Tatiane Nobre Pinto1†
Laura M. A. Oliveira1†
Gisela L. da Costa2†
Natália Silva Costa1†
Elaine Cristina Francisco3†
Tatiana C. A. Pinto1†
Manoel M. E. Oliveira2*†- 1Laboratório de Cocos Patogênicos e Microbiota, Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
- 2Laboratório de Taxonomia, Bioquímica e Bioprospecção de Fungos, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil
- 3Laboratório Especial de Micologia, Universidade Federal de São Paulo, São Paulo, Brazil
In this study, we report the first isolation of Hanseniaspora opuntiae obtained from four pregnant women in Brazil. Clinical isolates were obtained from four samples taken between 35 and 37 gestational weeks, as part of the routine antenatal care for maternal colonization screening for Streptococcus agalactiae group B. The patients were immunocompetent, with two of them diagnosed with gestational diabetes mellitus. Species identification was performed by MALDI-TOF MS and rDNA sequencing. While Hanseniaspora species have not traditionally been considered a typical opportunist pathogen, our findings emphasize the importance of investigating and screening for Hanseniaspora in pregnant populations, highlighting H. opuntiae as a potential agent of human infections.
Introduction
The genus Hanseniaspora is part of the Saccharomycetaceae family, which is characterized by apiculate yeasts with bipolar budding cells and globose, warty ascospores (Cadez and Smith, 2011). Currently, the genus comprises approximately 20 species that can be divided into two major clades: the “fermentation clade,” known for its robust fermentation capability, and the “fruit clade,” consisting of species more adapted to fruit colonization (Čadež et al., 2019; van Wyk et al., 2024). While members of Hanseniaspora spp. have been extensively studied for their ability to enhance the sensory profile of wines and beer (Martin et al., 2018; Burini et al., 2021; Zhang et al., 2023), there is a significant gap in their clinical relevance.
Although rare, H. uvarum and H. opuntiae have been documented to play a pathogenic role in both immunocompetent and immunocompromised patients, with reports of superficial mycoses, gastroenteritis episodes, oral injuries, and invasive infections (Emmanouil-Nikoloussi et al., 1994; García-Martos et al., 1998; Karimi et al., 2015; González-Abad and Sanz, 2018; Jankowski et al., 2018). Recent studies have highlighted an increase in yeast colonization during pregnancy, and vaginal fungal infections in pregnant women have been associated with increased pregnancy complications (Disha and Haque, 2022). Notably, a significant rise in H. pseudoguilliermondii and H. meyeri has been noted in pregnant women with gestational diabetes mellitus (GDM) (Wu et al., 2022), emphasizing the necessity for a deeper understanding and assessment of Hanseniaspora species occurrence in pregnant individuals. In this study, we report the co-isolation of Hanseniaspora opuntiae and Streptococcus agalactiae in two pregnant women with a history of GDM who went to the Teaching Maternity of the Universidade Federal do Rio de Janeiro, Brazil.
Description of four cases
The four clinical isolates of Hanseniaspora were recovered from four pregnant women undergoing routine antenatal care procedures and maternal rectovaginal colonization surveillance for S. agalactiae (Group B or GBS) at the Teaching Maternity of the Federal University in 2020. A questionnaire was used during medical examinations to collect clinical data from the patients, who were aged between 13 and 37 at the time of collection. One patient reported a history of urinary tract infection during pregnancy plus vaginal discharge, and the use of the antibiotic cephalexin as treatment (Table 1). While two other patients reported the use of the antibiotic cephalexin during pregnancy, the specific period of usage was not documented. All patients were immunocompetent, with two of them having GDM.
Table 1 Demographic data and laboratory findings of the four Hanseniaspora opuntiae clinical isolates obtained from pregnant women.
The four samples were collected from the anus and vagina (anovaginal), during the 35–37 weeks of gestation, using a single swab for both the anal and vaginal regions while avoiding the cervix, in accordance with current guidelines (Filkins et al., 2020; Costa et al., 2022). Subsequently, the swabs were preserved in skim milk, tryptone, glucose, and glycerin (STGG) transport medium at −20°C. An aliquot of the STGG medium containing the swabs was inoculated onto blood agar and Sabouraud dextrose agar (BD Difco, Franklin Lakes, USA) plates supplemented with 0.05 g/L chloramphenicol. The plates were then incubated at 35°C for 48 h. Following the incubation period, colonies of S. agalactiae were observed in two clinical samples and yeast growth was observed from all four clinical samples. Yeast identification was conducted by MALDI-TOF MS using the Bruker Daltonics platform (Bruker Daltonics, Bremen, Germany). Protein extraction was performed according to the manufacturer’s recommendations with few modifications (Wang et al., 2021; Pinto et al., 2022). The isolates were tested in triplicate and data analysis was performed using the Biotyper™ 3.1 software (Bruker Daltonics), with log score ≥ 2.0 considered high confidence. Using the Bruker database, the four isolates were identified as H. opuntiae with a score ranging from 1.761 to 1.878 (Table 1).
The species was identified by sequencing the ITS1–5.8S-ITS2 rDNA region using a new DNA extraction method developed by our team. This methodology is an adaptation of the colony PCR technique described by Mirhendi et al. (2007) and is described for the first time in this paper. In our methodology, after 48 h of growth at 30°C, a small amount of each isolate was selected (approximately 106 yeast cells), transferred to PCR tubes, and manually smeared using a 10-μL pipette tip. The tubes were then heated in a microwave for 90 s at maximum power and immediately cooled to 0°C. The PCR mix was prepared to a final volume of 50 μL, containing 1 μg of DNA and 25 ng/μL of the ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) primer pair (Van Gerrits den Ende and de Hoog, 1999; Pinto et al., 2022; Costa, 2023; Oliveira et al., 2023). Sequencing was carried out using the ABI-3730 (Applied Biosystems) at the Network Technological Platforms of Fiocruz. Sequencing editing was performed using CodonCode Aligner (Genes Code Corporation, Ann Arbor, United States). The consensus sequences were cross-referenced with those available in the NCBI database (http://ncbi.nlm.nih.gov) using the BLASTn tool. Only sequences with a percentage of identity and sequence coverage of ≥98% and an E-value of <10−5 were considered. Phylogenetic analysis was performed by using the Neighbor-Joining method based on the Kimura two-parameter model with 1,000 bootstrap pseudo replicates in MEGA X (Kimura, 1980; Felsenstein, 1985; Saitou and Nei, 1987; Cadez et al., 2003; Kumar et al., 2018).
During the BLASTn analysis, the sequences of the four clinical isolates exhibited significant similarity to H. opuntiae and H. meyeri, with 100% coverage/identity and an E-value of 0.0 for both species. Through phylogenetic analysis, four isolates were accurately identified as H. opuntiae (Figure 1). The ITS rDNA sequences were deposited in the GenBank database under accession numbers OR641877 to OR641880. This study was approved by the research ethics committee of the Clementino Fraga Filho University Hospital of the Federal University of the State of Rio de Janeiro (CAAE 43389321.9.0000.5257). All patients were granted free and informed consent.
Figure 1 The phylogenetic analysis was inferred using the Neighbor-Joining method (Saitou and Nei, 1987). The optimal tree is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) is shown next to the branches (Felsenstein, 1985). The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Kimura 2-parameter method (Kimura, 1980). These distances are measured in the number of substitutions for each site. Four clinical isolates from our investigation and 14 sequences of reference strains retrieved from GenBank data formed the 18 nucleotide sequences that were analyzed. There were a total of 719 positions in the final dataset. Evolutionary analyses were conducted in MEGA X (Kumar et al., 2018).
Discussion
In the present study, the MALDI-TOF MS proved to be a reliable tool for identifying Hanseniaspora spp. exhibiting high-confidence log scores for genus-level identification (1.761–1.878). Despite the lack of success in providing species characterization with a high-confidence log score (≥2.0) among our isolates, the identification of Hanseniaspora spp. at the species level by using the Bruker platform has been observed in previous studies with promising results for H. guilliermondii. We highlight here the relevance of optimizing commercial databases with diverse reference protein spectra of Hanseniaspora species (Cassagne et al., 2016).
Based on our phylogenetic analysis, the four clinical isolates were accurately identified as H. opuntiae. However, the limited availability of sequences from H. opuntiae and from its closely related species, such H. lachancei, H. pseudoguilliermondii, and H. urvarum, may pose a potential challenge for the correct characterization of H. opuntiae in routine laboratories, even when using molecular investigation methods. Conventional physiological assays are not able to distinguish H. opuntiae from H. meyeri, H. uvarum, or H. guilliermondii, but investigation of 2-keto-d-gluconate as a single carbon source may be helpful to distinguish H. lachancei from other Hanseniaspora species, including H. opuntiae (Cadez et al., 2003).
Although the genus Hanseniaspora has not been considered a typical opportunist pathogen, its species have been documented in different spectra of human mycoses (Emmanouil-Nikoloussi et al., 1994; García-Martos et al., 1998; Karimi et al., 2015; González-Abad and Sanz, 2018; Jankowski et al., 2018). To the best of our knowledge, this is the first report in Brazil of H. opuntiae isolated from anovaginal samples from pregnant women. According to (Wu and colleagues (2022)), the increased prevalence of the genus in the gastrointestinal microbiome of pregnant women may be related to GDM (Costa et al., 2022). Similarly, in our study, two isolates of H. opuntiae were recovered from pregnant women with the same condition. Elevated blood glucose levels may represent a potential risk factor for infections caused by this species, given the evolutionary adaptation of H. opuntiae to sugar-rich environments (phylogenetic fruit clade).
We were unable to conduct antifungal susceptibility tests on the clinical isolates and to continue laboratory monitoring of the patients to track the colonization/infection profile of Hanseniaspora. However, the isolation of Hanseniaspora species in anovaginal samples provided by this study opens discussions regarding non-Candida yeasts as a potential pathogen implicated in vaginal infections among this population. In conclusion, our findings emphasize the importance of investigating and screening for Hanseniaspora spp. in pregnant women, with H. opuntiae as a potential agent of human infections. Further research are needed to explore the genus comprehensively, elucidating its natural history and its health implications in pregnant women and diabetic hosts.
Data availability statement
The datasets generated during the current study are available in the GenBank repository (https://www.ncbi.nlm.nih.gov/nuccore) under accession numbers OR641877, OR641878, OR641879.1, and OR641880.1.
Ethics statement
The study was approved by the Research Ethics Committee of Clementino Fraga Filho University Hospital of the Federal University of the State of Rio de Janeiro (CAAE 43389321.9.0000.5257).
Author contributions
TP: Formal analysis, Investigation, Methodology, Writing – original draft. LO: Methodology, Visualization, Writing – review & editing. Gd: Supervision, Visualization, Writing – review & editing. NC: Formal analysis, Investigation, Methodology, Writing – original draft. EF: Methodology, Visualization, Writing – review & editing. TP: Conceptualization, Writing – original draft, Writing – review & editing. MO: Conceptualization, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Visualization, Writing – review & editing.
Funding
The author(s) declare financial support was received for the research, authorship, and/or publication of this article. The study was partially supported by a grant received from the State Funding Agency Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ - Grants: JCNE E-26/201.433/2021–MMEO) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq - Grant Proc. 409227/2016–1).
Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Publisher’s note
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.
References
Burini, J. A., Eizaguirre, J. I., Loviso, C., Libkind, D. (2021). Levaduras no convencionales como herramientas de innovación y diferenciación en la producción de cerveza [Non-conventional yeasts as tools for innovation and differentiation in brewing]. Rev. Argent Microbiol. 53, 359–377. doi: 10.1016/j.ram.2021.01.003
Čadež, N., Bellora, N., Ulloa, R., Hittinger, C. T., Libkind, D. (2019). Genomic content of a novel yeast species Hanseniaspora gamundiae sp. nov. from fungal stromata (Cyttaria) associated with a unique fermented beverage in Andean Patagonia, Argentina. PloS One 14, e0210792. doi: 10.1371/journal.pone.0210792
Cadez, N., Poot, G. A., Raspor, P., Smith, M. T. (2003). Hanseniaspora meyeri sp. nov., Hanseniaspora clermontiae sp. nov., Hanseniaspora lachancei sp. nov. and Hanseniaspora opuntiae sp. nov., novel apiculate yeast species. Int. J. Syst. Evol. Microbiol. 53, 1671–1680. doi: 10.1099/ijs.0.02618-0
Cadez, N., Smith, M. (2011). “Hanseniaspora zikes (1912),” in The Yeasts, A Taxonomic study, 5th ed. Eds. Kurtzman, C., Fell, J. W., Boekhout, T. (Elsevier, Amsterdam), 421–434.
Cassagne, C., Normand, A. C., Bonzon, L., L’Ollivier, C., Gautier, M., Jeddi, F., et al. (2016). Routine identification and mixed species detection in 6,192 clinical yeast isolates. Med. Mycol. 54, 256–265. doi: 10.1093/mmy/myv095
Costa, N. S., Rio-Tinto, A., Pinto, I. B. F., dos Santos Silva Alvim, D. C., de Assis Rocha, A., Oliveira, L. M. A., et al. (2022). Changes in Group B Streptococcus Colonization among Pregnant Women before and after the Onset of the COVID-19 Pandemic in Brazil. Pathogens 11 (10), 1104. doi: 10.3390/pathogens11101104
da Costa, G. L. (2023). Candida palmioleophila: A new emerging threat in Brazil? J. Fungi 9 (7), 770. doi: 10.3390/jof9070770
Disha, T., Haque, F. (2022). Prevalence and risk factors of vulvovaginal candidosis during pregnancy: A review. Infect. Dis. Obstet Gynecol. 2022, 6195712. doi: 10.1155/2022/6195712
Emmanouil-Nikoloussi, E., Kanellaki-Kyparissi, M., Papavassiliou, P., Koliakos, K., Dermentzopoulou, M., Foroglou, C. H. (1994). Hanseniaspora uvarum» the ultrastructural morphology of a rare ascomycete, isolated from oral thrush. Bull. Group Int. Rech Sci. Stomatol Odontol 37 (1-2), 13–17.
Felsenstein, J. (1985). Confidence limits on phylogenies: an approach using the bootstrap. Evolution. 39, 783–791. doi: 10.2307/2408678
Filkins, L., Hauser, J. R., Robinson-Dunn, B., Tibbetts, R., Boyanton, B. L., Revell, P. (2020). American society for microbiology provides 2020 guidelines for detection and identification of group B streptococcus. J. Clin. Microbiol. 59, e01230–e01220. doi: 10.1128/JCM.01230-20
García-Martos, P., Hernández-Molina, J. M., Galán, F., Ruiz-Henestrosa, J. R., García-Agudo, R., Palomo, M. J., et al. (1998). Isolation of Hanseniaspora uvarum (Kloeckera apiculata) in humans. Mycopathologia. 144, 73–75. doi: 10.1023/a:1006900909455
González-Abad, M. J., Sanz, M. A. (2018). Isolation of Hanseniaspora opuntiae from blood cultures of an immunosuppressed patient: An infrequent and difficult to assess finding. Rev. Iberoamericana Micologia. Asociacion Espanola Micologia; 35, 168–169. doi: 10.1016/j.riam.2018.05.001
Jankowski, M., Jagielski, T., Misiak, G., Czajkowski, R. (2018). Hand dermatitis with Hanseniaspora uvarum as a plausible causative agent. Postepy Dermatologii i Alergologii 35, 641–643. doi: 10.5114/ada.2018.72854
Karimi, L., Mirhendi, H., Khodadadi, H., Mohammadi, R. (2015). Molecular identification of uncommon clinical yeast species in Iran. Curr. Med. Mycol. 1, 1–6. doi: 10.18869/acadpub.cmm.1.2.1
Kimura, M. (1980). A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol. 16, 111–120. doi: 10.1007/BF01731581
Kumar, S., Stecher, G., Li, M., Knyaz, C., Tamura, K. (2018). MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol. Biol. Evol. 35, 1547–1549. doi: 10.1093/molbev/msy096
Martin, V., Valera, M. J., Medina, K., Boido, E., Carrau, F. (2018). Oenological impact of the hanseniaspora/kloeckera yeast genus on wines—A review. Fermentation 4, 76. doi: 10.3390/fermentation4030076
Mirhendi, H., Diba, K., Rezaei, A., Jalalizand, N., Hosseinpur, L., Khodadadi, H. (2007). Colony PCR is a rapid and sensitive method for DNA amplification in yeasts. Iran J Public Health. 36, 40–44.
Oliveira, M. M. E., Lopes, A. P., Pinto, T. N., da Costa, G. L., Goes-Neto, A., Hauser-Davis, R. A. (2023). A Novel One Health Approach concerning Yeast Present in the Oral Microbiome of the Endangered Rio Skate (Rioraja agassizii) from Southeastern Brazil. Microorganisms 11, 1969. doi: 10.3390/microorganisms11081969
Pinto, T. N., Kohn, A., da Costa, G. L., Oliveira, L. M. A., Pinto, T. C. A., Oliveira, M. M. E. (2022). Candida guilliermondii as an agent of postpartum subacute mastitis in Rio de Janeiro, Brazil: Case report. Front. Microbiol., 13. doi: 10.3389/fmicb.2022.964685
Saitou, N., Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406–425. doi: 10.1093/oxfordjournals.molbev.a040454
Van Gerrits den Ende, A. H. G., de Hoog, G. S. (1999). Variability and molecular diagnostics of the neurotropic species Cladophialophora bantiana. Stud. Mycol 43, 151–162.
van Wyk, N., Badura, J., von Wallbrunn, C., Pretorius, I. S. (2024). Exploring future applications of the apiculate yeast Hanseniaspora. Crit. Rev. Biotechnol. 44, 100–119. doi: 10.1080/07388551.2022.2136565
Wang, J., Wang, H., Cai, K., Yu, P., Liu, Y., Zhao, G., et al. (2021). Evaluation of three sample preparation methods for the identification of clinical strains by using two MALDI-TOF MS systems. J. Mass Spectrometry 56, e4696. doi: 10.1002/jms.4696
Wu, N., Mo, H., Mu, Q., Liu, P., Liu, G., Yu, W. (2022). The gut mycobiome characterization of gestational diabetes mellitus and its association with dietary intervention. Front. Microbiol. 13. doi: 10.3389/fmicb.2022.892859
Keywords: Hanseniaspora, Streptococcus agalactiae, opportunistic pathogens, apiculate yeasts, MALDI-TOF MS
Citation: Pinto TN, Oliveira LMA, da Costa GL, Costa NS, Francisco EC, Pinto TCA and Oliveira MME (2024) Detection of Hanseniaspora opuntiae in anovaginal samples of pregnant women in Rio de Janeiro, Brazil—a case report. Front. Cell. Infect. Microbiol. 14:1394663. doi: 10.3389/fcimb.2024.1394663
Received: 25 March 2024; Accepted: 06 May 2024;
Published: 30 May 2024.
Edited by:
Patrícia Albuquerque, University of Brasilia, BrazilReviewed by:
Emeka Nweze, University of Nigeria, Nsukka, NigeriaTomasz Jagielski, University of Warsaw, Poland
Copyright © 2024 Pinto, Oliveira, da Costa, Costa, Francisco, Pinto and Oliveira. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Manoel M. E. Oliveira, manoel.marques@fiocruz.br
†ORCID: Tatiane Nobre Pinto, orcid.org/0009-0002-5811-1304
Laura M. A. Oliveira, orcid.org/0000-0002-7162-2013
Gisela L. da Costa, orcid.org/0000-0001-6038-0728
Natália Silva Costa, orcid.org/0000-0002-9194-6445
Elaine Cristina Francisco, orcid.org/0000-0002-5530-1332
Tatiana C. A. Pinto, orcid.org/0000-0002-7878-9377
Manoel M. E. Oliveira, orcid.org/0000-0001-7670-9225