FBA Ecological Guild: Trio of Firmicutes-Bacteroidetes Alliance against Actinobacteria in Human Oral Microbiome

In a pioneering study, Zaura et al. (2009) found that majority of oral microbes fall within the five phyla including, Firmicutes, Proteobacteria, Actinobacteria, Bacteroidetes and Fusobacteria. Subsequent studies further identified a set of microbes that were commonly shared among unrelated individuals (i.e., core). However, these existing studies may have not been designed to investigate the interactions among various core species. Here by harnessing the power of ecological network analysis, we identified some important ecological guilds in the form of network clusters. In particular, we found that the strongest cluster is an alliance between Firmicutes and Bacteroidetes against Actinobacteria (FBA-guild). Within the guild, we further identified two sub-guilds, the Actinobacteria-dominant sub-guild (ASG) and Firmicutes-dominant allied with Bacteroidetes sub-guild (FBSG). Furthermore, we identified so-termed guard nodes in both sub-guilds, and their role may be to inhibit the peer sub-guild given they held competitive interactions only with the outside nodes only but held cooperative interactions only with the internal nodes, which we termed civilian nodes given that they only held cooperative interactions. We postulated that FBA-guild might be to do with protection of oral health against some opportunistic pathogens from Corynebacterium and Actinomyces, the two major genera of Actinobacteria (target of FB alliance).

The network analysis approach. We adopted standard approach for correlation network analysis to construct and analyze the human oral microbiome network with the 16s-rRNA datasets originally reported by Zaura et al. 24,[36][37][38][39] . To reduce the noise effect of the OTUs with extremely low abundance and potentially spurious OTU reads, we filtered out the OTUs whose total reads in all 29 samples were less than 30, i.e., approximately one read per sample, equivalent to removing the so-called singleton, which is a common practice in ecological analysis. A total of 347 OTUs remain after the filtering operation, and their abundances were utilized to construct the species correlation network, based on Spearman's rank correlation coefficient (R). The correlation relationships with |R| ≥ 0.6 and p-value ≤ 0.05 (significance level) were set as criteria for selecting network edges (links).
Cytoscape software (Version 2.8.3) was used to visualize the network graphs and MCODE plug-in for Cytoscape for detecting network clusters (modules) 36,40,41 . MCODE (Molecular Complex Detection) is a graph-theoretic clustering algorithm, which was first introduced by Bader et al. (2003) to identify molecular complexes in large protein interaction networks 41 . The molecular complexes in a protein network can be considered as the locally dense regions or clusters in a graph. The core algorithm of the MCODE is to detect the clusters of vertex weighted based on the local neighborhood density or cliquishness, which can be measured by the clustering coefficient, where k i is the number of neighborhood vertices of vertex i, and n is the number of edges (links) in the neighborhood. There are three main steps in the MCODE algorithms. First, MCODE weights all vertices based on their local neighborhood density, generating the so-termed vertex weighted graph (VWG). Next, the locally highest weighted vertex in the VWG will be set as a seed for a candidate cluster, and the cluster will be isolated by outwardly traversing from the seed to find all the vertices whose weights are within a given threshold. The third step is post-processing to filter vertices in the candidate cluster according to the given parameter sets. The detail interpretation of the algorithm is referred to Bader et al. 41 .
In addition, iGraph R-package was utilized for computing the network properties 42 . We also identified the P/N (positive to negative links) ratio in the network, which is a network property proposed by Ma (2017) to measure the balance between cooperative and competitive interactions in the microbiome 43 .

Results and Discussion
Basic network properties. The oral microbiome network we reconstructed contained 335 nodes (OTUs) and 4335 links (3692 positive links and 643 negative links). Table 1 lists the basic network properties. As shown in Table 1, the ratio of positive to negative correlation relationships is approximately 5.7, which suggests that the oral microbiome network is predominantly cooperative 43 . Since these network properties do not offer much intuitive www.nature.com/scientificreports www.nature.com/scientificreports/ insights on the oral microbiome network, we focus on the detection of network clusters, which are equivalent to the guild in ecological community, through which we expect to deepen our understanding on the critical species interactions in the oral microbiome and to further shed light on the structure and functions of core oral microbiota or guilds.
fBA Guild-Firmicutes-Bacteroidetes ally against Actinobacteria. An ecological guild can be defined as a group of species that exploit the same resources or exploit different resources in related manners 44 . Guild members could be competing for resources and hence hold negative correlation relationships in their abundances in the species correlation network of the oral microbiome. They may also cooperatively exploit other resources and therefore hold positive correlation relationships. Although rigorously defining and identifying microbial guilds can be rather challenging at this stage of human microbiome research mainly because functional   www.nature.com/scientificreports www.nature.com/scientificreports/ studies on the microbiome are still scarce, we believe that the following exploration for bacterial guilds through network cluster detection technique is the best we can perform in order to deepen our understanding on the interspecific interactions and to further shed light on the structure and functions of core oral microbes.
The technique we use for detecting network clusters (modules or ecological guilds) is the MCODE plug-in for Cytoscape 40,41 . Table 2 lists the 11 clusters we detected with MCODE, including the cluster number, cluster score, number of nodes and number of edges for each cluster. The cluster score is a measure of the cluster density. The higher the cluster score is, and the stronger the corresponding cluster is. The strongest cluster (i.e., No. 1 cluster in Table 2) contains 55 nodes and 648 edges, which is nearly 1/6 of all OTUs in the oral microbiome network.
The strongest cluster primarily consists of the OTUs from three phyla: Actinobacteria, Firmicutes, and Bacteriodetes, and we term the strongest cluster as FBA-cluster with the initials of the three phyla (Fig. 1). As shown in Tables 3, 38.2% (21 out of 55) of the OTUs in the FBA-cluster belong to Actinobacteria, 25.5% (14 out of 55) to Firmicutes, and 16.4% (9 out of 55) to Bacteriodetes. As further illustrated below, FBA cluster or guild is a trio of Firmicutes-Bacteriodete (F-B) ally against Actinobacteria, in which Firmicutes and Bacteriodetes hold positive links and both hold negative links with Actinobacteria in oral microbiome network. In other words, the F-B coalition competes against Actinobacteria, and each of them holds negative relationship with their common 'enemy' .
A broadly defined ecological guild almost always contains constituent guilds or sub-guilds, and so does our FBA guild that consists of two sub-guilds. Table 3 shows the component taxa of the two sub-guilds (sub-clusters) FBA contains. One is the Actinobacteria-dominant sub-guild (ASG), in which nearly 2/3 (67%) of species belong to Actinobacteria, and no Firmicutes exist and the number (only 2) of species from Bacteroidetes is negligible in the ASG sub-guild. Another is the Firmicutes-dominant sub-guild, in which more than 40% of the species are from the phylum of Firmicutes, and 21% are from Bacteroidetes in this sub-guild. Given the significant presence of Bacteroidetes in the second sub-guild, we term it FBSG (Firmicutes Bacteroidetes sub-guild). Figure 1 illustrates the topological structure of the two sub-guilds, the left side is the ASG sub-guild and the right side is the FBSG sub-guild. We consider FBSG sub-guild as an 'alliance' between Firmicutes and Bacteroidetes against ASG sub-guild. Our justifications include: (i) all interactions between F & B are cooperative, as illustrated   www.nature.com/scientificreports www.nature.com/scientificreports/ in the all positive relationships in the FBSG sub-guild (green edge in the left sub-cluster); (ii) all interactions between the FBSG and ASG sub-guilds are competitive, as illustrated in the all negative relationships between the two sub-clusters (the intermediate red edges); (iii) the numbers of members (network nodes) in both sub-guilds (i.e., 21 A in ASG vs. 14 F + 7B in FBSG) are also on a par with each other. Figure 2A,B were drawn to facilitate the visualization of the relationships mentioned above by deconstructing graph (Fig. 1) of FBA cluster into two regional blocks. Figure 2A shows the ASG sub-guild, i.e., the left block in Fig. 1. Figure 2B shows the FBSG sub-guild, i.e., the right block in Fig. 1. Figure 3 shows the interactions between both the sub-guilds. While Fig. 2A,B are self-evident, more insights can be revealed by further analyzing the interactions between both the sub-guilds. In remaining part of this section, we focus on further exploring those interactions (in the forms of inter-species and inter-guilds) to complete the objective set for this article as introduced previously.
fBA Guild-further analysis of the inter-species and inter-guild interactions. In the previous sub-section, we observed the two sub-guilds of the FBA guild, i.e., sub-guild ASG dominated by Actinobacteria, and sub-guild FBSG dominated by Firmicutes and its ally Bacteroidetes. Both sub-guilds compete with each other. The interactions (correlations) within each sub-guild are cooperative (positive), but the interactions between the two sub-guilds are competitive (negative).
To further explore the inter-species and inter-sub-guild interactions, we introduce the concept of 'guard' nodes. We define guard nodes of a sub-guild as nodes that have negative relationships with the nodes in another sub-guild, but with full positive relations with nodes within its own sub-guild. That is, guard nodes "guard against" their counterparts in another sub-guild, but are 'friendly' to their own sub-guild-members (i.e., their relationships with other sub-guild members are cooperative). Figure 3 shows the interactions between guard nodes from both ASG and FBSG sub-guilds. In Fig. 3, the left column exhibits the 14 guard nodes in the ASG, in which 7 species are from Cornebacterium genus, 2 species from Actinomyces genus, and the remaining 5 guard species from other small phyla but none from Firmicutes or Bacteriodetes. The right column of Fig. 3 displays the 16 guard nodes in the FBSG, in which 7 species are from Firmicutes, 4 from Bacteriodetes, 2 from Actinobacteria, and 3 from other phyla.
The taxonomic information FBA-guild is listed in Table 4, which is tabulated based on Figs. 1 and 3. In Table 4, nodes are classified into two types: one type is the guard node that is always 'hostile' (negative interactions) to its counterparts in another sub-guild but always 'friendly' to its civilian nodes within the same sub-guild; the other type is, what we called, civilian node who may be 'friendly' to any node in the whole FBA guild (or any sub-guild). The distinction between civilian nodes and guard nodes suggests the possible functional differentiations among the nodes in each sub-guild. It should be the differentiation that shape or even determine the interactions between two sub-guilds. Two types of nodes may play rather different roles in the interactions. The role (function) of guard nodes should be to protect their home-sub-guild against invasions from foreign guards, while they should never compete with any nodes in their homeland. Using an analogy, civilian nodes in both sub-guilds, although they have their own citizenships, can 'friendly' interact with any nodes regardless of their citizenship. Using another analogy, FBA guild is like a global village, where civilians may friendly trade with each other, but each sub-guild still preserves their military forces (guards) and 'fight' each other to keep order. This reminds us that, in the FBA triangle relationship, although FB (Firmicutes & Bacteriodetes) is united against A (Actinobacteria), the competition between FB & A only occurs in military sector and two sides (sub-guilds) cooperate with each other in civilian sectors. Table 5 further lists all negative interactions (correlations) between both the sub-guilds of the FBA guild. Table 6 further lists the number of positive, negative and total interactions, respectively, between Firmicutes, Bacteriodetes and Actinobacteria. Table 6 also computed the P/N (positive to negative) ratio of links between the three phyla according to Ma (2017) P/N ratio approach 43 . Table 6 indicates that in the F-B alliance against A, F plays a larger role than B does, given that P/N ratio between F & A is approximately ½ that between B & A and small P/N ratio is resulted from larger number of competitive interactions (the denominator).   Table 5. Brief information on the negative interactions between the two sub-guilds of FBA guild*. *The negative interactions only occurred between the guard nodes distributed in two separate sub-guilds.  24 . While the distributions of Firmicutes, Bacteroidetes and Actinobacteria are rather aggregated in the sense that they form strongly connected sub-clusters, the distributions of Proteobacteria and Fusobacteria are rather dispersed in the sense that they are distributed all over the place (all sub-clusters), and they do not dominate in any sub-clusters. Furthermore, "the others" do not seem to have a special or fixed pattern in their interactions with Firmicutes, Bacteroidetes and Actinobacteria. For example, while most interactions are cooperative, competitive relationships also exist. Using an analogy, we characterize "the others" as "nomads" of small "ethnic groups" in the FBA guild. Although further investigation on "the others" could be interesting, we believe the results should not affect the validity of the findings discussed in previous sections.

Sub-Guild
As a side note, we conducted similar examinations of other clusters detected with MCODE and listed in Table 2, but failed to find similarly interesting structures or interactions. Since majority of the core oral microbes (i.e., Firmicutes, Proteobacteria, Actinobacteria, Bacteroidetes and Fusobacteria) identified by Zaura et al. (2009) are contained in the FBA guild, which is the largest (also the strongest) cluster we detected, the failure should not be surprising 24 . Hence, the structure and species-interaction mechanism of FBA guild revealed in this article also represent those of core oral microbes.
The mission of FBA guild in the oral microbiome-a new hypothesis. In previous sections, we have showed the structure and inter-species interactions within the FBA. Nevertheless, at this stage, we cannot fully explain the underlying mechanisms leading to this interesting triangular relationship, which requires   www.nature.com/scientificreports www.nature.com/scientificreports/ experimental investigations beyond the scope of this article. Here, we propose a new hypothesis to explain the observed phenomenon, and hope to stimulate the further studies on this obviously rather important phenomenon.
First, both Firmicutes and Bacteroidetes have been the dominant players in the gut microbiome and have attracted extensive attentions in recent years, in particularly, their implications to obesity. The ratio of Firmicutes to Bacteroidetes (F/B) has been suggested as an index of the health of gut microbiome. Both the phyla are the most abundant taxa of gut microbiome, although the inter-individual variations are huge and their dynamics is rather dramatic 30,[45][46][47] . For example, the F/B ratio could decrease from approximately 10.9 in middle-age adults to 0.6 in the elderly 45,48 . In the oral microbiome, Zaura et al. (2009Zaura et al. ( , 2014Zaura et al. ( , 2015 studies also suggested the dominance of both phyla, and contributed approximately 50% (36% Firmicutes and 12% Bacteroidetes) to the oral microbiome, and were two of the five major phyla in the oral microbes [the other three were: Proteobacteria (22%), Actinobacteria (24%), and Fusobacteria (4%)] 23,24,33 . Since oral and gut environments are well connected, and bacteria may freely disperse but environment would select who can stay and who can only be by-passers or nomads 49,50 . Therefore, it can be expected that the oral and gut microbiomes should be of certain level of similarity. Therefore, the dominance of F & B in the oral environment can be expected, but we are puzzled by the fact that there were not any negative interactions between F & B ( Table 5, Fig. 4). It might be just that the gut environment allows for the competition between the both because both F & B may be competing for the fermentation niche, one of the three metabolic niches (the other twos are sulfate reduction and methanogenesis) gut microbes compete for in the gut ecosystem 51 . Existing literature reveals that majority of species in F & B are involved in fermentation 51 . However, healthy oral environment is not a fermentation habitat in general, and therefore F & B lose the battle ground for competing, instead they may turn to cooperation (positive correlations), possibly forming an alliance against Actinobacteria as we discovered previously. But this leads to another question, which we try to answer below, why do F & B both do not like Actinobacteria?
Second, note that in the Actinobacteria-dominant sub-cluster, Cornebacterium and Actinomyces are the two primary genera. Existing literatures suggest that these two genera include some of the notorious pathogens, especially opportunistic pathogens. For example, C. diphtheriae causes diphtheria. Other pathogenic species in humans include: C. amicolatum, C. striatum, C. jeikeium, C. urealyticum, and C. xerosis 5,[52][53][54][55] . Certain species of Actinomyces are known to be opportunistic pathogens, particularly, when the immune system of host is weak [56][57][58][59][60][61] . Of course, there are innocuous species in these genera, given that oral microbiome and its environment (host) usually live harmoniously and their interactions are cooperative in large [62][63][64][65][66] . We conjecture that the potentially suppression of F-B alliance against A exhibited by the FBA guild, as the primary component of core oral microbiome, should be important for maintaining a healthy oral microbiome and protect humans from many opportunistic infections.
A major limitation of this study is that the dataset used to reconstruct the oral microbiome network was published a decade ago, and the 29 samples were collected from three healthy individuals only (Zaura et al. 2009). Therefore, the findings from our reanalysis of the datasets should be validated with more extensive datasets in future. A primary motivation for us to publish our results was to demonstrate the potentially important application of the concept of ecological guild in microbiome studies. The dataset originally reported by Zaura et al. (2009), which we reanalyzed, offered us an excellent opportunity to pursue our objective because of its multi-site nature and high-quality sequencing experiments.