The 113 larva L3 individuals used for this study were taken from ten different fish species (Additional file 1: Table S1): Micromesistius poutassou (9), Merluccius merluccius (76), Conger conger (2), Gadus morhua (2), Lepidorhombus boscii (9), Lophius budegassa (5), Lophius piscatorius (5), Phycis blennoides (2), Scomber scombrum (1), Thunnus thynnus (2). The Anisakids taxa resulted as: Pseudoterranova sp (5), Anisakis simplex (100), A. pegreffii (7) and hybrid haplotypes from A. simplex and A. pegreffii (1). The asymmetric representation of these variables in the survey avoid the use of standard parametric statistic. However, the use of a multifactorial approach it is possible to detect the importance of each of the variables related to microbiota, even if they are spurious.
A total of 2689113 sequences were generated (average of 23797 sequences per analyzed sample) at the different taxonomical level (phyla, class, order, family, genus) of associated microbiota based on the 16S rRNA gene sequences for OTUs (Additional file 1: Table S2). The bacterial lineages which have been detected based on 16S rRNA gene sequencing follow a conservative taxonomy. Fourteen phyla have been taxonomically characterized through 1803 different sequences quoted to OTUs of which, one hundred five are “unclassified” (5.82% of the whole sequences). In total, 14 phyla, 31 classes and 52 orders were unambiguously identified (Table 1) except four orders from the phylum Cyanobacteria (from classes MLE1-12, Subsection IV and Subsection III in addition to other unclassified classes). Table 2 (a, b and c) shows the systematics of orders, families (129) and genera (187) (only unambigous genera are considered; 6 with very scarce representation and 13 unclassified genera were excluded). For most of the Cyanobacteria it was not possible to classiffied them at phylum level. Proteobacteria (Table 2a) is represented by 92 genera (40% are members of the Class Alphaproteobacteria), Firmicutes (Table 2b) (75 genera, from which 68% are members from the order Clostridales) and Actinobacteria (Table 2b) (46 genera, from which 74% are from the order Actinomycetales) are the most diverse phyla detected in this study. The genera of these three phyla represent 81% of the total associated genera to Anisakids. Thaking this into account, the class Alphabacteria represented by 7 orders and 55 genera should be the most important from Proteobacteria; Rhizobiales (20 genera), Rhodospirales (14 genera) and Sphingomonadales (at least 9 genera) are the most important orders from Proteobacteria. Class Clostridia would be the most important from Firmicutes, especialy the order Clostridiales (51 genera) and class Actinobacteria represented by order Actynomycetales (34 genera) from the phylum Actinobacteria.
The presence (frequency) and abundance of phyla and orders in the individuals of Anisakids (samples) is summarized in Table 1: Actinobacteria, Bacteroidetes, Firmicutes, Fusobacteria, Proteobacteria and Cyanobacteria are present in all Anisakids specimen. Other important presence is for Acidobacteria (present in 76% of samples), Chloroflexi (74.33%), Planctomycetes (63.72%) and Deinococcus-Thermus (61.94%). Minor presence is for Verrucomicrobia (23.9%), Chlamydiae (15.93%), Synergistetes (10.62%) and Aquificae (2.65%). The most abundant phyla into the Anisakids are Proteobacteria (with an average of 9291 bacteria per L3 larvae), Firmicutes (average 5659) and Actinobacteria (average 2838). Fusobacteria (average 755), Bacteroidetes (average 705) and Cyanobacteria (average 297) could be considered of medium abundance into the L3 larvae comparing with rest of the phyla wich range from 9 to 76 bacteria per L3 larva: Aquificae (average 76), Synergistetes (average 73), Chloroflexi (average 66), Acidobacteria (average 59), Planctomycetes (average 35), Deinococcus-Thermus (23), Verrucomicrobia (average 17) and Chlamydiae (average 9).
Orders with higher presence (frequency) are ranging between 75–100% considering the total individuals (samples). These are: Acidobacteriales, Actinomycetales, Bacteroidales, Flavobacteriales, Sphingobacteriales, Deinoccocales, Bacillales Lactobacillales, Clostridiales, Fusobacteriales, Caulobacterales, Rhizobiales, Rhodobacterales, Rhodospirillales Sphingomonadales, Burkholderiales, Neisseriales,
Campylobacteriales Enterobacteriales, Pasteurellales, Pseudomonadales and Vibrionales. Orders present in the samples (Anisakids individuas) with a frequency between 40–75% are: Acidimicrobiales, Bifidobacteriales, Coriobacteriales, Erysipelothrichales, Planctomycetales, Aeromonadales, Alteromonadales and Xanthomonadales. Orders with a frequency between 10–40% are: Rubrobacterales, Chlamydiales, Anaerolineales, Chloroflexales, Bdellovibrionales, Myxococcales, Cardiobacteriales and Oceanospirillales, Finally, orders with a frequency below 10% in the total of samples are: Aquificales, Caldilineales, Thermales, Mycoplasmatales, Thermoanaerobacteriales, Phycisphaerales, Parvularculales, Rickettsiales, Desulfobacterales, Desulfovibrionales, Desulfuromonadales and Cardiobacteriales. The most abundant orders (number of bacteria per L3 larva) are clearly diferenciated from the rest: Actinomycetales (2625), Clostridiales (4450), Rhizobiales (2472) and Sphingomonadales (2692). There are other group of orders whose abundance into the Anisakids individuals range between 300 to 800 bacteria per juvenil L3 such as: Lactobacillales, Fusobacteriales, Caulobacterales and Pseudomonadales. Rest of the orders have and average of abundance under 300 bacteria per L3 larva.
In order to avoid redundance, the statistical analysis is applied to phylum, order, family and genus levels where taxonomy is well established.
The rarefaction curves per each sample indicate that most of the OTUs in the “sampled community” have not been observed because new OTUs would be found with additional sampling. According our data the “asymptotic stage” would be reached in 80% if we would have taken 2500 samples (Fig. 1a) (values in Additional file 2: Table S3). In the present study, based only in 113 samples (L3 individuals), we have found an average of 123 OTUs per L3 individual (minimum 20 OTUs, maximum 281 OTUs). All of this indicates the extraordinary diversity of bacteria associated to Anisakids nematodes and also than this study can be considered representative of that diversity. The diversity ordering by mean of Renyi´s index family curves (Fig. 1b) shows indepent differences in OTUs diversity comparing all Anisakids individuals demonstrating than diversity among them are non-comparable (most of the diversity profiles intersect among them). The use of Shannon and Simpson Diversity Indices scatter graphically the samples (Additional file 2: Fig. S1a-b) indicating again an extraordinary diversity of bacteria at OTUs level. These values range from 0.3 to 4.7 for Shannon and from 2 to 45 for Simpson diversity index (the inverse option better represents diversity). Both indices clearly detected clusters of samples which are not significantly related with fish hosts or anisakid taxa at the phyla or order levels. This implies that any relationship would exist at a genus or family level without the influence of fish species. The lack of relationships of the microbiome composition with hosts of Anisakids taxa has to be necessarily expresssed by the association among bacterial OTUs or other taxonomical levels which are ranging from OTUs to phylum level.
Microbiota structure associated to Anisakids
Discriminant Cluster analysis (Fig. 2a-c) of samples based in microbiome composition reflects almost an identical structure of samples through phylum and order levels (Fig. 2a, b). At a class level the cluster distribution of the samples is identical to phylum (data at class level not shown); both are practically the same cluster than at order level. The only difference of cluster based in order level with that based on phylum level is that some samples formerly present in a group (G2 or G3) in phylum cluster are “moving” to other group in order cluster (sample 115 from G2 to G1 and samples 86A, 102A and 105PS from G3 to G1). At the family and genus level it is almost identical (data at families level is not shown), however the structure of the cluster at a family and genus levels (Fig. 2c) is very different to the former clusters phylum to order (Fig. 2a,b).
Groups of samples are statistically compared by means of Discriminant Analysis reflecting the differences among them and determining which are the most important variables (bacteria) included in the analysis (Table 3). Actinobacteria (total average = 2838,283), Aquificae (2,017699), Firmicutes (total average = 5659,035) and Proteobacteria (total average = 9291,938) are the phyla whose abundance determines the sample groups in the clusters. Aquificae, exclusively represented by the genus Hydrogenobacter (total average = 2,0176), is absent in G1, with very scarce representation in G3. The statistical values are very significant and also the percentage of classification for each group of samples; total percent of correct classification is 96.46% (Table 3a). When orders are included (Table 3b) the statistical significance as well as the percent of correct classification is also very high (97.34%). Actinobacteria is exclusively represented by the order Actinomicetales (34 genera, including some unclassified), Firmicutes is represented by the orders Bacillales (5 genera including some unclassified) and Clostridiales (51 genera including some unclassified) and Proteobacteria is represented by the orders Campylobacterales (3 genera: Arcobacter, Campylobacter and Sulfurospirillum), Caulobacterales (4 genera: Caulobacter, Brevundimonas, Phenylobacterium and Asticcacaulis). Table 3b also includes the orders Anaerolineales (one unclassified genus) and Caldilineales (genus Caldilinea) from the phylum Cloroflexi and the order Bacteroidales (6 genera) from phylum Bacteroidetes which did not reach the established level of significance (p < 0.05) in Table 3a. At the family and genus levels (Fig. 2c) the clusters of samples maintain three different groups (G1, G2 and G3). But the new G1 is mainly formed with samples which were formerly at Order level from G1, five samples of G2 (97, 101 s, 101p,107, 109, 151) and one sample from from G3 (98). The Discriminanta Analysis was not applied at this level due the number of “empy cells” in the data matrix.
Factor Analysis allows to define the number of clusters of bacteria in addition to their association (microbiota association) in spite that the percentage of representation is low when the number of variables is high as is the case at the genus level. This method, combines the bacteria (active variables) explaining the total variability of the factorial space, with the bacteria which explain the variability into each cluster. At the phylum level (Fig. 3) the percentage of explanation for the first three factors is 46.62% of the total. The correlations of variables clusters with what are the primary and secondary factors are very high indicating the existence of at least three clusters of phyla: cluster 1(Cyanobacteria and Proteobacteria), cluster 2 (Bacteriodetes, Aquificae, Firmicutes, Fusobacteria and Synergistetes) and Cluster 3 (Verrucomicrobia, Planctomicetes, Chlofoflexi, Deinococcus-Thermus; Acidobacteria, Actinobacteria, Chlamidiae;). The association defined by cluster 1 (Cyanobacteria, Proteobacteria) should be more specific of samples from group 1 (G1); the association defined by cluster 2 (Bacteriodetes, Aquificae, Firmicutes, Fusobacteria and Synergetes) are more related to samples from group 2 (G2) while the other association from cluster 3 are mainly related to Group 1 and 2. Group 3 of samples gather all the associations. The most important phyla to establish the association among them are: Proteobacteria and Cyanobacteria (Cluster 1); Aquificae, Bacterioidetes and Firmicutes (Cluster 2) and Actinobacteria, Chloroflexi, Deinococcus-Thermus and Planctomycetes (Cluster 3).
The total variability at the order level (Fig. 4) is explained by 25.75% for the three first factors. One of the reasons could be that it was not possible to classify any order from Cyanobacteria. However, five bacterial clusters can be detected in the hierarchical analysis that involves at least five main factors for ordering the microbiota in the factorial space (36.78% of total explanation). The clustering of associated microbiota detected at phylum level it is also detected at order level confirming the relationships. The main influence for conforming the five associated clusters of orders are due to fourteen of them: Two orders of Bacteroidetes (Flavobacteriales and Bacteroidales), one of Firmicutes (Clostridiales), the only order of Aquificae (Aquificales, due to its central position in the factorial space), one of Planctomycetes (Planctomycetales), six of Proteobacteria (Chromatiales, Neisseriales, Parvularculales, Pasteurellales, Sphingomonadales, Rhodobacterales), one of Actinobacteria (Bifidobacteriales), one of Verrucomicribia (Opitutales) and one of Chloroflexi (Chloroflexales). Regarding the clusters of orders (Fig. 4), they can be related with groups of samples (Fig. 2b): cluster 1 is mainly associated to samples in group 1, cluster 2 is mainly associated to samples in group 2, clusters 3 and 4 could be associated to any group of samples, and cluster 5 is mainly related to samples of group 3. Factor Analysis and its hierarchical analysis also identified three clusters of bacterial association when it is applied to the genus level (Fig. 5). In spite the large number of variables (187 genera), the clusters are quite well defined with very high correlations (values ranging from 0.88 to 0.948) among clusters and variables. Comparison with Fig. 2c, Cluster 1 is clearly associated to samples in group 2, cluster 2 with samples of group 1 and 3 and cluster 3 with samples of group 1. In table 2 (a,b,c) the genera which conform the three clusters are highlighted by three colors (black, green and red) and those which are of highest influence in the factorial space are marked with asterisk.
Genus level associations (clusters description): The ultimate test is the significant structure (Fig. 5) which represents the microbiota association at genus level.
The Cluster 1 merges independently 28 genera which are thermofiles or mesofiles, anaerobics (facultative or strict). This cluster is mainly related with the set of samples from G2 (Fig. 2c). In this cluster Wolbachia, genus of Rickettsiales which is an endosymbiont of artropods and nematodes. It is estimated to infect more than 65% of insect species and considered an extensive symbiont in Nematoda [30]. At least 60.71% of the cluster members are considered typical periodiontal and oral bacteria of mammals (some of them include species causing gingival or dental disease in humans) such as: Campylobacter, Capnocytophaga, Eubacterium, Fusobacterium, Porphyromonas, Prevotella, Tannerella [31]; Selenomonas, Leptotrichia and Peptococcus [32, 33]; Bulleida [34]; Mogibacterium [35 ]; Catonella, Johnsonella and Dialister [36]; Filifactor [37] and Parvimonas [38, 39]. Species of genus Carnobacterium are not known to be pathogenic in humans; C. divergens and C. maltaromaticum may cause disease in fish [40]. Succinivibrio is an obligate anaerobe which was firstly considered inhabitants of the rumen of cattle and sheep [41]. the rest of the bacteria are considered part of the Human gastrointestinal microbiome: Olsonella [42], Slackia [43], Collinsella [44] and Peptococcus [45]. From what can be considered members of a generalistic human microbiome we found: Anaerococcus [46] and other opportunistic human pathogens causing nosocomial infections such as Finegoldia [47], Peptoniphilus [48] and Dolosigranulum with only one known species, D. pigrum [49].
The Cluster 2, associated to samples which have defined the Fig. 2c is characterized by 158 genera (unclassified genera has not been included in factorial analysis). Except three genera (Wolbachia, Campylobacter and Succinivibrio) all the recorded genera from phylum Proteobacteria are represented in this Cluster by: classes Alphaproteobacteria (37 genera), Betaproteobacteria (4), Epsiloproteobacteria (2), Deltaproteobacteria (6) and Gammaproteobacteria (10).
Alphabacteria are represented by 7 orders: Rhizobiales (13 genera) some of which are endosymbions of animals [50] such as Devosia [51], endosymbionts of plants such as Bradyrhizobium, Bosea and Mesorhizobium [52], but most of the Rhizobiales which have been found in this study are common components of soil and water microbiota: Hyphomicrobium used in the denitrification of sewage [53], Methylobacterium [54], Angulomicrobium, Pedomicrobium [55], Rhodobium [6], Chelatococcus [57], Parvibaculum [58], Nordella [59] and Ochrobactrum (also isolated from the intestinal tract in human) has been associated to nosocomial infections in immunocompromised patients by [60, 61].
In this cluster 2 there are the four genera of the order Caulobacterales (Asticcacaulis, Brevundimonas, Phenylobacterium and Caulobacter) usually described as freshwater bacteria [62]. The Order Parvularculales is represented by the marine genus Parvularcula [63]. Order Rhodobacterales is represented by water eutrophic bacteria Paracoccus and Roseobacter sensu lato clade, with important functions in the marine biogeochemical cycle (25% of coastal marine bacteria are members of the Roseobacter clade [64]). All the genera found in this study from the order Rhodospirillales are memebers of Cluster 2. They are typical bacteria of aquatic and soil environment producing acetic acid (Gluconobacter, Roseomonas, Acetobacter, Acidocella, Acidiphilium) or what are considered purple non sulfur bacteria (Skermanella, Azospirillum, Caenispirillum, Magnetospirillum and Defluviicoccus [65]). Although they are considered mainly as environmental bacteria (air, soil and water), some of these especies such as Roseomonas are isolated frequently from wounds, abscesses and genitourinary track [66]. The Order Rickettsiales is represented by the genus Rickettsia, which is a symbiont of Eukaryota and can induce disease in humans [67]. Sphingomonadales (Sphingomonas, Novosphingobium, Sphingobium, Sphingopyxis and Erythrobacter) are common aerobic bacteria isolated from a wide range of environments, including temperate and polar soils, marine sediments and plant surfaces and tissues [68].
Genera of the class Betaproteobacteria are Janthinobacterium, Alcaligenes, Ralstonia and Neisseria. Some species have clinical importance for humans [such as Neisseria [69], can be agents of nosocomial and opportunistic infections (Alcaligenes [70]), plant pathogens (Ralstonia [71]) or have antifungal properties able to control Batrachochytrium dendrobatidis when the bacteria is symbiont of amphibians (i. e. Janthinobacterium [72]).
Arcobacter and Sulfurospirillum are the genera of class Epsiloproteobacteria which are in this cluster; Arcobacter comprises some species which can be human and animal pathogens widely distributed in many environment including marine ones [73] while Sulfurospirillum is found in contaminated sediments, wastewater plants, marine environments or on biocathodes [74]. Class Deltaproteobacteria is represented by the following orders and genera: Bdellovibrionales (Peridibacter and Bdellovibrio), Desulfobacterales (Desulfobulbus), Desulfuromonadales (Geobacter) and Myxococcales (Halianqium). All of these genera are quite uniform from an ecological and physiological point of view (sulfur-reducing bacteria). These genera are very common in anaerobic aquatic and marine sediments [75].
Finally, class Gammaproteobacteria (excluding the unclassified genera) is represented by orders Aeromonadales (Aeromonas), Alteromonadales (Shemanella, Psychromonas), Enterobaceriales (Enteric Bacteria cluster), Oceanospirillales (Marinomonas), Pseudomonadalles (Pseudomonas, Psychrobacter, Acinetobacter), Vibrionales (Photobacterium) and Xanthomonadales (Nevskia). Aeromonas is an ubiquistous genus present in continental and marine water, in which most of their species have been associated to human diseases (as opportunistic in immunocompromised patients, gastroenteritis and infected injuries); it is also considered a pathogen in fishes, amphibians and reptiles [76]. Shewanella and Psychromonas are marine genera from cold waters [77, 78]. Bacteria from “Enteric_Bacteria_cluster” [79] (order Enterobacteriales) includes medically important genera and species which are part of human and animal microbiota. Its distribution is widespread along any environment. Marinomonas is a marine bacteria [80] with important ecological implication. Order Pseudomonadales is represented by three genera (Psychrobacter, Acinetobacter and Pseudomonas). Psychrobacter is recorded frequently in marine environments [81] but also in terrestrials and some species has been isolated from foods [82]. Acinetobacter is a typical bacteria from soils [83]. Pseudomonas is a well known genus with at least eight different phylogenetic groups of species [84]; its distribution is ubiquitous and present in all type of ecological niches from terrestrial to marine. Some species are pathogens of animals and plants. Pseudomonas and Acinetobacter are considered pathogens for immunocompromised patients [85]. Vibrionales (Photobacterium) and Xantomonadales (Nevskia) are present in this cluster. Most of the species of Photobacterium are symbionts of marine organisms [86] and it is demonstrated that also some of them are pathogens of fishes [87]. Nevskia is an aquatic bacteria living at the air-water interface (the epineuston) forming hydrophobic surface films [88] and constitutes a clade of sister species into Xantomonadales [89].
Phylum Actinobacteria is represented in this cluster 2 by two Classes and 33 genera (unclassified genera are not included): Rubrobacteria [order Rubrobacterales (1 genus)], Actinobacteria [orders Actinomycetales (29) and Bifidobacteriales (2)] and Coriobacteria [order Coriobacteriales (1)]. Rubrobacterales is represented by the extremophilic genus Rubrobacter considered to be radiotolerant [90]. From Coriobacteriales we have recorded the genus Atopobium [91] a common member of human pathogenic biota [92, 93]. The Order Actinomycetales has a complex taxonomy; they can be found mostly in soil and organic matter and also in animals. In addition, they constitute symbiotic nitrogen fixing associations with over 200 species of plants; they can serve as biocontrol agents, or be plant pathogens. Genera of this order are also component of the human urogenital tract or the oral digestive system. They also have wide medicinal (antibiotic production; i.e. Streptomyces spp) and botanical applications [94]. In cluster 2 is represented by the 29 genera recorded in Table 2b. It can be considered as the most important genera: Brachybacterium which has been isolated from sea water [95]; Dietzia considered as opportunistic human pathogen [96]; Rothia which causes a wide range of serious infections, especially in immunocompromised hosts [97]; Mycobacterium, a genus free-living in soil and water widely distributed but the major habitat for some species is the diseased tissue of warm-blooded hosts (M. tuberculosis and M. leprae are the most known pathogens of this genus for humans) [98]; some species of Nocardia cause disease in immunocompromised patients [99].
Phylum Bacteroidetes is represented in cluster 2 by orders Flavobacteriales (Flavobacterium, Cloacibacterium, Chryseobacterium, Polaribacter, Wautersiella, Amoebinatus ) and Sphingobacteriales (Pedobacter, Sphingobacterium, Hymenobacter, Dyadobacter, Chitinophaga ). Flavobacteriales have been isolated in many environments, including soil, marine water, plants, and animal gut. They play an important role in in aquatic and terrestrial environments, accounting for more than 20% of microbial communities. Some flavobacterial species (Flavobacterium spp) are also responsible for severe fish disease [100, 101]. Sphingobacteriales are all considered as “environmental” bacteria with a wide ecological niche, from water to soil [102]. Phylum Cloroflexi is represented by orders Caldilineales (Caldilinea) and Cloroflexales (Roseiflexus). Both genera are filamentous thermophilic and found in hot springs [103, 104]. All members of phylum Cloroflexi are considered to be the origin of photosynthesis [105].
The phylum Firmicutes is represented by Classes Bacilli (14 genera), and Clostridia (22 genera). The genera of Bacilli are members of two orders [orders Bacillales (Listeria, Ammoniphillus, Kurthia, Staphylococcus and Laceyella) and Lactobacillales (Desemzia, Alloiococcus, Streptococcus, Lactococcus, Leuconostoc, Weissella, Lactobacillus, Flacklamia, Aerococcus)]. From Bacillales Listeria, Kurthia and Ammoniphillus can be found in soil, but the former also is found in vegetablse and animals where can be considered a pathogen [106, 107, 108]. Eleven species of Staphylococcus can be isolated from humans as commensals, some of them pathogenic in persons debilitated by chronic illness, traumatic injury, burns or immunosuppression [109] ; Laceyella is considered to be an environment thermophilic bacteria [110]. From Lactobacillales, Desemzia is a lactic acid bacteria (as the rest of the order) common in fish gut considered to be sometimes abundant in the intestine, notably in freshwater fish [111]; Alloiococcus has only one known species with implications as a secondary pathogen in human otitis [112]. The rest of Lactobacillales of this cluster 2 are ubiquitous bacteria, usually found in decomposing plants, milk products and foods in fermentation (including fish) contributing to the healthy microbiota of animals and humans [113].
The genera of Clostridia is represented by two orders [Thermoanaerobacterales (Caldicellulosiruptor and Thermoanaerobacter) and Clostridiales (Helcococcus, Eubacterium, Sedimentibacter, Shuttleworthia, Oribacterium, Moryella, Butyrivibrio-Pseudobutyrivibrio, Fastidiosipila, Ethanoligenens, Acetivibrio, Sporacetigenium, Desulfosporosinus,Veillonella, Mitsuokella Megamonas, Clostridium, Caloramator, Acetobacterium)]. Both species of Thermoanaerobacterales are thermophilic and anaerobic showing adaptation to survive in elevated temperatures (up to 80–90 °C) without oxygen [114]. Regarding Clostridiales, all genera are strict anaerobic bacteria; the ability to form spores makes them highly stable in the environment being natural soil inhabitants as saprophytic bacteria [115]. However, genera of this order have also common presence as members of gut microbioma with clinical and healthfunctions [116].
The rest of the genera from this Cluster 2, are members of the phyla Acidobacteria (Chloroacidobacterium, Acidobacterium, Edaphobacter, Koribacter and Solibacter), Aquificae (Hydrogenobacter), Deinococcus-Thermus (Truepera and Deinococcus), Fusobacteria (Psychrilyobacter and Sneathia), Planctomycetes (Singulisphaera, Planctomyces, CL500-3 and Phycisphaera) and Synergistetes (Jonquetella). The genera of Acidobacteria are considered soil inhabitants with a ubiquitous distribution along many ecosystems [117, 118]. Aquificae (Hydrogenobacter) encompas a set of bacteria able to live in harsh environments (these bacteria have been found in springs, pools, and oceans) [119, 120]. Truepera and Deinococcus from phylum Deinococcus-Thermus are considered as typical extremophiles bacteria [121, 122, 123]. From Fusobacteria, Psychrilyobacter was isolated from marine sediment from the Atlantic Ocean [124] and Sneathia appears to be a significant, emerging opportunistic pathogen that may play a significant role in urogenital track for humans (male and female); as a common component of the vaginal microbiota that can affect vaginal and reproductive health [125]. Planctomycetes (Singulisphaera, Planctomyces, CL500-3 and Phycisphaera) are aquatic bacteria (brackish water mass, fresh water and marine) [126, 127] but also present in wastewater and terrestrial soils [128]. Genus Jonquetella is considered to be a bacterium of anaerobic environments including soil, oil wells, and wastewater treatment plants and animal gastrointestinal tracts; as other members of Synergistetes they are also associated to human pathology such as cysts, abscesses, and periodontal diseases [129].
The Cluster 3, associated to samples from G1 (Fig. 2c) is formed by 22 genera from phyla Proteobacteria (Labrys and Cardiobacterium), Deinococcus-Thermus (Meiothermus), Verrucomicrobia (Opitutus), Actinobacteria (Micrococcus, Arthrobacter, Alloscardovia, Adlercreutzia and Eggerthella), Bacteroidetes (Bacteroides, Parabacteroides and Odoribacter) and Firmicutes (Solobacterium, Roseburia, Coprococcus, Blautia, Dorea, Lachnospira, Faecalibacterium, Subdoligranulum, Ruminococcus and Oscillibacter). This cluster groups bacteria mesofiles aerobic or anaerobic. The genus Labrys (mesophile, 30ºC) contains soil inhabiting bacteria species (Rhizobiales) including aerobes or facultative anaerobes bacteria [130]. Cardiobacterium is a part of normal human microbiome (specially oropharyngeal zone) (aerobes and enriched CO2 atmosphere) [131]. Meiothermus (thermophilic, 35–68 ºC) aerobic and facultative anaerobes genus [132]. Opitutus behave as an obligate anaerobe in oxygen-tolerance test growing at temperatures of 10 to 37 ºC [133]. Genus Micrococcus (strict aerobe), include species generally mesophile but some may be also cryophiles; although this genus have been isolated from human skin and animals, it is considered to be a normal environmental inhabitant, especially water, dust, and soil [134]. Arthrobacter is a strict aerobe genus, usually found in soil. Although Alloscardovia, Adlercreutzia and Eggerthella have been isolated from human and animal digestive tracts [135, 136, 43], could potentially be present in soil and water as the other Actinobacteria. Alloscardovia, shows high aerotolerance while some species of Adlercreutzia and Eggerthella are anaerobic. As Bacteriodites (order Bacteroidiales) Bacteroides, Parabacteroides and Odoribacter are widely distributed in the environment, including sea, soil, water and digestive tract of humans and animals [137]; they are anaerobic genera. Firmicutes of this cluster are represented by orders Erysipelotrycales by means of Solobacterium (anaerobe, family Erysipelotrichaceae) considered to be a common member of the microbiome of human salivary glands [138] and two families of order Clostridiales (Lachnospiraceae and Ruminococcaceae). In general bacteria of the family Lachnospiraceae are considered to be very abundant in rumens [139] and the human gut microbiota [140]: Roseburia (anaerobe) is associated to the digestive tract (cecum) of mammals [141, 142]; Coprococcus, anaerobic, is part of the human faecal microbiota [143]; Blautia from humans, cattle and chickens [144], Dorea isolated in human faeces [145] and Lachnospira found in the rumen of bovine animals since [146] and intestine of others such as pigs [147]. Regarding Ruminococcaceae, all members of this family are obligate anaerobes and mesophiles [148]. Faecalibacterium prausnitzii the unique species of the genus is very abundant in the human gut microbiota [149]. Members of Ruminococcus and Subdoligranulum are also found in the human gut and or faeces [150, 151] while the genus Oscillibacter, first isolated from the clam Corbicula japonica [152] has been also recorded in the rumen of cattle [153].