Within the variables recorded in the sampling associated with olive trees, the maximum temperature recorded was 25 ºC and a minimum temperature of 9 ºC. In addition, an increase in relative humidity (maximum of 75.8%) was recorded in the municipality of Sutamarchán During July. The most recurrent direct light indices were low, with a value of 0.0394 and indirect light 0.0173.
Microbiological identification
The results of the microbiological identification showed that 45 of 46 presented a capsule. In addition, 35 of the 46 isolates were positive for the urease test, and 42 of the 46 isolates grew at 37ºC, except for three species of C. albidus (AM-0277, AM-0286, and AM-0323), and one of C. saitoi (AM-0329). All isolates corresponding to C. gattii (AM-0310, AM-0316, AM-0317 and AM-0333) were positive in CGB (L-canavanine-glycine-bromothymol blue) medium, as were three of the four isolates of the species C. laurentii (AM-0313, AM-0314 and AM-0315). The phenoloxidase test was negative for C. gattii AM-316 and AM-317 after ten days of incubation on SSA plates. On the other hand, the C. neoformans AM-310 isolate started pigmentation on the tenth day of incubation; however, it did not have full pigmentation (Fig. 2). Those three isolated negatives for phenoloxidase were urea positive.
Table 1
Collection number, data, locality, source, species, molecular type, and GenBank accession numbers of Cryptococcus spp.
Collection number UBCHM | Collected data (Month/Year) | Locality | Source | Taxon | Molecular type | Sequence length (LROR/LR5) | Gene Bank accession number LSU | Sequence length (ITS1/ITS4) | Gene Bank accession number ITS | |
AM-0272 | Jan-18 | Tunja | Pigeon feces | C. laurentii (Papilotrema laurentii) | N/A | 769 | OP076773 | 511 | OP060634 | |
AM-0275 | feb-18 | Tunja | Pigeon feces | Cryptococcus neoformans_var_grubii | VNI | 859 | OP076774 | 543 | OP060635 | |
AM-0276 | Tunja | Pigeon feces | Cryptococcus neoformans_var_grubii | VNI | 871 | OP076775 | 541 | OP060636 | |
AM-0277 | mar-18 | Tunja | Pigeon feces | Cryptococcus albidus (Naganishia albida)1 | N/A | 782 | OP076776 | 588 | OP060637 | |
AM-0279 | Tunja | Pigeon feces | Cryptococcus neoformans var grubii | VNII | 807 | OP076777 | 534 | OP060638 | |
AM-0282 | Tunja | Pigeon feces | Cryptococcus neoformans var grubii | VNII | 802 | OP076778 | 530 | OP060639 | |
AM-0283 | Tunja | Pigeon feces | Cryptococcus neoformans var grubii | VNII | 766 | OP076779 | 533 | OP060640 | |
AM-0284 | Tunja | Pigeon feces | Cryptococcus neoformans var grubii | VNII | 861 | OP076780 | 544 | OP060641 | |
AM-0285 | Tunja | Pigeon feces | Cryptococcus neoformans_var_grubii | VNII | 865 | OP076781 | 527 | OP060642 | |
AM-0286 | Tunja | Pigeon feces | Cryptococcus albidus (Naganishia albida)1 | N/A | 843 | OP076782 | 593 | OP060643 | |
AM-0288 | apr-18 | Tunja | Pigeon feces | Cryptococcus neoformans var grubii | VNI | 891 | OP076783 | 547 | OP060644 | |
AM-0289 | Tunja | Pigeon feces | Cryptococcus neoformans var grubii | VNI | 806 | OP076784 | 531 | OP060645 | |
AM-0290 | Tunja | Pigeon feces | Cryptococcus neoformans var grubii | VNI | 861 | OP076785 | 527 | OP060646 | |
AM-0291 | Tunja | Pigeon feces | Cryptococcus neoformans var grubii | VNI | 848 | OP076786 | 535 | OP060647 | |
AM-0292 | Tunja | Pigeon feces | Cryptococcus neoformans var grubii | VNI | 851 | OP076787 | 532 | OP060648 | |
AM-0295 | Tunja | Pigeon feces | Cryptococcus neoformans var grubii | VNI | 896 | OP076788 | 527 | OP060649 | |
AM-0297 | Tunja | Pigeon feces | C. saitoi (Naganishia globosa) | N/A | 862 | OP076789 | 610 | OP060650 | |
AM-0298 | Tunja | Pigeon feces | C. saitoi (Naganishia globosa) | N/A | 827 | OP076790 | 605 | OP060651 | |
AM-0299 | Tunja | Pigeon feces | Cryptococcus neoformans var grubii1 | VNI | 771 | OP076791 | 532 | OP060652 | |
AM-0300 | Tunja | Pigeon feces | C. saitoi (Naganishia globosa) | N/A | 858 | OP076792 | 607 | OP060653 | |
AM-0301 | may-18 | Tunja | Pigeon feces | Cryptococcus neoformans var grubii | VNI | 859 | OP076793 | 471 | OP060654 | |
AM-0302 | Tunja | Pigeon feces | Cryptococcus neoformans var grubii | VNI | 871 | OP076794 | 542 | OP060655 | |
AM-0303 | jun-18 | Tunja | Pigeon feces | Cryptococcus neoformans var grubii | VNII | 865 | OP076795 | 542 | OP060656 | |
AM-0304 | Tunja | Pigeon feces | Cryptococcus neoformans var grubii | VNI | 870 | OP076796 | 526 | OP060657 | |
AM-0305 | Tunja | Pigeon feces | Cryptococcus neoformans var grubii | VNI | 876 | OP076797 | 525 | OP060658 | |
AM-0306 | Tunja | Pigeon feces | Cryptococcus neoformans var grubii | VNI | 800 | OP076798 | 523 | OP060659 | |
AM-0307 | Tunja | Pigeon feces | Cryptococcus neoformans var grubii | VNI | 766 | OP076799 | 523 | OP060660 | |
AM-0308 | jul-18 | Tunja | Pigeon feces | Cryptococcus neoformans var grubii | VNI | 863 | OP076800 | 526 | OP060661 | |
AM-0309 | Tunja | Pigeon feces | Cryptococcus neoformans var grubii | VNII | 867 | OP076801 | 542 | OP060662 | |
AM-0310 | jun-19 | Sáchica | Hollowness | Cryptococcus gattii | VGIII | 874 | OP076802 | 531 | OP060663 | |
AM-0312 | Sáchica | Debris | Cryptococcus albidus (Naganishia albida)1 | N/A | 799 | OP076803 | 593 | OP060664 | |
AM-0313 | Sáchica | Ground | Cryptococcus laurentii (Papilotrema laurentii) | N/A | 885 | OP076804 | 510 | OP060665 | |
AM-0314 | Sáchica | Ground | Cryptococcus laurentii (Papilotrema laurentii) | N/A | 868 | OP076805 | 508 | OP060666 | |
AM-0315 | Sáchica | Cortex | Cryptococcus laurentii (Papilotrema laurentii) | N/A | 872 | OP076806 | 507 | OP060667 | |
AM-0316 | Sáchica | Leaves | Cryptococcus gattii | VGIII | 776 | OP076807 | 526 | OP060668 | |
AM-0317 | Sáchica | Leaves | Cryptococcus gattii | VGIV | 868 | OP076808 | 495 | OP060669 | |
AM-0319 | jul-19 | Sáchica | Hollowness | Cryptococcus flavescens (Papiliotrema flavescens) | N/A | 801 | OP076809 | 515 | OP060670 | |
AM-0322 | Sutamarchán | Debris | Cryptococcus saitoi (Naganishia globosa)1 | N/A | 781 | OP076810 | 613 | OP060671 | |
AM-0323 | Sutamarchán | Cortex | Cryptococcus albidus (Naganishia albida)1 | N/A | 871 | OP076811 | 593 | OP060672 | |
AM-0325 | Sutamarchán | Leaves | Cryptococcus neoformans var grubii1 | VNI | 800 | OP076812 | 523 | OP060674 | |
AM-0326 | Sutamarchán | Hollowness | Cryptococcus neoformans var grubii | VNI | 804 | OP076813 | 531 | OP060673 | |
AM-0328 | Aug-19 | Sáchica | Hollowness | Cryptococcus saitoi (Naganishia globosa) | N/A | 866 | OP076814 | 597 | OP060675 | |
AM-0329 | Sáchica | Hollowness | Cryptococcus saitoi (Naganishia globosa) | N/A | 802 | OP076815 | 600 | OP060676 | |
AM-0330 | Sáchica | Debris | Cryptococcus saitoi (Naganishia globosa) | N/A | 874 | OP076816 | 604 | OP060677 | |
AM-0331 | Sáchica | Cortex | Cryptococcus saitoi (Naganishia globosa) | N/A | 874 | OP076817 | 604 | OP060678 | |
AM-0333 | oct-19 | Sáchica | Hollowness | Cryptococcus gattii | VGIII | 864 | OP076818 | 526 | OP060679 | |
1The MALDI-TOF results were inconsistent with LSU and ITS sequencing results. (N/A): not applicable.
MALDI-TOF identification.
Sixty-four microorganisms were identified, from which 46 specimens of the genus Cryptococcus were isolated; 11 corresponded to other yeast species and seven to bacteria. Of the stool samples taken from the city of Tunja, 29 (315.18%) positive isolates belonging to the genus Cryptococcus were obtained, of which 23 (24.8%) corresponded to C. neoformans var grubii, 3 (3.22%) to C. saitoi, 2 (2.2%) C. albidus, 1 (1.07%) to C. laurentii. In addition to finding, other yeasts such as Candida albicans (1), Candida guillermondi (5), Candida parapsilosis (1), Candida tropicalis (1) and Rhodotorula mucilaginosa (1), and some bacterial species such as Bacillus subtillis (4) and Patoea agglomerans (1). On the other hand, for the samples taken in the Ricaurte Alto region, a lower number of positive isolates was recorded, with a total of 17 (1.43%) positive samples for different species of the genus Cryptococcus, including C. gattii (4), C. neoformans (2), C. laurentii (3), C. albidus (2), C. saitoi (5) and C. flavescens (1), in addition to finding yeasts such as Rhodotorula mucilaginosa (2) and two species of Pseudomonas; Pseudomonas jesseni (1) and Pseudomonas oryzihabitans (1).
Molecular typification by RFLP of the URA5 gen. The molecular pattern was determined for the isolates identified as species of the C. neoformans and C. gattii complex. Of the 23 isolates identified by MALDI-TOF as C. neoformans var grubii, 16 had a VNI molecular pattern, and 7 had a VNII molecular pattern. Likewise, for the four isolates identified as C. gattii, three presented molecular pattern VGIII and one molecular pattern VGIV (Fig. 3).
Identification by sequencing LSU gen. Amplification of DNA samples with primers LROR and LR5 resulted in approximately 1,000 bp. The sequences were deposited in GenBank, and accession numbers were obtained (Table 1). Forty- six isolates belonging to the genus Cryptococcus; C. neoformans var grubii (n = 25), C. saitoi (n = 8), C. albidus (n = 4), C. bacillisporus (n = 4) (Cryptococcus gattii VGIII), C. laurentii (n = 4), currently Papiliotrema laurentii (Kuff., Xin Zhan et al. 2015) and C. flavescens (n = 1). Also, nine species non-Cryptococcus genus, including Meyerozyma guilliermondii (n = 3) (Kurtzman and Suzuki 2010), (anamorph Candida guilliermondii, Langeron & Guerra (1938)), Meyerozyma (Pichia) guilliermondii (n = 1), Candida albicans (n = 1), Candida tropicalis (n = 1), Meyerozyma caribbica (n = 1) and Rhodotorula mucilaginosa (n = 2).
Concatenated ITS and LSU sequences with high bootstrap values generated by neighbor-joining analyses supported the differentiation of the sixclades: 1. C. albidus (bootstrap values 100), 2. C. saitoi (boot values 100), 3. C. laurentii (bootstrap values 94.5), 4. C. flavescens (boot values 99.7) 5. C. gattii (bootstrap 97.2 values). 6. C. neoformans (boot values 100) (Fig. 4).
Redundancy Analysis (RDA) for stool samples of C. livia. It is essential to mention that the data collected in Tunja were organized according to the sampling months, as can be seen in Fig. 5a for the stool samples. It is highlighted that the results indicate that the first three components explain the variability of 93.47% of the data.
The redundancy analysis established that the main variables that intensify the appearance of Cryptococcus species were relative humidity, temperature, and precipitation. Relative humidity (RH) and temperature (T) positively correlated with the appearance of the species C. albidus in March. The other environmental variables are positively related to the appearance of this species. Likewise, these environmental variables (HR, T, and P) negatively correlated with the C. laurentii species. As these variables increase, the probability of finding this species in the environment is lower.
For the C. neoformans species, the variables that have a positive correlation and favor its appearance in the environment are solar brightness (BS) with 76.46% and evaporation (EV_mm) with 80.3%, contrary to the negative relationship that was evidenced with the precipitation variable.
Finally, the species C. saitoi presented a more significant relationship with the precipitation variable (PR_mm); it is more likely to find this species when precipitation is lower since its correlation with this analysis was 39%.
It should be noted that for the sampling carried out in the city of Tunja, direct and indirect light records were not made, only those mentioned above, which IDEAM provided.
Redundancy Analysis (RDA) for olive tree samples. It is essential to mention that to carry out this analysis, data was organized with codes where J was assigned to July, A to August, S to September, O to October, and N to November, as can be shown in Fig. 5b, for the data taken in Sutamarchán, Sáchica and Villa de Leyva.
The redundancy analysis allowed us to establish the main variables that intensify the appearance of Cryptococcus species; they were relative humidity and temperature.
The relative humidity (HR) is inversely related to the variables of indirect light (ISite), direct light (Dsite), average temperature (Tprom), and precipitation (Prec). According to the data collected, the relative humidity increases when ISite, Dsite, Tprom, and Prec variables decrease; then, the relative humidity decreases when the mentioned variables increase (Fig. 5b). The species C. albidus, C. laurenteii and C. flavencens are found to a greater extent when the relative humidity of the medium is lower. In contrast, C. neoformans, C. gattii, and C. saitoi were isolated more frequently when the relative humidity was higher.
Likewise, the maximum temperature is also related to the appearance of species of the genus Cryptococcus, presenting a directly proportional relationship with the species of C. neoformans and C. gattii and, to a lesser extent, C. saitoi. These species were found between 20°C to 25°C, reported in July and August in the municipality of Sáchica. It should be noted that the variables of direct and indirect light, precipitation, minimum temperature, and average temperature were inversely related to the presence of all the species above.
Multiple correlations analysis from samples obtained from stool samples. As shown in Fig. 6a, there is a positive correlation (78%) between the isolates of C. neoformans and precipitation; that is, precipitation contributed positively to obtaining isolates of this species; therefore, they are directly found related. Contrary to the negative correlation (30%) evidenced in the variable of solar brightness, that says, as the solar brightness increases, the probability of isolating C. neoformans in the environment decreases. Unlike what was found for C. saitoi, which presents a positive correlation (62%), as the solar brightness increases, the probability of finding this species in the environment increases.
On the other hand, for C. laurentii a negative correlation of 80% with relative humidity was determined; that is, it is an inversely proportional relationship; therefore, as relative humidity increases, it is unlikely to find this species in the environment.
Multiple correlations analysis from samples obtained from the Ricaurte Alto region. The variables of direct light (direct Site) and indirect (indirsite) present a positive correlation of 100%, with a relationship percentage of 1, which reflects that they are directly related. Similarly, C. gattii has a slight positive correlation of 40% with relative humidity, which means that it is more likely to find this species when the relative humidity is higher. In the same way, a negative correlation of 30% can be observed between the appearance of C. neoformans and the minimum temperature; this is an inversely proportional relationship. As the temperature increased, it was more likely to isolate C. neoformans from the environment (Fig. 6b).
For the other species of Cryptococcus, it is essential to point out that C. laurentii has a positive correlation of 40% with the average temperature, thus indicating that this particular species is associated with average temperatures that range between 14°C or 15°C, which would indicate an association with cold to temperate places. In the case of the C. albidus and C. saitoi species, they present an inversely proportional correlation of 50% with the minimum temperature; according to our results, it is more likely to be found at temperatures above 15°C.
When performing the logistic regression test, it was established that the specie that was most influenced by the environmental variables was C. albidus with a p-value of 0.0078.