Within the last decade, many studies have highlighted the radical changes in the components of indoor and outdoor dust. Humans consistently face dermal, respiratory, and dietary exposures to these particles either indoors or outdoors.
A growing body of evidence from human and animal studies has revealed a link particularly between fungal exposure and lung diseases. This is not surprising, first, in consideration that we are exposed to the external air, that might include fungal spores through our airways [
] and secondly because fungi are ubiquitous in indoor and outdoor environments [
While many of these fungal spores are innocuous, some have the potential to germinate and cause invasive lung diseases [
]. The most known respiratory disease linked to fungi is asthma, but fungal spores can contribute to several other pathological conditions such as bronchial pulmonary allergic aspergillosis (ABPA), pneumonia and lung cancer [
genus includes some of the most dangerous toxigen fungi common in the Mediterranean environment, able to colonize different crops, including maize, grapes and dried fruits [
], and to produce mycotoxins, such as aflatoxin, ochratoxin A and fumonisins [
], when host plants are stressed by extreme temperature or moisture conditions, poor soil fertility or insect damage. Our group recently described the presence of
species in lung cancer patients using as a matrix coming from the airways, the exhaled breath condensate sample already validated in the study of airways microbiota [
]. Other fungi were also found in our oncologic population as
spp., whose possible role in the development of cancer and other airways diseases is not yet known. Notwithstanding the recognized dangers for human health of
and other fungi, our knowledge showed an absence of contamination of airways [
] and no studies are available that prove the possible contamination of airways of healthy subjects with outdoor fungal spores. Furthermore, there is still limited evidence of possible physiological impact of fungal contamination on the respiratory system [
Moreover, epidemiological studies often rely on broad microbiota exposure but fail to identify the taxonomic composition of the microbial community [
]. With this study, we want to give a preliminary contribution to this lacking field of research, giving a view of the incidence and nature of possible fungal contaminations in healthy subjects from Puglia. Toward this goal, we tried to achieve a better understanding of species/taxon diversity and population dynamics of the fungal microbial community present outdoors in the Puglia region, giving a survey of fungal microbiota of airways of healthy volunteer subjects.
Previous studies with sequencing different fungal genera in indoor dust and outdoor air samples showed that significant proportions of
along with some contribution from
and the human commensal
], and that the indoor air microbial communities are thought to be a function of dispersal from the outdoors, and growth and resuspension from the indoor environment [
]. Visagie et al. [
] and Flannigan et al. [
] listed 100 fungal species common in indoor environments, including
A. fumigatus, A. sydowii, P. brevicompactum
, classified as common in collected house dust, but the origin of common indoor species is difficult to determine.
The present study is the first to take a real picture of the incidence and nature of fungal contamination in healthy subjects from the Puglia Region of Italy. We tested fungal microbiota of exhaled breath condensate of healthy subjects and used the DNA sequencing approach for fungal species identification. ITS is the most commonly sequenced gene for fungi and was recently accepted as the official DNA barcode [
], but it does not distinguish among all species, because some species share identical sequences [
], even though it provides valuable information on sectional classification and often provides enough information for species identification. In order to compensate for the lack of variability in ITS, we also used BenA, as a secondary identification marker.
The presence of moulds was detected in 37,03% of enrolled healthy subjects (
Aspergillus sydowii, Cladosporium spp, Cladosporium herbarum, Penicillum brevicompactum, Penicillum expansum, Penicillum glabrum, Penicillum olsonii, Penicillum bilaiae, Alternaria infectoria, Alternaria alternata). The fungal positivity in airways didn’t correlate with any analysed variables (sex, age, BMI, smoking habit, pack years, area of residence, job).
However, the fungal contamination of airways of healthy subjects that we reported in this study was very high (37,03%) and, also supposing a high concentration of fungi in ambient air, we were surprised that almost half of the subjects enrolled had contaminated airways. This data was unexpected, especially in consideration that in our previous study [
] we didn’t find fungal contamination in healthy subjects. Surely, the enrollment conditions were completely different because in the previous study subjects were taken from the outpatient clinic and samples were collected in the clinic while, in this real life study, subjects were enrolled during the annual public regional meetings “Fiera del Levante”, held in the city of Bari that subjects visited as tourists.
is one of the most common species in collected samples and the species is generally considered as widespread. The species is often isolated from soil [
], and is very common on mouldy gypsum wallboard, dust, paint and various foods [
] and is commonly found in marine environments where it acts as an opportunistic pathogen of sea corals [
]. The source or origin of this species is still unknown, even though most studies indicate it as a terrestrial soil-borne fungus and shows its ability to grow in such a wide range of niches, suggesting the need for further studies that might help in understanding their possible role in underestimated pathologies.
However, the possibility of a high outdoor contamination of fungi is coherent with the season. Enrollment of patients took place in September, one of the hottest and most humid months in southern Italy and we know that fungal spore counts properly peaked during warm months [
Furthermore, the days of enrollment were particularly windy, a climatic condition that further contributes to the large diffusion of fungal spores.
However, it is virtually impossible in this study to determine the fact that the inhalation of fungal spores surely leads to fungal presence in airways after exposition. In the same manner the presence of fungi does not automatically determine future contamination. Indeed, this study has been designed just to take a survey of fungal microbiome in airways of healthy subjects from the Puglia Region and therefore enrolled patients just once.
A limit of the study was not to have repeated the collection of exhaled breath condensate and subsequent fungal analysis of this airway’s sample from healthy volunteers a short time after exposure. It would be very useful to see what happens after 1 day, 1 week-end, 1 month from exposure to a contaminated outdoor environment.
Another important limit of this study, due to the conditions previously explained, was to have collected clinical data of patients only with an anamnesis. Subjects were volunteers who came to our respiratory stand at the “Fiera del Levante” just to test their airways contamination.
A spirometer or other clinical instruments were not available in order to test lung function and to diagnose possible respiratory diseases. Therefore we were only able to identify a respiratory condition that might justify fungal contamination in airways.
An important point of this study was to have used a non-invasive method to analyse the airways of healthy subjects that otherwise would not have undergone more invasive techniques of sampling airways. Our group previously demonstrated the suitability of the EBC as non-invasive sample for the study of fungal microbiome of airways and this study further confirms its value [
We were unable to find any correlation between fungi positivity and sex, age, BMI, smoking habit, pack years, area of residence, or job. However, the number of subjects enrolled in this study was low and justified our results, which we intend to verify on a larger population.
Furthermore, important analyses should also be addressed to identify the fungal genotypes isolated, assess their ability to produce toxins, and above all, to evaluate the effective presence in human fluids, such as the EBC, of mycotoxins, potentially produced by isolated
Aspergillus species or other fungi. This was just a preliminary study that will be followed by a genomic and epigenomic characterization and mycotoxin analysis of airways of healthy subjects.
Methods for taxonomic identification of microbial communities through metagenomics approaches to DNA sequencing are rapidly gaining importance in fungal biodiversity research, allowing both generation of barcode markers and identification of isolates to species level [
], but reference databases are mostly incomplete, and, mostly developed for purposes other than the study of the relation of the environmental microbiome to human physiologic or health outcomes. Thus, EBC has the potential to study a more complete view of fungal microbial communities, or even previously unstudied individual taxa that may influence human health. However, the EBC microbiota may still be an interesting avenue of study despite the fact that the small quantities of bacterial DNA in these samples leave them more vulnerable to contamination, and any future studies would have to be designed with this in mind.