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Molecular typing of environmental Cryptococcus neoformans isolated from

pigeon droppings in Tripoli, Libya

1Mohamed S Ellabib, 1Mohamed A Aboshkiwa, 2Roberta DAmicis and Massimo Cogliati

1Department of Medical Microbiology and Immunology Faculty of Medicine, Tripoli

University, Libya 2Lab. MicologiaMedica, Dipartimento di

ScienzeBiomediche per la Salute, UniversitdegliStudi di Milano

Aim of Study This study was aimed to obtain an overview of the presence of C. neoformans in pigeon droppings in Tripoli urban area as well as to identify the molecular type and the mating type of the collected isolates.

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Back Ground: Cryptococcus neoformans is the principal etiological agent of cryptococcosis that has a worldwide distribution. The pathogen occurs as a saprobe in a wide variety of natural substrates including avian excreta, soil and wood. The yeast Cryptococcus neoformans has currently been classified according to its perfect state, Filobasidiella. Single species has been described, composed by two varieties and five serotypes: Filobasidiella neoformans variety neoformans (serotypes A, D, and A/D) and F. neoformans variety bacillispora (serotypes B and C). The varieties neoformans (serotypes A, D, A/D) and gattii (serotypes B and C), which belong to teleomorph F. neoformans, have been correlated (Cryptococcus neoformans). More recently, the variety grubii (serotype A) and the new specie, C. bacillispora (var gattii), have been proposed. The yeast has been frequently recovered from pigeon droppings in many countries of the world but not Libya. Therefore, the present study was conducted on the environmental distribution, variety and mating types of C. neoformans in Libya.

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Materials and Methods Sampling Collection and Processing:

One hundred samples of dried and old pigeon droppings were collected during year 2013 with wooden spatulas and placed in clean polythene bags from four different locations in Tripoli, the capital of Libya. They were processed in the laboratory of Medical College. One gram of specimen from each sample was suspended in sterilized glass bottle containing 9 ml of sterile physiological saline (0.85% NaCl) supplemented with chloramphenicol (10 g/ml). The mixture was left at room temperature for about 10-15 min, then shaken manually for 4-5 min and later incubated at 37C for one hour. Ten fold serial dilutions were made and 0.1 of each dilution was plated onto duplicate plates of sunflower seed agar supplemented with antibiotics and biphenyl. The plates were incubated at 30C for one week.

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Pigeon droppings samples collected from various sites

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Species Identification: Identification of C. neoformans was performed on the basis of melanin pigment production on sunflower seed agar, presence of a capsule observed in India ink preparation, urease production on urea agar medium, and ability to grow at 37 C. Vitek2 compact system was used to confirm C. neoformans identification.

Molecular Typing and Mating Type Allelic Pattern:

The 32 strains molecular type and mating type were carried out at Lab. Medical Mycology, Department of Biomedical Sciences for Health, University of Milan by multiplex PCR as described elsewhere (Cogliati et al., Med. Mycol. 2000; 38: 97-103,

Esposto et al., Clin. Microbiol. Infect. 2004; 10:1089-

1104)

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Figure Legend

Determination of mating type and molecular type by

multiplex PCR.The pictures show the results obtained for

nine representative isolates from pigeon droppings. H99

strain was included as reference strain VNI A.

Results

Cryptococcus neoformans was isolated from pigeon dropping samples collected from two out of four sites selected. Out of 100 samples collected, 32 (32%) samples were positive for Cryptococcus neoformans. The samples from site four were showed the highest rate of contamination 24 out of 50 (48%), while site one 8 sample were positive out of 35 (22.9%). Only 10 and 5 samples were obtained from 2 and 3 sites respectively. These two sites did not show any contamination to Cryptococcus neoformans as shown in Table 1 and Figure 1. .

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Site Number N NP Positive %

1 35 8 23

2 10 0 0

3 5 0 0

4 50 24 48

Table.1 Distribution of C. neoformans isolated from

pigeon droppings in Tripoli region

N=Number of Sample, NP= Positive sample

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Growth of Cryptococcus neoformans on

Sunflower Seed Agar shown brown color

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Urease test for Cryptococcus neoformans

(Positive) and Candida albicans (Negative).

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Figure 1: Percentage of Cryptococcus neoformans in pigeon droppings.

-10%

0%

10%

20%

30%

40%

50%

60%

0 1 2 3 4 5

positive sample %

positive sample %

Site number

% of positivity

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Molecular and Mating Type

Multiplex PCR amplifications reveal that all isolates belonged to molecular type VNI (C. neoformans var. grubii) and mating type alpha A, as shown in Figure 2.

Figure 2: Multiplex PCR amplifications of isolate strains

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Discussion: This study provides for the first time information concerning the ecology and genotypes of C. neoformans in the Tripoli region of Libya. In this study, 32 isolates of Cryptococcus neoformans were obtained from samples of pigeon feces of Tripoli, in which all of them reveal that they were, belong to molecular type VNI (C. neoformans var. grubii) and mating type alpha A. Conclusion

This study provides for the first time information concerning the ecology and genotypes of C. neoformans in the Tripoli region of Libya Recommendation:

Epidemiology surveying study on large scale is needed for confirming presence or absence of other Cryptococcus neoformans varieties and their genotypes.

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Acknowledgment:

I would like to express my very great appreciation and thanks to Dr Massimo Cogliati and Roberta DAmicis for their valuable and constructive molecular analysis work. I would also like to thank the staff of all places for enabling me to collect the sample from their sites.