Application of MALDI-TOF MS to species complex differentiation and strain typing of food related fungi: Case studies with Aspergillus section Flavi species and Penicillium roqueforti isolates
Introduction
Fungi are frequently involved in food spoilage and represent a major cause of food and economic losses. Indeed, among food losses and waste which represent 1 billion tons each year (FAO, 2011), it is estimated that 5–10% of them are due to fungal spoilage (Filtenborg et al., 1996; Pitt and Hocking, 2009). Fungi can spoil a large variety of feeds and foods, causing organoleptic properties deterioration such as visible growth on the product surface, off-flavor production, texture and color changes. Moreover, a large number of species such as Penicillium and Aspergillus spp. are potential mycotoxin producers, and may represent a great hazard for human health (Waśkiewicz, 2014). Hence, rapid and reliable identification of filamentous fungi is a key step for a better management of food safety and quality.
For several years now, MALDI-TOF MS has been successfully applied to microorganism identification, from bacteria (Basile et al., 1998) to fungi (Welham et al., 2000) including food-related fungi (Quéro et al., 2019). In the latter study, a spectral database comprising 619 strains belonging to 136 species of food interest was built and 90% correct identification at the species level were achieved after external validation. Several commercial instruments and databases are available (Deak et al., 2015) for routine identification. Besides identification at species level, MALDI-TOF MS has been shown as a powerful tool to discriminate fungal species complex and cryptic fungal species. As an example, Al-Hatmi et al. (2015) were able to correctly identify species of clinical interest belonging to the Fusarium fujikuroi complex, some of which being cryptic species. Allen et al. (2005) defined species complex as a cluster of related isolates which individuals may represent more than one species while cryptic species are morphologically indiscernible biological/phylogenetic units that are only revealed using DNA-based molecular analysis (Hawksworth, 2006). In most cases, the identification of such species requires the analysis of several specific genes and expertise in data analysis (Balasundaram et al., 2015). Species complex are an issue not only in clinical context but also in the food context, particularly regarding mycotoxin production. For example, in the Aspergillus genera, and more particularly in the Flavi section which contains several cryptic species and currently comprises 33 phylogenetically distinct species (Frisvad et al., 2019), species have different mycotoxin production abilities, some species being able to produce B1, B2, G1 and G2 aflatoxins (e.g. A. nomius, A. novoparasiticus, A. parasiticus) while others only produce B1 and B2 aflatoxins (A. flavus, A. pseudotamarii and A. togoensis) or no aflatoxins (A. caelatus, A. subflavus and A. tamarii) (Frisvad et al., 2019). Another challenge within this section is the discrimination between the toxigenic Aspergillus flavus and A. parasiticus and the non-toxigenic A. oryzae and A. sojae, the latter being used in the production of numerous fermented products like sake or soy sauce (Gibbons et al., 2012).
In order to discriminate Aspergillus species, MALDI-TOF MS could be a good alternative to molecular techniques, because of its accuracy, high-throughput and low cost per analysis. Several studies already pointed out its use for different Aspergillus sections including the Flavi section. For example, Alanio et al. (2011) used MALDI-TOF MS for discriminating 10 species of Aspergillus section Fumigati while Hettick et al. (2008) and De Carolis et al. (2012) could differentiate A. flavus from A. parasiticus, and A. parasiticus, A. flavus and A. oryzae, respectively. In a more extensive study, Rodrigues et al. (2011) could identify 9 species of Aspergillus section Flavi but could not distinguish aflatoxigenic and non-aflatoxigenic isolates of A. Flavus. More recently, Imbert et al. (2019) assessed MALDI-TOF MS identification accuracy of Aspergillus cryptic species on a very large dataset of 1477 isolates using the freely available mass spectrometry identification (MSI) platform. After sequencing a subset of 245 cryptic species isolates for confirmation, they showed that, while very good identification (99.6%) could be achieved with MALDI-TOF MS at the section level, only 66.1% of isolates were correctly assigned at the species level indicating that the database needed further improvement for cryptic species.
To another extent, MALDI-TOF MS could also be of great interest for differentiating strains from a same species, i.e. for strain typing. In the past 10 years, it has been applied to bacteria of different genera and species such as Salmonella enterica (Kuhns et al., 2012), Staphylococcus aureus (Ueda et al., 2015), Legionella spp. (Fujinami et al., 2011) or Arthrobacter spp. (Vargha et al., 2006) and could be in some cases as effective as traditional typing methods such as multi-locus sequence typing (MLST) or Pulsed-Field Gel Electrophoresis (PFGE). In a recent study, Kern et al. (2014) showed the ability of MALDI TOF MS to differentiate Lactobacillus brevis isolates at the strain level, and correlations could also be made between spectra classification and strain physiological properties. MALDI-TOF MS typing was also applied to yeasts of clinical interest, for which it could be as powerful as microsatellite markers for monitoring the spread of nosocomial infections (Pulcrano et al., 2012). This technique was also recently applied for the typing of brewing yeast strains allowing their classification into different major beer types (Lauterbach et al., 2017) as well as for the typing of 33 wine yeasts which could be sorted according to their genetic background (Usbeck et al., 2014). One of the main challenges of fungal typing is that, as compared to bacteria, their phylogenetic relationships are more complex and species boundaries are not easily drawn (Bader, 2013). Nevertheless, a rapid and reliable method for fungal typing would be of great interest in several contexts, e.g., to help understanding the domestication process and history of numerous species used in industry, for source-tracking of spoilage fungi in the food industry, to differentiate toxigenic and atoxigenic strains of a same species, to discriminate the different strains involved in natural fermentation processes for the selection of starter cultures and for deciphering the tenuous limits between contaminant and biotechnological isolates (Belén Flórez et al. 2007). In the past 15 years, several studies focused on strain-level classification of filamentous fungi with technological interest such as those used in cheese manufacture. Belén Flórez et al. (2007) and Fontaine et al. (2015), using randomly amplified polymorphic DNA (RAPD)-PCR, could discriminate, at the intraspecies level, P. roqueforti isolates from cheese and environmental origins. Microsatellite markers were also used to investigate the genetic diversity within Penicillium roqueforti isolates (Ropars et al., 2014; Gillot et al., 2015), and these studies allowed the differentiation of isolates in several genetically divergent populations. For instance, Gillot et al. (2015), using 4 polymorphic microsatellite markers, distinguished 28 haplotypes among a worldwide collection of 164 P. roqueforti isolates from cheese and other environments. Furthermore, these 28 haplotypes could be clustered into three well-defined genetically differentiated populations.
While there is a strong body of evidence that MALDI-TOF MS can be applied to discriminate closely-related bacterial species and bacterial strains, only few studies have evaluated MALDI-TOF MS as a rapid tool to differentiate closely-related fungal species and strains or genetic populations from a same species. Therefore, the aim of this study was first to evaluate the potential of MALDI-TOF MS to accurately identify 23 closely-related species of Aspergillus section Flavi, and then to assess whether this technique could be used for discriminating Penicillium roqueforti isolates previously shown by Gillot et al. (2015) to belong to three distinct genetic populations.
Section snippets
Fungal strains and cultivation
Sixty-eight strains belonging to 23 species from the Aspergillus section Flavi were obtained from different culture collections. They are listed in Table 1. Strain identification was performed by DNA sequencing in previous studies (Frisvad et al., 2019; Carvajal-Campos et al., 2017; Quéro et al., 2019) or by the culture collection providing the isolates. Sixty-three P. roqueforti isolates obtained from the Université de Bretagne Occidentale Culture Collection (UBOCC, Plouzané, France, //www.univ-brest.fr/ubocc
Evaluation of MALDI-TOF MS for discriminating Aspergillus section Flavi species
The distances between spectra of all tested species can be visualized in Fig. 1, which shows the results of MDS analysis for all spectra (Fig. 1A), spectra of the A. flavus-clade (Fig. 1B) and spectra from the other clades (Fig. 1C). Overall, most species were quite well separated from the others while some did not (Fig. 1A). Based on spectra similarity, 3 groups of species could be distinguished except for spectra of A. pseudotamarii which stood apart from these groups (Fig. 1A). The first two
Conflicts of interest
LQ, PC, BC and VG are employees of bioMérieux, a company developing and selling in vitro diagnostic assays including the VITEK MS used in this study.
Acknowledgements
This work was done as part of a CIFRE PhD funded by bioMérieux and the French Association for Research and Technology (ANRT) [Convention #2015/0821] in collaboration with the LUBEM laboratory.
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