Molecular identification of yeasts in oral cavity in immunocompetent teenagers from the North of Mexico

doi:10.21203/rs.3.rs-1995629/v1

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Molecular identification of yeasts in oral cavity in immunocompetent teenagers from the North of Mexico

https://doi.org/10.21203/rs.3.rs-1995629/v1

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Human oral cavity is normally colonized for microorganisms including bacteria, fungi, archaea, viruses and protozoa. The aim of this study was to determine the frequency of Candida albicans in de oral cavity in a group of medicine students from the north of Mexico. 240 oral samples from healthy students were analyzed by traditional microbiology cultures and DNA sequencing. 57 samples grew in media cultures. Sequence analysis identified the yeast as follow: 28 Clavispora lusitaniae, 20 Candida albicans, 5 Pichia kudriavzevii and 4 Candida parapsilosis. Our finding revealed that 23.75% of healthy population has a potential pathogen in its mouth. Surprisingly Candida albicans is not the predominant yeast, instead of others non-Candida species are the colonizers of the oral cavity in immunocompetent individuals. Oral cavity is colonized by potential pathogen yeast being Clavispora lusitaniae, Candida albicans, Pichia kudriavzevii and Candida parapsilosis.

opportunistic

microbiota

mycobiome

pathogen

multi drug resistant

The human oral cavity is normally colonized for a wide range of microorganisms including bacteria, fungi, archaea, viruses and protozoa¹. The bacterial 16S rRNA gene sequencing has provided information about the oral microbiome and has allowed the creation of the Human Oral Microbiome Database (HOMD). Bacterial microbiome is more studied that the fungal one^{2, 3}. Fungi constitute a little part of oral microbiome, it is also named oral fungal microbiome or mycobiome⁴. Yeast infections are one of the most common diseases caused by opportunistic pathogens in both immunocompromised and immunocompetent host⁵. Candida species are opportunistic fungal pathogens and are common members of human mycobiome. Candida albicans is an early colonizer acquired at or near birth primarily by physical contact⁶. Candida genus causes a wide range of pathologies, their severity varies depending the pathogen and the host’s immune condition. Candida infection begins by mucous membrane colonization. It happens by a microbiota misbalance, being Candida albicans the most predominant microorganism, then Candida albicans disseminates and contributes to acquire other oral diseases⁷. In the last decades, isolation of non-Candida albicans species has significantly increased^{6, 8}. Four Candida species, such as: C. albicans, C. glabrata, C. tropicalis and C. parapsilosis encompasses ~ 95% of identifiable Candida infections. Although C. albicans is the most common causative agents, its incidence is declining while the frequency of the other species is increasing⁹. Other clinical relevant non-Candida albicans species are: Clavispora lusitaniae (anamorph: Candida lusitaniae), Meyerozyma guilliermondii (anamorph: Candida guillermondii), Candida krusei (anamorph: Pichia kudriavzevii), Candida dubliniensis, Debaryomyces hansenii (synonym: Candida famata) and Lodderomyces elongisporus^{10, 11}. The genus Clavispora was proposed by Rodrigues de Miranda and comprised two species: C. lusitaniae and C. opuntiae¹². Clavispora lusitaniae (anamorph of Candida lusitaniae) has been isolated from animals and human samples showing its ability to stablish as a persistent commensal on the skin, genitourinary and respiratory tracts of hospitalize patients^{13, 14, 15}. The microorganism identification in the human gut has been poorly explored. One of the first studies showed the presence of C. albicans, C. glabrata, Trichosporon asahii, Clavispora lusitaniae, Devariomyces hanseii, Malazzesia restricta and Malazzesia sp using molecular and traditional culture methods¹⁶.

Opportunistic pathogenic such as Candida species, contradictorily also are normal in human oral cavity. Candida switches from non-pathogen to pathogen avoiding immune response and local or systemic infection can arise¹⁷. Pichia kudriavzavii is other example of yeast that can switch from non-pathogen to pathogen. Pichia kudriavzavii has been isolated from food, fruits, sourdoughs and fermented butter-like products¹⁸. Pichia kurdriavzevii also was isolated from fecal samples in healthy individuals¹⁹. Nosocomial sepsis, also known as late-onset sepsis or healthcare-associated sepsis is a very common complication in neonates hospitalized in the neonatal intensive care unit (NICU). Fungi are the most common cause on late-onset sepsis associated with substantial morbidity and mortality. Candida species are the commonest cause of late-onset fungal sepsis known to be transmitted horizontally resulting in outbreaks. However very few studies have demonstrated clusters of fungal sepsis due to teleomorphic form of non-albicans species identified by DNA sequencing. Nagarathnamma et al in India, reported an outbreak of fungal sepsis in a NICU that was caused by the rare pathogen Pichia kudriavzevii (teleomorph of Candida krusei)²⁰. Although oral C. albicans still remain at the forefront of Candida infections, recently non-albicans species have attracted a lot of attention due to an increase epidemiological changes and development of antifungal resistance²¹. Being C. albicans the most prevalent Candida species in oral cavity of healthy and sick individuals, the aim of this study was to determine the frequency of Candida albicans in de oral cavity in a group of medicine students from the north of Mexico.

Oral sample collection

This study was approved by the research committee and ethic in research committee of the Vicerrectoria de Ciencias de la Salud de la Universidad de Monterrey under reference number 08102018-CEI. Oral samples were collected from 240 students at private University in the North of Mexico from January to December 2019. All of them signed consent informative letter. No specific pathology was reported in any of these teenagers. Oral samples were transferred to Sabouraud dextrose agar (SDA) broth in a tube and incubate at 30° C for 48 hours. Growth in SDA broth was plated in CHROMagar Candida medium (Microbiology, Paris, France) at 30° C for 48 hours to obtain pure colonies and presumptive identification of Candida spp.

Molecular identification

Genomic DNA was extracted from pure culture of presumptive Candida spp isolates using the ZR Fungal/Bacterial DNA MiniPrep Kit (Zymo Research Irvine, CA). DNA was used to amplify the ITS1-5.8S-ITS2 ribosomal gene region, using primer and an amplification program described previosly^{22, 23}. The amplifications products were sequences by the Sanger method in a Genetic Analyzer 3130 sequencer (Applied Biosystems, Foster City, CA). The sequences obtained in our study were compared with sequences deposited in the public genomic databases (GenBank, NCBI, USA).

A total of 240 teenager students participated voluntary. The range age was 18–20 years old. 57 students were positive to yeasts presence in oral cavity by CHROMagar Candida medium (Microbiology, Paris, France). Thus, 57 sequences were analyzed. This analysis identified the yeast as follow: 28 Clavispora lusitaniae, 20 Candida albicans, 5 Pichia kudriavzevii and 4 Candida parapsilosis. Their homology respect to those reported in the GenBank was 99–100% except to Pichia kudriavzevii homology was lower (77–95%). Sequences from Clavispora lusitaniae, Candida albicans and Candida parapsilosis were deposited in the GenBank (Table 1). Pichia kudriavzevii sequences did not accepted in the GenBank due to their low quality.

Table 1

Accession numbers from the sequences obtained in this study.
Sample number	Accession number	Organism	Source	Host
25	MT814255	Clavispora lusitaniae	Oral cavity	immunocompetent
26	MT814256	Clavispora lusitaniae	Oral cavity	immunocompetent
27	MT814257	Clavispora lusitaniae	Oral cavity	immunocompetent
28	MT814258	Clavispora lusitaniae	Oral cavity	immunocompetent
29	MT814259	Clavispora lusitaniae	Oral cavity	immunocompetent
30	MT814260	Clavispora lusitaniae	Oral cavity	immunocompetent
31	MT814261	Clavispora lusitaniae	Oral cavity	immunocompetent
32	MT814262	Clavispora lusitaniae	Oral cavity	immunocompetent
33	MT814263	Clavispora lusitaniae	Oral cavity	immunocompetent
34	MT814264	Clavispora lusitaniae	Oral cavity	immunocompetent
35	MT814265	Clavispora lusitaniae	Oral cavity	immunocompetent
36	MT814266	Clavispora lusitaniae	Oral cavity	immunocompetent
37	MT814267	Clavispora lusitaniae	Oral cavity	immunocompetent
38	MT814268	Clavispora lusitaniae	Oral cavity	immunocompetent
39	MT814269	Clavispora lusitaniae	Oral cavity	immunocompetent
40	MT814270	Clavispora lusitaniae	Oral cavity	immunocompetent
41	MT814271	Clavispora lusitaniae	Oral cavity	immunocompetent
42	MT814272	Clavispora lusitaniae	Oral cavity	immunocompetent
43	MT814273	Clavispora lusitaniae	Oral cavity	immunocompetent
44	MT814274	Clavispora lusitaniae	Oral cavity	immunocompetent
45	MT814275	Clavispora lusitaniae	Oral cavity	immunocompetent
46	MT814276	Clavispora lusitaniae	Oral cavity	immunocompetent
47	MT814277	Clavispora lusitaniae	Oral cavity	immunocompetent
48	MT814278	Clavispora lusitaniae	Oral cavity	immunocompetent
50	MT814279	Clavispora lusitaniae	Oral cavity	immunocompetent
55	MT814280	Clavispora lusitaniae	Oral cavity	immunocompetent
56	MT814281	Clavispora lusitaniae	Oral cavity	immunocompetent
58	MT814282	Clavispora lusitaniae	Oral cavity	immunocompetent
1	MZ562454	Candida albicans	Oral cavity	immunocompetent
2	MZ562455	Candida albicans	Oral cavity	immunocompetent
5	MZ562456	Candida albicans	Oral cavity	immunocompetent
6	MZ562457	Candida albicans	Oral cavity	immunocompetent
7	MZ562458	Candida albicans	Oral cavity	immunocompetent
8	MZ562459	Candida albicans	Oral cavity	immunocompetent
9	MZ562460	Candida albicans	Oral cavity	immunocompetent
10	MZ562461	Candida albicans	Oral cavity	immunocompetent
11	MZ562462	Candida albicans	Oral cavity	immunocompetent
12	MZ562463	Candida albicans	Oral cavity	immunocompetent
13	MZ562464	Candida albicans	Oral cavity	immunocompetent
14	MZ562465	Candida albicans	Oral cavity	immunocompetent
15	MZ562466	Candida albicans	Oral cavity	immunocompetent
16	MZ562467	Candida albicans	Oral cavity	immunocompetent
17	MZ562468	Candida albicans	Oral cavity	immunocompetent
18	MZ562469	Candida albicans	Oral cavity	immunocompetent
19	MZ562470	Candida albicans	Oral cavity	immunocompetent
21	MZ562471	Candida albicans	Oral cavity	immunocompetent
23	MZ562472	Candida albicans	Oral cavity	immunocompetent
54	MZ562473	Candida albicans	Oral cavity	immunocompetent
20	MZ566837	Candida parapsilosis	Oral cavity	immunocompetent
22	MZ566838	Candida parapsilosis	Oral cavity	immunocompetent
24	MZ566839	Candida parapsilosis	Oral cavity	immunocompetent
57	MZ566840	Candida parapsilosis	Oral cavity	immunocompetent

The oral microbiota is the most complex and diverse microbial community. This study was focused on isolating oral yeasts in healthy individuals. Our finding revealed that 23.75% of healthy population has a potential pathogen in its mouth. Surprisingly Candida albicans is not the predominant yeast, instead of others non-Candida species are the colonizers of the oral cavity in immunocompetent individuals²⁴. Interestedly Clavispora lusitaniae was the most frequent yeast. This microorganism has generated significant scientific attention due to its isolation from diverse clinical samples in invasive infections. Also, there are reports in which Clavispora lusitaniae had acquired multidrug resistance. The present study increased our knowledge regarding Candida albicans and Clavispora lusitaniae as living microorganisms in the oral microbiota.

Funding

The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.

Competing Interests

The authors have no relevant financial or non-financial interests to disclose.

Author Contributions

All authors contributed to the study conception and design. Samples recovery, microbial culture, DNA extraction and data collection were performed by Olga C. Rojas, Cintia Amaral-Montesino, Soraya Mendoza-Olazaran and Alexandra M. Montoya. DNA sequence analysis and the first draft of the manuscript was written by Rafael González-Álvarez. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Data Availability

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

Acknowledgments

Authors grateful to sample donors

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No competing interests reported.

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Molecular identification of yeasts in oral cavity in immunocompetent teenagers from the North of Mexico

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Abstract

Introduction

Materials And Methods

Oral sample collection

Molecular identification

Results

Discussion

Declarations

References

Additional Declarations

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