This paper has been updated to correct a spelling error concerning an author name. It previously showed as Giuseppe Griseo, however the correct spelling is Giuseppe Criseo.

In order to elucidate the distribution of Cryptococcus neoformans and C. gattii in the Mediterranean basin, an extensive environmental survey was carried out during 2012-2015. A total of 302 sites located in 12 countries were sampled, 6436 samples from 3765 trees were collected and 5% of trees were found to be colonized by cryptococcal yeasts. Cryptococcus neoformans was isolated from 177 trees and C. gattii from 13. Cryptococcus neoformans colonized 27% of Ceratonia, 10% of Olea, Platanus and Prunus trees and a lower percentage of other tree genera. The 13 C. gattii isolates were collected from five Eucalyptus, four Ceratonia, two Pinus and two Olea trees. Cryptococcus neoformans was distributed all around the Mediterranean basin, whereas C. gattii was isolated in Greece, Southern Italy and Spain, in agreement with previous findings from both clinical and environmental sources. Among C. neoformans isolates, VNI was the prevalent molecular type but VNII, VNIV and VNIII hybrid strains were also isolated. With the exception of a single VGIV isolate, all C. gattii isolates were VGI. The results confirmed the presence of both Cryptococcus species in the Mediterranean environment, and showed that both carob and olive trees represent an important niche for these yeasts.

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Fundamental niche prediction of Cryptococcus neoformans and Cryptococcus gattii in Europe is an important tool to understand where these pathogenic yeasts have a high probability to survive in the environment and therefore to identify the areas with high risk of infection. In this study, occurrence data for C. neoformans and C. gattii were compared by MaxEnt software with several bioclimatic conditions as well as with soil characteristics and land use. The results showed that C. gattii distribution can be predicted with high probability along the Mediterranean coast. The analysis of variables showed that its distribution is limited by low temperatures during the coldest season, and by heavy precipitations in the driest season. C. neoformans var. grubii is able to colonize the same areas of C. gattii but is more tolerant to cold winter temperatures and summer precipitations. In contrast, the C. neoformans var. neoformans map was completely different. The best conditions for its survival were displayed in sub-continental areas and not along the Mediterranean coasts. In conclusion, we produced for the first time detailed prediction maps of the species and varieties of the C. neoformans and C. gattii species complex in Europe and Mediterranean area.

A total of 476 European isolates (310 Cryptococcus neoformans var. grubii, 150 C. neoformans var. neoformans, and 16 C. gattii species complex) from both clinical and environmental sources were analyzed by multi-locus sequence typing. Phylogenetic and population genetic analyses were performed. Sequence analysis identified 74 sequence types among C. neoformans var. neoformans (VNIV), 65 among C. neoformans var. grubii (56 VNI, 8 VNII, 1 VNB), and 5 among the C. gattii species complex (4 VGI and 1 VGIV) isolates. ST23 was the most frequent genotype (22%) among VNI isolates which were mostly grouped in a large clonal cluster including 50% of isolates. Among VNIV isolates, a predominant genotype was not identified. A high percentage of autochthonous STs were identified in both VNI (71%) and VNIV (96%) group of isolates. The 16 European C. gattii species complex isolates analyzed in the present study originated all from the environment and all belonged to a large cluster endemic in the Mediterranean area. Population genetic analysis confirmed that VNI group of isolates were characterized by low variability and clonal expansion while VNIV by a higher variability and a number of recombination events. However, when VNI and VNIV environmental isolates were compared, they showed a similar population structure with a high percentage of shared mutations and the absence of fixed mutations. Also linkage disequilibrium analysis reveals differences between clinical and environmental isolates showing a key role of PLB1 allele combinations in host infection as well as the key role of LAC1 allele combinations for survival of the fungus in the environment. The present study shows that genetic comparison of clinical and environmental isolates represents a first step to understand the genetic characteristics that cause the shift of some genotypes from a saprophytic to a parasitic life style.

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The present investigation, carried out in laboratory microcosms, regards the effects of some bioremediation treatments of a polluted soil and the use of specific parameters to study the evolution of biochemical processes which take place in the soil decontamination.The bioremediation treatments were the following: (1) a mixture of microorganisms-enzymesnutrients (MEN); (2); compost alone (C); (3) compost with earthworms (Eisenia fetida) (CL) and (4) control soil (without treatment) (BN).Chemical, physico-chemical, biological and biochemical parameters were determined to study the soil metabolic processes in order to assess the efficiency of the bioremediation process involved in the degradation of hydrocarbons.The study showed an intense microbiological activity expressed as carbon dioxide evolution during the time, with a tendency to stabilize at the end of the experiments. The highest carbon dioxide release was found in the two compost treatments, showing the availability of organic substrate characterising the compost.The organic substrate reduction during the time caused a decrease of hydrolytic enzyme activities representative of Carbon (β-glucosidase), Nitrogen (protease) and Phosphorus (phosphatase) cycles. However, the enzyme activities showed their highest values in the treatments with compost. Finally, the greatest reduction of hydrocarbons was found in the treatments with compost, in particular with earthworms that also contributed to regulate the biochemical equilibrium of the soil.

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