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

Abstract

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.

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

“…A 1.5% nutrient agar (NA, Sigma-Aldrich, St. Louis, MO, USA) was used to estimate and identify the cultivable fraction of airborne microorganisms, as is common in microbiology and has been used in previous studies in Slovenian caves [3,4] (Mulec, 2014;Mulec et al, 2017). A portion of the liquid samples was evenly distributed on the NA plates, and then, the plates were incubated at 37 • C for 48 h and at 20 • C for 7 days.…”

“…Karst caves are becoming increasingly interesting for tourists precisely because of their uniqueness. Human impact poses a major threat to caves and their conservation in some cases, through climatic changes (e.g., increased CO 2 levels), inputs of organic nutrients and allochthonous microorganisms [1,2,4]. The presence of people in the cave can lead to irreversible changes in the composition of the biocoenosis [5,6].…”

“…These include metagenomic-based approaches, such as advanced molecular genetic techniques and high-throughput sequencing technologies, which greatly expand our picture of the structure and dynamics of microbial populations [16][17][18][19][20]. However, other culture-independent methods, such as flow cytometry, detection of cell wall and membrane structural components and ATP-based luminescence methods, have also proven to be very informative in cave microbiology [4,15,[21][22][23]. In the study of show caves, flow cytometry alone or in combination with optical microscopy has been used for biofilm analyses, especially for phototrophic biofilms and also for aerobiological studies [21,[24][25][26].…”

“…In the study of show caves, flow cytometry alone or in combination with optical microscopy has been used for biofilm analyses, especially for phototrophic biofilms and also for aerobiological studies [21,[24][25][26]. To the best of the authors' knowledge, the detection of cell wall and membrane structure components of bacteria and fungi in caves has so far only been carried out in the Postojnska jama [4]. These components, mainly lipopolysaccharide (LPS) and β-D-glucan (BDG), are known triggers of innate immunity and have immunomodulatory effects [27,28].…”

“…Their detection in clinical samples is included in current diagnostic guidelines, as they play an important role in the pathobiology of many infectious diseases and intoxications [33]. Since LPS and BDG have also been detected in cave air [4], these two molecules could be used as indicators of aerosolised bacteria and fungi. The aim of this study was to assess the anthropogenic effect of mass tourism on the air microbiota and consider its potential impact on human health in Postojnska jama and Škocjanske jame.…”