Introduction

The microbiota is a complex ecology of an organism that varies greatly with time and lifestyles and between individuals, making it difficult to measure its implications in the organism’s immune response. Numerous model organisms (fruit fly, nematode, zebrafish, honey bee, hydra, squid)1 have been used to investigate the implications of the microbiota in the antimicrobial response. To date, there is increasing evidence that microbiomes play a role in animal immune function. Indeed, axenic animals show reduced expression of several immune effectors and are more susceptible to microbial pathogens than non-axenic animals. The efficiency of the immune response of the animal differs with the composition of the microbiota. Some microbial taxa or microbial communities promote inflammation, while others foster immune tolerance and host health2,3. Similarly, the diversity of the bacterial population of the microbiota causes the microbiota to play various roles in chronic inflammatory disease and autoimmune diseases4,5, in oncogenesis and tumour progression6, and in limiting colonization and invasion by microbes (bacteria, fungi, parasites) through the alteration of nutrient metabolite production or by secreting antimicrobial peptides7,8. In addition, microbiota flora participate in the production of various metabolites9,10, which are an important source of energy that improve the function of the intestinal barrier11,12.

Immortal planaria (Plathyhelminthes) are known for its ability to regenerate an entire organism from a tissue fragment. The regeneration ability of planarians has been intensively investigated13,14,15. Planarians have a worldwide distribution and live in a wide range of natural habitats, such as lakes, ponds, and rivers16. In the environment, planarians exist primarily as flesh-eating animals, though they also feed on detritus, fungi, and bacteria17. Due to their diet and habitat, planarians are exposed to a wide range of microbes and are able to survive this exposure. The ease of handling planarians and the application of loss-of-function genetic approaches in these organisms make them a valuable model system to investigate immune response18.

The use of the laboratory planarian species Schmidtea mediterranea has shown that planarians are powerful tools to identify evolutionarily conserved antibacterial response mechanisms. Indeed, the use of Schmidtea mediterranea has revealed that planarians represent a remarkable system with an unmatched capacity to fight infectious agents, including Staphylococcus aureus, Mycobacterium tuberculosis, and Legionella pneumophila, indicating the presence of remarkably efficient but uncharacterized innate immunity18. Notably, it has also been reported that planarians are able to overcome fungal infection19. Several new components of the innate immune system that are conserved in humans and absent from Ecdysozoa (e.g., flies and nematodes) were discovered by studying this model organism18,20. As an example, Membrane Occupation and Recognition Nexus (MORN) repeat-containing-2 (MORN2) has been identified to be a component of LC3 (light chain 3)-associated phagocytosis18. Also the role of leukotriene A4 hydrolase, a pro-inflammatory lipid mediator21 timeless (circadian clock machinery)22 in S. aureus clearance has been demonstrated. In-silico analysis revealed that despite the identification and characterization of the repertoire of TIR domain-containing proteins in planarian species S. mediterranea, TLRs are absent from S. mediterranea23, and planarian stem cells are able to drive a sensitized antibacterial response through axis PGRP-2/setd8 methyltransferase signalling24. Very recently, it has been demonstrated that GILT (gamma interferon inducible lysosomal thiol reductase) is crucial to the antimicrobial response of planarians against gram-negative microbes25.

However, the use of planarians for studies of antimicrobial response requires a deeper knowledge of their microbial repertoire. Interestingly, all these studies have been performed using planarians kept in the laboratory for several years18,19,22,23,24. The contribution of the microbiota to the antimicrobial response of laboratory strains of planarians has not been investigated.

Microbiota can be investigated using metagenomics and/or culturomics. Metagenomics allows uncultured bacteria to be identified. It connects microbial signatures with a physiological condition or disease because the abundance of each taxon can be determined. Moreover, microbial species can be grouped without taxonomic assignment. Shotgun sequencing enables us to define the function of microbial communities via the analysis of genomes and coding capacity. Although metagenomics enables uncultured bacteria to be identified, in contrast to culturomics, metagenomics cannot allow functional analysis since bacteria are not cultivated. New species cannot be taxonomically validated since functional analysis cannot be performed, and bacterial strains cannot be officially deposited. Moreover, methods used for metagenomics are limited by the heterogeneity of the protocols used. As Lagier et al.26 wrote, “discrepant results can be obtained depending on the method used to extract DNA or the primers that are used for amplification. The variety of methodologies proposed for bioinformatics analyses (for example, operational taxonomic unit clustering, taxonomic assignment or statistical analysis) can substantially affect the results. Sequencing methods cannot discriminate between live bacteria and transient DNA, and despite recent progress, they cannot easily detect minority populations”.

In contrast to metagenomics, culturomics is a culture-based approach that uses multiple culture conditions, matrix-assisted laser desorption-ionization time of flight-mass spectrometry (MALDI-TOF MS) and complete 16S rRNA gene sequencing for the identification of bacterial species26. In addition, since bacteria are cultivated, culturomics allows for functional analysis, new species can be taxonomically validated, and new bacterial species can be officially deposited.

The goal of our study is to report the cultivable bacteria comprising the microbiota of a 10-year-old laboratory planarian reference species, Schmidtea mediterranea, to define a standard microbiota. This will, in the near future, give the scientific community the possibility to investigate, through the use of laboratory Schmidtea mediterranea, the implications of the cultivable bacteria detected in the antimicrobial response of planarians. For this purpose, the microbiota of laboratory Schmidtea mediterranea strains was analysed by a culturomic approach.

Our data highlight the presence of 40 bacterial species in the 10-year-old laboratory planarian reference species Schmidtea mediterranea used to investigate antimicrobial response. One species remains to be identified, and 4 species have been described to date only in planarians. The predominant phylum in the microbiota flora of S. mediterranea is Proteobacteria, and the most common genus is Pseudomonas. The flora of laboratory Schmidtea mediterranea strain is shared with several simple animal models, along with the environment, fish, and humans.

Results

Using the culturomics approach to determine the bacterial communities of S. mediterranea starved for 2 weeks allowed us to isolate and identify (by MALDI-TOF or 16 s RNA sequencing) a total of 40 bacterial species after 1 to 4 days of culturing at temperatures of 19 °C, 28 °C, and 37 °C on the following media: LB agar, COS, and BCYE. These bacterial species corresponded to 4 bacterial phyla: Proteobacteria, Bacteroidetes, Actinobacteria, and Firmicutes (Fig. 1A, Table 1, Table S1). The phenotypic characteristics of the colonies of the identified bacterial species are reported in Table S2 along with their growth conditions. In the laboratory S. mediterranea, the dominant phylum, accounting for 60% of all phyla, is Proteobacteria, and the less represented phyla (10%) are Actinobacteria and Firmicutes. The phylum Bacteroides accounted for 20% (Fig. 1A, Table 1). There was a predominance of gram-negative bacteria (82.5%), while gram-positive bacteria represented 17.5% of the bacterial population (Fig. 1B). The most common genus found in the bacterial population was Pseudomonas, accounting for 15% (Fig. 1C, Table 1).

Figure 1
figure 1

Bacterial composition of the microbiota of the laboratory strain S. mediterranea starved for 2 weeks. (A) Culturomics analysis of S. mediterranea followed by identification by MALDI TOF and 16S RNA sequencing revealed the phyla of the bacteria forming the microbiota of the animals (see also Table S1). (B) The nature of the bacterial membrane was determined by gram staining colouration. (C) The representativeness of each bacterial species is illustrated. Five experiments were each performed on ten individual worms.

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Table 1 Bacterial composition of the microbiota of the laboratory strain S. mediterranea starved for 2 weeks.
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The bacterial communities of the planarian S. mediterranea were distributed as follows: 72.5% of bacterial species colonized the gut, 5% the mucus, and 22.5% both the mucus and gut (Fig. 2A, Table S3). Two gram-negative bacterial species, Vogesella urethralis and Shinella zoogloeoides, both of which are from the phylum Proteobacteria, are specific to the S. mediterranea mucus (Fig. 2B,C, Table S3). Whereas four phyla (29 bacterial strains in total) were found within the gut of S. mediterranea, only three phyla (Proteobacteria, Bacteroidetes, and Firmicutes, represented by 9 bacterial strains) were present in both the gut and mucus (Fig. 2B,C, Table S3). Among the 40 bacterial species isolated, 1 is a nonclassified or putative new species of Pseudomonas from the phylum Proteobacteria and must be characterized by taxonogenomic methods in the near future. In addition, 4 bacterial species have been recently identified as new bacterial species: Chryseobacterium schmidteae27, Pedobacter ghigonii28, and Pedobacter schmidteae29 from the phylum Bacteroidetes, as well as Metabacillus schmidteae from the phylum Firmicutes30.

Figure 2
figure 2

Bacterial distribution of the laboratory strain S. mediterranea starved for 2 weeks. (A) Distribution of the bacterial communities of the planarian S. mediterranea, (B) Venn diagram highlighting the bacterial number present in the gut and mucus epidermal (noted here as mucus), and the gut and epidermal mucus combined of S. mediterranea, (C) representation of the distribution of the bacterial phyla in the gut, epidermal mucus (noted here as mucus), and the gut and epidermal mucus combined of S. mediterranea. Five experiments were each performed on ten individual worms (see also Table S3). Identical results were obtained for each experiment and each worm tested.

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Next, we analysed the abundance of the identified bacterial species (Fig. 3, Table S4). Fifteen species were detected in 40% of the S. mediterranea planarians tested, and 11 were detected in 20% of S. mediterranea planarians. The other species are detected in between 60 and 100% of the planarians. This abundance was not correlated with the phylum or the localization of the bacteria within planarians (Fig. 3).

Figure 3
figure 3

Abundance of the bacterial species identified in the laboratory strain of S. mediterranea starved for 2 weeks. The abundance is expressed as a percentage of the bacterial species. Phyla are shown as follows: Proteobacteria in red, Firmicutes in green, Bacteroidetes in blue, and Actinobacteria in black. Five experiments were each performed on ten individual worms (see also Table S4). Identical results were obtained for each experiment and each worm tested.

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Next, we compared the microbiota of S. mediterranea starved for 1 week, 1 weeks, and four weeks (Table 2). After 1 week of starvation, we identified 14 bacterial strains. This is in contrast to 2 and 4 weeks of starvation, after which we found 40 bacterial strains. Bacterial strains detected after 1 week of starvation were also detected after 2 and 4 weeks of starvation. The bacterial strains identified after 2 and 4 weeks of starvation were the same. After 1 week of starvation, the phyla Proteobacteria and Bacteroidetes represented 50 and 43%, respectively, of the bacterial strains present, while Actinobacteria represented less than 10%. After 1 week of starvation, the Firmicutes are not represented like they are after 2 weeks and 4 weeks of starvation.

Table 2 Bacterial composition of the microbiota of the laboratory strain S. mediterranea starved for different duration.
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Finally, we analysed whether the microorganisms identified in the laboratory S. mediterranea strain were shared with the environment, invertebrates, fish, and humans (Fig. 4, Table 3). We found that 32.50% of the bacteria are shared with the environment (freshwater, soil, brackish water, and plants), 20% are shared with the environment and humans, 15% are shared with humans31,32,33,34,35,36,37,38,39,40,41,42,43,44,45, 12.5% are shared with fish and have also been described as fish pathogens31,32,33,34, 12.50% are shared with invertebrates27,28,29,30,46,47,48, and 2.5% (Herminiimonas sp. Marseille-P9896) cannot be categorized because they are uncharacterized bacterial species. This last point illustrates the complexity of the microbiota of the laboratory S. mediterranea strain.

Figure 4
figure 4

The bacterial species identified in the laboratory strain S. mediterranea starved for 2 weeks are shared with the environment, vertebrates, and invertebrates. Bibliographic analysis allowed us to illustrate the potential sharing of the bacterial strains forming S. mediterranea microbiota (see also Table 3).

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Table 3 The bacterial species identified in the laboratory strain S. mediterranea starved for 2 weeks are shared with environment, vertebrates and invertebrates.
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Taken together, these data show that the predominant phylum in the S. mediterranea laboratory planarian flora is Proteobacteria, that the most common genus is Pseudomonas, and that planarians share microorganisms with both invertebrates and vertebrates.

Discussion

Analysis of the microbiome of the laboratory strain planarian species S. mediterranea using culturomics methods allowed us to identify 40 cultivable bacterial species forming the microbiota repertoire of S. mediterranea, most of which are found in the planarian gut. Among the 40 isolated bacterial species, we identified one non classified bacterial species (Pseudomonas sp.) that needs to be taxonomically and biochemically characterized in further work, four bacterial species (Pedobacter schmidteae, Pedobacter ghigonii, Chryseobacterium schmidteae, Metabacillus schmidteae) that to date have been identified only in planarians27,28,29,30, and one bacterial species (Comamonas aquatilis) that has been identified in both pond water and S. mediterranea46,49. In the calf liver used to feed S. mediterranea, we detected several bacterial species, including Brochothrix thermosphacta, Lactococcus piscium, Pseudomonas frederiksbergensis, Pseudomonas gessardii, Serratia proteamaculans, Staphylococcus hominis, and Pseudomonas azotoformans (Table S5). None of these were found in the planarian S. mediterranea, suggesting that they were thus eliminated by planarians. The division of bacterial species between the planarian gut and epidermal mucus remains difficult to interpret. We cannot be sure of 100% bacterial species repartition because bacteria can be regurgitated by planarians through the pharynx. This phenomenon most likely occurs for the following bacteria, which are primarily from the phylum Proteobacteria: Aeromonas veronii, Chryseobacterium scophthalmum, and Pseudomonas brennerii, along with Pseudomonas anguilliseptica bacteroidetes, because this bacterial strain are detected in water containing planarians (Table S6). Notably, no bacteria were detected in the water control, which does not contain planarians.

We also observed a variation in the microbiota composition as a function of starvation time. Indeed, whereas only 14 bacteria were detected after 1 week of starvation, 40 bacteria were detected after 2 and 4 weeks of starvation. Interestingly, the bacterial composition remained the same between 2 and 4 weeks of starvation, and the bacteria detected after one week of starvation were also found in the microbiota after 2 and 4 weeks of starvation. The bacterial distribution remained unchanged after 2 weeks of starvation. This microbiota evolution can be explained in several ways. First, after 1 week of starvation, the number of bacteria present in the worms was too low to allow for the detection of all the bacterial strains. Second, the bacteria present in the liver, although eliminated by planarians, can shape the composition of the microbiota after feeding by promoting the growth of different bacterial strains. Third, the 14 bacteria detected in planarians after 1 week of starvation promoted the growth of the other strains. Fourth, feeding induces the growth of planarians, thus modulating tissue homeostasis and regeneration; it cannot be ignored that feeding allows the development of several bacterial strains from planarian microbiota.

Firmicutes and Actinobacteria represent only 10% of the bacterial population found in the planarian S. mediterranea; Proteobacteria (60%) and Bacteroidetes (20%) are the predominant phyla in the microbiota. Proteobacteria is one of the largest bacterial phyla, with six classes and more than 116 families having been recognized (http://www.bacterio.net/). Proteobacteria are gram-negative bacteria and play numerous roles in diverse microbial ecosystems (aquatic, soil, plant, animal). Proteobacteria are involved in maintaining homeostasis of the gastrointestinal tract anaerobic environment and, thus, in the stability of the strictly anaerobic microbiota. Members of the phylum Bacteroidetes are known to be involved in the synthesis of short-chain fatty acids such as butyrate, propionate, and acetate, which are rich sources of energy for the host50,51. They also participate in carbohydrate metabolism by expressing enzymes such as glycosyl transferases, glycoside hydrolases, and polysaccharide lyases. Interestingly, Bacteroidetes have been shown to synthesize conjugated linoleic acid, which is reported to have immunomodulatory properties52,53,54. In addition, the bacteria present in the planarian microbiome are mostly gram-negative, and gram-negative organisms produce antimicrobial peptides55. Thus, it can be hypothesized that Bacteroidetes and other gram-negative bacteria, such as Proteobacteria bacteria, participate strongly in the control of the antimicrobial properties of planarians via antimicrobial peptides and immune regulation.

It is difficult to consider and discuss each bacterial strain found in S. mediterranea and to compare them with the microbiota of other simple animal models or humans. However, it has been reported that the Proteobacteria is the dominant microbial phylum found in Danio rerio, Apis melifera, and Caenorhabditis elegans1,56. The predominance of Proteobacteria has also been reported in the microbiota of another planarian species called Dugesia japonica57, as well as in the fruit fly, Drosophila. melanogaster58, and Hydra oligactis59. Notably, Firmicutes are also the predominant phyla for C. elegans. The other phyla, Bacteroidetes and Actinobacteria, were not found in the simple animal models cited above. Stenostomum leucops, belonging to Catenulida within the phylum Platyhelminthes, are tiny planarians of the phylum Platyhelminthes that reproduce asexually and have a lifestyle close to S. mediterranea. They also share several microorganisms with S. mediterranea, including S. epidermidis, A. tumefaciens, E. adhaerens, P. fluorescens, and V. paradoxas60.

In contrast to that of planarian species S. mediterranea, the normal human gut microbiota is predominantly composed of two major phyla, Bacteroidetes and Firmicutes, followed by Actinobacteria and Verrucomicrobia. Verrucomicrobia were not detected in S. mediterranea. In humans, the gut microbiota contains only a minor proportion of the phylum Proteobacteria. It has been shown that an increase in the Proteobacteria phylum is a potential signature of dysbiosis and indicates a higher risk of disease61. In humans, the presence of Proteobacteria within the microbiota is associated with an adaptation of the gut microbial community to the host’s diet, which could improve the ability of the host to harvest energy from indigestible polysaccharides61. Accordingly, we can hypothesize that the presence of large amounts of Proteobacteria in S. mediterranea might be associated with the capacity of planarians to adapt their body size to food availability. Indeed, in the absence of food, planarian size decreases, whereas in the presence of sufficient amounts of food, their body size increases greatly62,63.

In the planarian S. mediterranea, the major genus found is Pseudomonas, unlike in Drosophila, where the most common genus is Klebsiella64. Several bacterial species found in planarians are common to the Drosophila microbiota, such as Micrococcus luteus, Micrococcus yuennansis, and Microbacterium oxydans. In S. mediterranea, Aeromonas veronii was also detected. Although its function remains unknown, it has been shown that Aeromonas veronii plays a crucial role in the immune response of D. rerio for upregulating neutrophil abundance, which leads to a downmodulation of inflammation1.

In 2016, Arnold et al.65 reported that planarian S. mediterranea microbiota analysed by metagenomics contain more than 300 bacterial strains. Here, in our study, we reported 40 bacterial strains, among which we identified and cultivated 14 bacterial strains described by Arnold et al., including M. luteus, M. yunnanensis, S. capitis, S. epidermitis, A. guillouiae, A. tumefaciens, C. testosteroni, D. acidovorans, P. anguilliseptica, P. brenneri, P. fluorescens, P. gessardii, S. ginsenosidimutans, and V. paradoxus. Such a disparity can be explained by the methodology used; metagenomics highlights any nucleotide sequence related to a bacterial strain, but the cultivability of the strain remains unknown. In addition, as shown by Arnold et al., the methods of culturing S. mediterranea can easily change the microbiota composition65. The role of the feeding (beef liver vs. calf liver) cannot be excluded. Thus, the composition of microbiota from the same species of planarians, here S. mediterranea CI4W, but kept in another laboratory will be affected by the methodology of bacterial detection and the culture conditions. A similar situation has been described for the microbiota of D. melanogaster66.

We have also observed that the bacteria comprising the microbiota of S. mediterranea are shared with the environment (soil, water, brackish water, sewer, and plants) as well as with fish, which is in accordance with the lifestyle of planarians. Indeed, planarians are zoophages and live in water. We also found bacteria that are shared with humans. Indeed, 16 bacterial strains have been described as opportunistic human pathogens, which represent 40% of the S. mediterranea flora and are responsible for diarrhoea, bacteremia, and endocarditis. Some of them have been reported to cause nosocomial infections, such as Acinetobacter guillouiae, or infection in immunocompromised people, such as Micrococcus luteus, which is involved in bacteraemia associated with intravascular catheters and endocarditis, peritonitis, ocular infections, and urinary tract infections. We also identified Aeromonas veronii, which is commonly hosted by leeches and is known to be responsible for gastroenteritis in humans67. The virulence and opportunistic capacity remain unclear for some of the bacterial species identified, such as Pseudomonas fluorescens and Comamonas testosterone, which might be a cause of bacteraemia or gastroenteritis68,69. As in D. melanogaster, we detected bacteria that have been described to be opportunistic human pathogens, such as Micrococcus luteus. This is also an important pathogen for aquatic animals70, but it is also a probiotic which promotes the growth of Nile tilapia Oreochromis niloticus”71. We also found several Staphylococcus species responsible for bacteraemia, sepsis, and nosocomial infection. The planarian S. mediterranea microbiota flora also includes Aeromonas veronii, which is commonly hosted by leeches. Cases of infection, such as gastroenteritis, have been reported in people using leech therapy procedures35 or eating contaminated fishes72. Similarly, Mycobacterium marinum infections have been reported to be associated with the exposure of damaged skin to polluted water from fish pools or objects contaminated with infected fish73. Planarians are known to be fish tank invaders. Thus, although the planarian S. mediterranea is a free-living flatworm, it cannot be ignored that planarians are a reservoir or host of several human microbial pathogens that might be transmitted to predators of planarians or released in fish tanks contaminated with planarians.

Several studies suggest the role of probiotics in tissue homeostasis, as well as in tissue regeneration74, and that manipulation of the microbiome could be a way to resolve some tissue homeostasis deficiencies75. It has been shown that antibiotic treatment affects the planarian microbiota, which leads to an alteration of the regeneration process of S. mediterranea65. In Dugesia japonica, metabolites such as indole produced by the bacteria Aquitalea sp. delay the regeneration of the tissue after amputation57. Although there are contradictory and controversial findings, it appears that commensals, symbionts, and pathogens from the human cutaneous microbiome can play an important role in the resolution of nonhealing wounds75.

The role of the microbiota in the antimicrobial capacity of S. mediterranea and in their ability to have trained immunity remain to be elucidated. For this purpose, it is important to have information concerning the composition of the microbiota of the laboratory strain used for the experiments and to consider that a divergence in results can be caused by the mode of culture.

Materials and methods

Culture of the planarian species Schmidtea mediterranea

The S. mediterranea asexual clonal line ClW424 was maintained at 18 °C in water. The water was first filtered through charcoal and ceramics with pores of 0.2 µm (manufactured by Fairey Industrial Ceramics Limited) and through a membrane of 0.2 µm (Thermo Scientific Nalgene Filtration Products) for 10 years. Microbiological analysis of the filtered water was performed by inoculation of 5% sheep blood-enriched Columbia agar plates (bioMérieux, Marcy l’étoile, France) with 25, 50, or 100 µL of filtered water. Inoculated plates were then incubated at 19, 28, or 37 °C for 4 days. Any bacteria were detected.

S. mediterranea were fed once per week with homogenized calf liver (batch 20118-5814, origin: Saprimex (a local supermarket), 13310 St Martin de Crau) and then starved 2 weeks prior to experiments. In some experiments, worms were starved for 1 or 4 weeks. The bacterial constituents of homogenized calf liver were defined by inoculation of 5% sheep blood-enriched Columbia agar plates (bioMérieux, Marcy l’étoile, France) with 25, 50, or 100 µL of homogenized calf liver. Inoculated plates were then incubated at 19, 28, or 37 °C for 1, 2, 3, and 4 days.

Culturomics

Two-week starved planarians 0.4–0.6 mm in length were used for the experiments. Selected worms were placed on agar plates (13%) and pressed slightly to collect their epidermal mucus (also denoted as mucus in the manuscript). The recovered epidermal mucus (one planarian per sample) was mixed with sterile phosphate-buffered saline (PBS), and then 100 µL of sample was inoculated on 5% sheep blood-enriched Columbia agar (bioMérieux, Marcy l’étoile, France), buffered charcoal yeast extract (BCYE) (Oxoid Deutschland GmbH, Wesel, Germany), and lysogeny broth (LB) under anaerobic and aerobic conditions and incubated at 19, 28, or 37 °C for 1, 2, 3, and 4 days76. The whole microbiota (gut and mucus) was then characterized by grinding one two-week-starved animal in PBS (one worm per sample). Homogenates were inoculated on 5% sheep blood-enriched Columbia agar (bioMérieux, Marcy l’étoile, France), BCYE (Oxoid Deutschland GmbH, Wesel, Germany), and lysogeny broth (LB) under anaerobic and aerobic conditions and then incubated at 19, 28 or 37 °C for 1, 2, 3, and 4 days.

MALDI-TOF MS and bacterial identification

Individual bacterial colonies were collected every day for 4 days, and then each colony was identified by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) (Microflex Spectrometer; Bruker Daltonics, Bremen, Germany) as previously described77. The obtained MALDI-TOF MS spectra were imported into MALDI Biotyper 3.0 software (Bruker Daltonics) and analysed against the reference bacterial spectral database. The MALDI Biotyper RTC software interprets the results according to predefined values, i.e., values between 2.00 ≤ species identified ≤ 3.00; of 1.70 ≤ probably identified ≤ 1.99 and 0.00 ≤ no identification ≤ 1.69. The unidentified colonies (with values from 0.00 to 1.99) were sequenced using the complete 16S rRNA gene.

Sequencing of the 16S rRNA gene and bacterial identification

The unidentified bacterial colonies were cultured under the appropriate conditions, and the genomic DNA of each bacterium was extracted using an EZ1 automate (BioRobot) and the EZ1 DNA tissue kit (Cat No./ID: 953034, Qiagen, Hilden, Germany) according to the manufacturing protocol. The genomic materials were quantified using a Qubit assay (Life Technologies, Carlsbad, CA, USA) and then amplified by standard PCR. The standard PCR protocol was performed in a Thermal Cycler Peltier PTC200 cycler thermal model (MJ Research Inc., Watertown, MA, USA). Each reaction was conducted in a final volume of 50 μL, containing 5 μL of DNA from each sample, 25 HotstarTaq—AmpliTaq Gold (Life Technologies, Carlsbad, CA, USA), 1.5 μL of primers (Fd1-AGAGTTTGATCCTGGCTCAG; Rp2-ACGGCTACCTTGTTACGACTT (Eurogentec, Angers, France))78 and 17 μL DNAse/RNAse-free water. The amplification was performed as follows: an initial denaturation step at 95 °C for 15 min, 40 cycles of denaturation at 95 °C for 30 s, step hybridization at a temperature of 52 °C for 30 s, and elongation at 72 °C for 60 s. All PCR products were resolved in 0.5× Tris Borate EDTA buffer (Ref. ET020-A, EUROMEDEX, Souffelweyersheim, France) and 1.5% agarose (Ref. LE-8200-B, EUROMEDEX, Souffelweyersheim, France), purified using NucleoFast 96 PCR plates (Macherey–Nagel EURL, Hoerdt, France), and sequenced using the Big Dye Terminator Cycle sequencing kit (Perkin Elmer Applied Biosystems, Foster City, CA, USA) with an ABI Prism 3130xl Genetic Analyser capillary sequencer (Applied Biosystems, Bedford, MA, USA). The following primers were used for the sequencing of the complete 16S rRNA: Fd1-AGAGTTTGATCCTGGCTCAG; Rp2-ACGGCTACCTTGTTACGACTT; F536-CAGCAGCCGCGGTAATAC; R536-GTATTACCGCGGCTGCTG; F800-ATTAGATACCCTGGTAG; R800-CTACCAGGGTATCTAAT; F1050-TGTCGTCAGCTCGTG; and R1050-CACGAGCTGACGACA (Eurogentec, Angers, France). CodonCode Aligner software was used for alignment and assembly and to correct the sequence (https://www.codoncode.com/). A consensus sequence was generated after analysis. BLASTn searches were performed against the nr database to check the similarity of the sequence (https://blast.ncbi.nlm.nih.gov/Blast.cgi). A sequence similarity threshold of 98.65% by comparison with the phylogenetically closest species with standing in the literature was used to delineate species79.