of the spectrum when excited at 405 nm. Microbial mats that colonize surfaces are probably frequently overlooked in caves because they are difficult to observe when they have no pigmentation and the contrast with their surroundings is low. Additional studies are needed to aid the understanding of the role of pigmented biofilms and their interactions with underlying substrata in respect of the evolution of substrate micromorphology.

ABSTRACT Cryptococcosis is a major fungal disease caused by members of the Cryptococcus gattii and Cryptococcus neoformans species complexes. After more than 15 years of molecular genetic and phenotypic studies and much debate, a proposal for a taxonomic revision was made. The two varieties within C. neoformans were raised to species level, and the same was done for five genotypes within C. gattii. In a recent perspective (K. J. Kwon-Chung et al., mSphere 2:e00357-16, 2017, https://doi.org/10.1128/mSphere.00357-16 ), it was argued that this taxonomic proposal was premature and without consensus in the community. Although the authors of the perspective recognized the existence of genetic diversity, they preferred the use of the informal nomenclature “C. neoformans species complex” and “C. gattii species complex.” Here we highlight the advantage of recognizing these seven species, as ignoring these species will impede deciphering further biologically and clinically relevant differences between them, which may in turn delay future clinical advances.

In the last two decades prevalence of fungal infections is increasing for various reasons. One of them is the advance of medical science and the associated longer life expectancy in some patient groups. This includes cystic fibrosis patients who encounter fungal diseases already in their childhood. Fungal pathogens isolated in high frequencies from the respiratory tract include Aspergillus fumigatus, Candida albicans and Scedosporium apiospermum. In the case of cystic fibrosis, these organisms normaly colonise the respiratory and intestinal mucosae and can cause hipersensitivity reactions and invasive diseases. The fungus-patient interactions are complex and depend on several different factors which determine what course will the colonisation/infection take.

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.

(1) Background: Airborne microbes are an integral part of a cave ecosystem. Cave allochtonous airborne microbiota, which occurs mainly during aerosolization from an underground river, from animals, and from visitors, is particularly pronounced in show caves. The impacts of tourists and natural river aerosolization on the cave air were estimated in large cave spaces within the Škocjan Caves; (2) Methods: Simultaneously with the measurements of atmospheric parameters, cultivable airborne bacteria were impacted, counted and identified using MALDI-TOF MS (Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry); (3) Results: A mix of bacteria typically associated with humans and with natural habitats, including a large percentage of non-identified isolates, was found in the cave air. Few of the isolates were attributed to Risk Group 2. A strong positive correlation between tourist numbers and the rise in the concentration of airborne bacteria was indicated. Concentration of airborne bacteria rises to particularly high levels close to the underground river during periods of high discharge. A 10-times lower discharge reflected an approximately 20-times lower concentration of airborne bacteria; (4) Conclusions: Caves that are open and visited contain a diverse airborne microbiota originating from different sources. Enormous cave chambers that display relatively dynamic cave climate conditions do not normally support the enhancement of airborne bacterial concentrations.

The incidence of fungal infections is increasing together with the growing number of immunocompromised patients. For successful treatment it is necessary to select the optimal dose of a specific antifungal agent based on the in vitrosusceptibility testing according to the microdilution method by CLSI (Clinical and Laboratory Standards Institute) or EUCAST (European Committee on Antimicrobial Susceptibility Testing). All of the susceptibility testing results in Slovenia were processed and interpreted according to CLSI, but gradually we will start using the EUCAST system, which brings certain differences in methodology that shorten the incubation time by 24 hours and bring some changes in the interpretation of MICs (Minimal Inhibitory Concentration).