# Eucalyptus Tree: A Potential Source of Cryptococcus neoformans in Egyptian Environment

Authors: Mahmoud Elhariri; Dalia Hamza; Rehab Elhelw; Mohamed Refai
Journal: International Journal of Microbiology
 (2016)

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## Abstract

In Egypt, the River Red Gum (Eucalyptus camaldulensis) is a well-known tree and is highly appreciated by the rural and urban dwellers. The role of Eucalyptus trees in the ecology of Cryptococcus neoformans is documented worldwide. The aim of this survey was to show the prevalence of C. neoformans during the flowering season of E. camaldulensis at the Delta region in Egypt. Three hundred and eleven samples out of two hundred Eucalyptus trees, including leaves, flowers, and woody trunks, were collected from four governorates in the Delta region. Thirteen isolates of C. neoformans were recovered from Eucalyptus tree samples (4.2%). Molecular identification of C. neoformans was done by capsular gene specific primer CAP64 and serotype identification was done depending on LAC1 gene. This study represents an update on the ecology of C. neoformans associated with Eucalyptus tree in Egyptian environment.


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## Body

## 1. Introduction

The basidiomycetes yeast of genusCryptococcus includesC. neoformans/C. gattii species complex, which is composed of two separate species,C. neoformans  and C. deneoformans, and five species within C. gattii. The most clinically relevant complex species were recently revised based on phenotypic and genotypic diversity, supported by the presence of distinct and consistent lines, and it proposes to recognizeC. neoformansvar.grubii (represented by genotypes VNI and VNII) andC. neoformans (VNIII and VNIV) as separate species, as well as five species ofC. gattii (represented by genotypes VGI, VGII, VGIII, and VGIV) [1, 2].C. neoformans has a worldwide distribution and has been recovered from pigeon droppings (Columba livia), urban environments, and soil. Many reports have shown the presence ofC. neoformansin the hollows of different tree species, proposing that trees play a major role inCryptococcus infection [3, 4]. The most common isolate responsible for this fungal infection isC. neoformansvar.grubii serotype A [1, 5–7].C. gattii has been proposed to have a specific ecological association with a number ofEucalyptus species [8].E. camaldulensis is a well-known tree in Egypt since it was imported by Mohamed Ali, the Governor of Egypt (1805–1848), for fixing the River Nile banks in the 19th century. It is one of the most widely distributed trees in most of the arid and semiarid areas. This kind of tree exists on almost every roadside in Egypt, but there are no data about its role as potential carrier of fungal elements.In Egypt, the incidence ofCryptococcus neoformans fromEucalyptus trees and pigeon droppings has been reported [9]. In this report, the author depended on the conventional differentiation methods to determineC. neoformans varieties. There is no recent noticeable information aboutthe Eucalyptus tree role in the ecology ofC. neoformans in Egyptian environment. Therefore, the present study was aimed at determining the possible role of this tree as a potential dispersing source ofC. neoformansin Delta region’s environment.


## 2. Materials and Methods

### 2.1. Study Area and Sampling Collection

The study area in Delta region covered 240 kilometers (150 mi) of Mediterranean coastline of Egypt. A total 311 samples out of 200Eucalyptus trees, including leaves, flowers, and woody trunks, were collected from four different governorates in the Delta region (Cairo, Giza, Elmenofia, Al-Sharqia) by the rate of fifty samples for each region (Table 1). The samples were stored on ice in clean, sterile plastic bags and transferred to the Microbiology Department laboratory, Faculty of Veterinary Medicine. The samples were rinsed in sterile distilled water, then immersed in sterile saline solution supplemented with chloramphenicol (10.0 mg/mL), and homogenized with ultrahomogenization for 4 min. The bottle was left for 30 min at room temperature to settle the sediment.Table 1
Eucalyptus tree collected samples in the Delta region.
Governorate
Tree number
Samples type
Total
Leaves
Flowers
Wood trunks
Giza
50
40
25
30
95
Cairo
50
31
13
15
59
Al-Sharqia
50
50
30
30
110
Elmenofia
50
17
20
10
47
Total
200
138
88
85
311


### 2.2. Isolation and Identification

From the supernatant fluid of each homogenized sample, a loopful was streaked onto plates of Sabouraud dextrose agar with chloramphenicol and incubated at 30°C for 48 hours. The colonies suspected to beC. neoformans were streaked onEucalyptus leaves agar media [12]. The isolates were identified by classical mycological procedures ofC. neoformans [13].


### 2.3. Molecular Characterization

#### 2.3.1. DNA Extraction

The yeast cells from SDA slants were collected after 48-hour incubation with sterile PBS. The collected pellets were mixed into a microtube with 500μL TES (100 mM Tris, pH 8.0, 10 mM EDTA, and 2% SDS); 50–100 μg Proteinase K from an appropriate stock solution was added and then incubated for 30 min (minimum) up to 1 h at 55°–60°C with occasional gentle mixing. The lysate mix salt concentration was adjusted to 1.4 M with 5 M NaCl (=140 μL); 1/10 vol (=65 μL) of 10% CTAB was added and incubated for 30 min at 65°C. The lysate was mixed gently and then incubated for 30 min at 0°C; finally, the mix was centrifuged for 10 min at 4°C, at 15000 rpm. The supernatant was transferred to a 1.5 mL tube followed by the addition of 0.55 vol isopropanol (=510 μL) to precipitate DNA followed by immediate centrifugation for 5 min, at 15000 rpm.


### 2.4. Molecular Identification by Capsular Gene

Detection ofC. neoformans was done by using specific capsular gene primersCAP64. The primers forCAP64 were designed on the basis of DNA sequences (Table 2) [10].Table 2
Primers used in this study.
Primer
Primer sequence 3′-5′
PCR product
Reference
CAP64
GCCAAGGGAGTCTTATATGGGCAAAGGGTTCACCAAATCG
400 bp
[10]
LAC1
GGAACAGCAACCACACTACTGCATATTGGGTGGCATCTTACTGAGGGACCAGGGAACATGTTGTTGACGTTGTGGAAGGCAAAGAAAC
250 bp760 bp880 bp
[11]


### 2.5. Molecular Differentiation of Serotypes

This was applied by subjecting genomic DNA of identified strains byCAP64 gene to multiplex PCR amplification using a set of four primers of the laccase gene (LAC1) (Table 2) which were used for differentiating four major serotypes, A, D, B, and C, ofC. neoformans [11].


## 2.1. Study Area and Sampling Collection

The study area in Delta region covered 240 kilometers (150 mi) of Mediterranean coastline of Egypt. A total 311 samples out of 200Eucalyptus trees, including leaves, flowers, and woody trunks, were collected from four different governorates in the Delta region (Cairo, Giza, Elmenofia, Al-Sharqia) by the rate of fifty samples for each region (Table 1). The samples were stored on ice in clean, sterile plastic bags and transferred to the Microbiology Department laboratory, Faculty of Veterinary Medicine. The samples were rinsed in sterile distilled water, then immersed in sterile saline solution supplemented with chloramphenicol (10.0 mg/mL), and homogenized with ultrahomogenization for 4 min. The bottle was left for 30 min at room temperature to settle the sediment.Table 1
Eucalyptus tree collected samples in the Delta region.
Governorate
Tree number
Samples type
Total
Leaves
Flowers
Wood trunks
Giza
50
40
25
30
95
Cairo
50
31
13
15
59
Al-Sharqia
50
50
30
30
110
Elmenofia
50
17
20
10
47
Total
200
138
88
85
311


## 2.2. Isolation and Identification

From the supernatant fluid of each homogenized sample, a loopful was streaked onto plates of Sabouraud dextrose agar with chloramphenicol and incubated at 30°C for 48 hours. The colonies suspected to beC. neoformans were streaked onEucalyptus leaves agar media [12]. The isolates were identified by classical mycological procedures ofC. neoformans [13].


## 2.3. Molecular Characterization

### 2.3.1. DNA Extraction

The yeast cells from SDA slants were collected after 48-hour incubation with sterile PBS. The collected pellets were mixed into a microtube with 500μL TES (100 mM Tris, pH 8.0, 10 mM EDTA, and 2% SDS); 50–100 μg Proteinase K from an appropriate stock solution was added and then incubated for 30 min (minimum) up to 1 h at 55°–60°C with occasional gentle mixing. The lysate mix salt concentration was adjusted to 1.4 M with 5 M NaCl (=140 μL); 1/10 vol (=65 μL) of 10% CTAB was added and incubated for 30 min at 65°C. The lysate was mixed gently and then incubated for 30 min at 0°C; finally, the mix was centrifuged for 10 min at 4°C, at 15000 rpm. The supernatant was transferred to a 1.5 mL tube followed by the addition of 0.55 vol isopropanol (=510 μL) to precipitate DNA followed by immediate centrifugation for 5 min, at 15000 rpm.


## 2.3.1. DNA Extraction

The yeast cells from SDA slants were collected after 48-hour incubation with sterile PBS. The collected pellets were mixed into a microtube with 500μL TES (100 mM Tris, pH 8.0, 10 mM EDTA, and 2% SDS); 50–100 μg Proteinase K from an appropriate stock solution was added and then incubated for 30 min (minimum) up to 1 h at 55°–60°C with occasional gentle mixing. The lysate mix salt concentration was adjusted to 1.4 M with 5 M NaCl (=140 μL); 1/10 vol (=65 μL) of 10% CTAB was added and incubated for 30 min at 65°C. The lysate was mixed gently and then incubated for 30 min at 0°C; finally, the mix was centrifuged for 10 min at 4°C, at 15000 rpm. The supernatant was transferred to a 1.5 mL tube followed by the addition of 0.55 vol isopropanol (=510 μL) to precipitate DNA followed by immediate centrifugation for 5 min, at 15000 rpm.


## 2.4. Molecular Identification by Capsular Gene

Detection ofC. neoformans was done by using specific capsular gene primersCAP64. The primers forCAP64 were designed on the basis of DNA sequences (Table 2) [10].Table 2
Primers used in this study.
Primer
Primer sequence 3′-5′
PCR product
Reference
CAP64
GCCAAGGGAGTCTTATATGGGCAAAGGGTTCACCAAATCG
400 bp
[10]
LAC1
GGAACAGCAACCACACTACTGCATATTGGGTGGCATCTTACTGAGGGACCAGGGAACATGTTGTTGACGTTGTGGAAGGCAAAGAAAC
250 bp760 bp880 bp
[11]


## 2.5. Molecular Differentiation of Serotypes

This was applied by subjecting genomic DNA of identified strains byCAP64 gene to multiplex PCR amplification using a set of four primers of the laccase gene (LAC1) (Table 2) which were used for differentiating four major serotypes, A, D, B, and C, ofC. neoformans [11].


## 3. Results

### 3.1. Recovery Rate ofC. neoformans fromEucalyptus camaldulensis

In this study,E. camaldulensis trees acted as a potential refuge forC. neoformans. A total of 13 (4.2%)C. neoformans isolates out of 311 examined samples in Delta region were recovered during the flowering season ofEucalyptus tree. All the recoveredCryptococcus isolates were identified asC. neoformans strains based on all conventional and physiological characters ofC. neoformans (Figure 1). Among these, 7 isolates (7.9%) were recovered from 88Eucalyptusflowers, 5 isolates (3.6%) were recovered from 138Eucalyptus tree leaves, and 1 isolate (1.1) was recovered from 85 woody trunks (Table 3).Table 3
Recovery rate and distribution ofC. neoformans in tested Eucalyptus trees sampled in the Delta region.
Total samples
Giza
Cairo
Al-Sharqia
Elmenofia
Total
Recovery rate
Number ofC. neoformansisolates
Leaves
138
1
1
2
1
5
3.6
Flowers
88
1
1
3
2
7
7.9
Woody trunks
85
1
0
0
0
1
1.1
Total
311
3
2
5
3
13
4.2Figure 1
Brown color effect ofC. neoformansonEucalyptus leaves agar media; white colonies ofC. albicansnegative control and brown pigmented colonies of positive control and environmental isolates.


### 3.2. Molecular Identification and Differentiation ofC. neoformans

All tested isolates and reference strain were produced (400 bp) byCAP64 specific capsular gene primer (Figure 2). Molecular typing ofC. neoformans isolates was done by four primers forLAC1 gene (Table 2) which were used for amplification; serotype A strains produced three DNA fragments with sizes of 0.88, 0.76, and 0.25 kb (Figure 3). All testedC. neoformans strains were identified asC. neoformansvar.grubiiand there are no other serotypes ofC. neoformansdetected.Figure 2
Agarose gel electropheresis ofCAP64 gene specific PCR of all the examinedC. neoformans isolates with production of amplicons of 400 bp, marker 100 bp DNA ladder (Jena Bioscience).Figure 3
Agarose gel electropheresis ofLAC1 gene specific PCR of all the examinedC. neoformans isolates with production of amplicons of 250, 760, and 880 bp, marker 100 bp DNA plus ladder (Jena Bioscience).


## 3.1. Recovery Rate ofC. neoformans fromEucalyptus camaldulensis

In this study,E. camaldulensis trees acted as a potential refuge forC. neoformans. A total of 13 (4.2%)C. neoformans isolates out of 311 examined samples in Delta region were recovered during the flowering season ofEucalyptus tree. All the recoveredCryptococcus isolates were identified asC. neoformans strains based on all conventional and physiological characters ofC. neoformans (Figure 1). Among these, 7 isolates (7.9%) were recovered from 88Eucalyptusflowers, 5 isolates (3.6%) were recovered from 138Eucalyptus tree leaves, and 1 isolate (1.1) was recovered from 85 woody trunks (Table 3).Table 3
Recovery rate and distribution ofC. neoformans in tested Eucalyptus trees sampled in the Delta region.
Total samples
Giza
Cairo
Al-Sharqia
Elmenofia
Total
Recovery rate
Number ofC. neoformansisolates
Leaves
138
1
1
2
1
5
3.6
Flowers
88
1
1
3
2
7
7.9
Woody trunks
85
1
0
0
0
1
1.1
Total
311
3
2
5
3
13
4.2Figure 1
Brown color effect ofC. neoformansonEucalyptus leaves agar media; white colonies ofC. albicansnegative control and brown pigmented colonies of positive control and environmental isolates.


## 3.2. Molecular Identification and Differentiation ofC. neoformans

All tested isolates and reference strain were produced (400 bp) byCAP64 specific capsular gene primer (Figure 2). Molecular typing ofC. neoformans isolates was done by four primers forLAC1 gene (Table 2) which were used for amplification; serotype A strains produced three DNA fragments with sizes of 0.88, 0.76, and 0.25 kb (Figure 3). All testedC. neoformans strains were identified asC. neoformansvar.grubiiand there are no other serotypes ofC. neoformansdetected.Figure 2
Agarose gel electropheresis ofCAP64 gene specific PCR of all the examinedC. neoformans isolates with production of amplicons of 400 bp, marker 100 bp DNA ladder (Jena Bioscience).Figure 3
Agarose gel electropheresis ofLAC1 gene specific PCR of all the examinedC. neoformans isolates with production of amplicons of 250, 760, and 880 bp, marker 100 bp DNA plus ladder (Jena Bioscience).


## 4. Discussion

In Egypt,Eucalyptus trees are in abundance mainly as windbreaks and for afforestation of the drains and canals or other watercourses, plus the highways and roads in rural or urban areas.E. camaldulensis has a potential role inC. neoformansecology, particularly var.gattii. In Australia (home country ofE. camaldulensis), Ellis and Pfeiffer, 1990 reported the first environmental isolation ofC. gattii from wood, bark, leaves, and plant debris ofEucalyptus trees [14]. AlthoughEucalyptus is present in many of the areas known to haveC. gattii cryptococcosis, the actual isolation ofC. gattii fromEucalyptus trees is rare outside Australia. Moreover, importedEucalyptus has not been associated with the environmental presence ofC. gattii in Spain, Central Africa, Canada, Papua New Guinea, Egypt, and Italy [15].On the African level, the isolation ofC. gattii from the environment is somewhat limited in comparison to the isolation ofC. neoformans. Only two cases were recorded in isolation ofC. gattii fromE. camaldulensis in African countries, Egypt [9] and Tunisia [16].In Egypt, earlier report of Mahmoud (1999) [9] depended on canavanine-glycine-bromothymol blue (CGB) agar to determineC. neoformans variety, which evoked a high need to investigate the environmental ecology of this fungus, depending on molecular techniques to determine the actual variety ofC. neoformansin relation toE. camaldulensis in order to establish a real surveillance program and applying the preventive measures for this pathogen infection.This study was applied onEucalyptus trees during the flowering season, as mostC. neoformans andC. gattii reported cases were associated withEucalyptus showing strong seasonality in its occurrence, which coincides with the periods of flowering [17].The results show that the isolation ofC. neoformans fromEucalyptus flowers is more frequent than from leaves and woody trunk (Table 3). All examined isolates were identified asC. neoformansvar.grubii with a recovery rate of 4.2% of the total examined samples. It is normally the high isolation rate of var.grubiias the global distributed isolate responsible for cryptococcal infection [1, 5–7]. Also, it is commonly the recovering ofC. neoformans from pigeon droppings, soil, and decaying wood in hollow trees [3].Ambitiously, the present study documents the first record for the isolation of var.grubiifromE camaldulensisleaves, flower, and woody trunks in Egypt. Most of the previous reports stated thatC. grubii association withEucalyptus trees or other types of trees is interpreted in one sentence: “C. neoformans presence might represent fecal contamination by birds inhabiting these trees” [9, 14, 16].Globally, many reports are recorded for isolation ofC. grubii fromEucalyptus tree parts or other types of trees. In India, more than one report states thatC. grubiitree association and its distribution differ from each part of tree or season or time of the study. The prevalence ofC. grubii (5.56%) andC. gattii (9.26%) from decayed wood inside trunk hollows of diverse tree species was reported [18].Recently,C. grubii was isolated from the bark ofEucalyptus trees followed by flower, bud, fruit, and detritus [19]. The prevalence ofC. grubii in this study (4.2%) is somewhat near to the rate of Nawange et al.’s (2006) [18] study (5.5%), while the recovery rate ofC. grubii is the highest from flowers (7.9%), then leaves (3.6%), and finally woody trunks (1.1%) (Table 3).In sunny countries, C. neoformans can escape from lethal effects of sunlight and drying by taking trees as a good natural habitat in the environment because these pathogens can live inside woody debris as well as trunk hollows.The result of the present study highlighted the potential role of tree parts ofE. camaldulensis in environmental ecology ofC. grubii.Eucalyptus flowers were the best natural habitat and a suitable transporting means for these pathogen infectious propagules in the surrounding environment. Flowering season ofEucalyptus tree is mainly in winter and spring from November to February. At this time of year in Egypt, the temperature is slightly low to temperate which gives a potential chance for isolation of this pathogen. The association betweenC. grubii and tree is controlled by many environmental factors including humidity, temperature, and solar radiation [13, 17].In Egypt, theEucalyptus tree exists almost along every roadside, especially in the Delta region around River Nile and its tributaries. These study results confirm the potential role ofEucalyptus trees as a major source forC. grubii in Egyptian environment which act as a high risk for immunocompromised patients.Most of the reported cases of human cryptococcosis were registered as cryptococcal meningitis. Cryptococcal meningitis in Egypt is rarely diagnosed, but this may be due to inadequate investigation rather than absence of definite epidemiological data about the organism in Egypt. More attention should be considered for human cases of unexplained chronic meningitis that is not responding to conventional therapy asC. neoformans could be the main cause of such fetal meningitis [20–23].The only survey for fungal meningitis was done in Egypt at NAMRU-3 during the period of 1998 to 2001 of 1000 CSF samples, where 10C. neoformans were recovered at a rate of 0.01%. All isolates belonged to serotype A (C. neoformansvar.grubii) [24]. Recently,C. neoformans serotype A is the most common variety in association of pet birds droppings in the Egyptian environment [25].This study’s findings come in the same direction with the previous surveillance of the main causes of cryptococcal infection in Egypt and it confirmed thatC. neoformansvar.grubii is the main etiological agent of cryptococcal infection in Egypt.Conclusively, this is the first record describing isolation ofC. neoformansvar.grubiifromE. camaldulensis in Africa and Egypt. The results highlighted the potential role and risk ofEucalyptustree as a carrier reservoir of one of the high pathogenic fungal elements in Egypt.



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*Source: 4080725-2016-01-13.xml*