Higher order taxa
Domain: Bacteria – Phylum: Firmicutes – Class: Clostridia – Order: Clostridiales – Family: Acidaminococcacceae – Genus: Dialister
Species: D. invisus Current strain types: CCUG 47026T, CIP108215T, DSM 15470T, E7.25T JCM 17566T (1).
Description and significance
In recent decades, with the advent of molecular sequencing techniques, great strides have been made in characterising and identifying many more proponents of the oral cavity’s microbial community. D. invisus identification was only possible in the wake of these technologies. Historical background For decades a general ‘group’ of bacteria was known to reside in the oral cavity, usually in tandem with some form of dental infection, and was characterised as a small coccobacilli, that was anaerobic, gram negative and generally undetectable by traditional biochemical tests. This unresponsiveness to metabolic characterisation, its apparent slow growth rates and difficulty to visualise under cultured conditions meant that the various strains within this group were lumped together under the more visible members; such as Dialister pneumosintes. This species was originally described in 1921 as Bacterium pneumosintes (Olitsky and Gates) but was renamed during a phylogenetic reengagement of the bacterial groups (Downes et al 2003). Later, culture independent studies and the application of 16S rRNA helped tease this genus apart and has identified a number of closely related taxa within it that have distinct niches around the human body (see Figure 2). One such study in 2003 was investigating a strain isolated from chronic endodontic lesions, known as Dialister E1 (Downes et al 2003). After completing a number of characterising tests the research group proposed the existence of a new distinct species within the Dialister genus – D. invisus. Its language root ‘invisus’ translates roughly to ‘unseen,’ thus the species is named for its apparent ‘lack’ of visibility when grown in broth conditions (Downes et al 2003).
Description The researchers described the species as an anaerobic, small coccobacilli (See Figure 1), gram-negative, asaccharide, non-motile and with a low biochemical reactivity under general tests. The species when grown on an FAA plate was able to be cultured, but the colonies were small, slow growing and difficult to visualise without microscopy. The strain E7.25T is susceptible to kanamycin and colistin antibiotics (Downes et al 2003). While these early studies only identified the bacterium in the oral cavity, subsequent studies have found it to also be widespread within the microflora gut communities. D. invisus has shown to carry several antibiotic genes which are indicated to play a possible role in the oral cavity’s biofilm communities (Al-Ahmad et al 2014). Yet, while it is considered to play a pathogenic role within the oral cavity (Rôças and Siqueira Jr. 2005), new research is indicating that within the gut of its host, it may have beneficial interactions. For example , potentially preventing Irritable Bowel Syndrome (IBS) (Nagao-Kitamoto and Kamada 2017).
Figure 1: (Downes et al 2003) microscopy of D. invisus.
Figure 2: Phylogenetic tree showing Dialister genus based on 16SrRNA. D. invisus is 2nd from bottom of the tree. Tree rooted with E.coli. Sourced from (Morotomi et al 2008).
The known structure and composition of D. invisus genome is limited as of yet with only one strain genome so far published in the NCIB (9) (see table 1). An additional strain providing the current partial 16S sequence of 1530bp, is currently available at Strain-Info and was denoted from strain JCM (10).
During the original characterisation of the species, the authors reported that the bacterium has a C+G content of 45-46 mol%, which was provided as means of species identification within its genus (1). The total genome is reported to be 1.89596 Mb (11).
Table 1: A summary of key linear genomic elements based on data obtained from strain DSM 15470T. (9).
Element Count Gene total 1829 Genes coding 1742 Genes RNAs 65 Pseudogenes 22 CRISPR arrays 4
Genes of interest Within the current published studies, several antibiotic resistance genes have been identified, including BlaTEM, ermC and tetM, which conveys resistance to TEM-type beta-lactamases, erythromycin and encodes ribosomal protection respectively (Rôças and Siqueira Jr. 2012). The incorporation of these genes within the genome vary across strains, with the study finding one strain carrying all the described resistance genes, while the additional four strains, only carried a variation of two (Rôças and Siqueira Jr. 2012).
Cell structure and metabolism
Cell structure D. invisus as a gram negative bacterium that has a thin peptidoglycan wall (see figure 1). This small coccobacilli species (0.3-0.4 x0.3-0.5 um) has no motility and can occur either singularly, in pairs, small clumps or in short chains (Downes et al 2003).
No specific studies have investigated the role D. invisus plays within the formation and function of oral cavity biofilms. However one study which investigated biofilms in relation to 42 species isolated from an infected root canal and their respective antibiotic resistance within these infections, used primers designed for D. pneumosintes (Al-Ahmad et al 2014). This Dialister species has a 93% sequence similarity to D. invisus so can therfore serve as an indicator of its possible functionality within biofilms. This study summarised that periodontitis (where D. invisus can be located in) can be classified as a biofilm induced disease. D. pneumosintes showed resistance to gentamycin and vancomycin but did not have the capacity to be a biofilm producer, scoring amongst one of lowest OD values published in the study (Al-Ahmad et al 2014). However just because this species is unable to establish a biofilm, doesn’t mean it’s incapable of being incorporated into a biofilm. In such a complex Dialister antibiotic genes may contribute to the structures resistance to antibiotic.
Using two distinct tests, one growth dependent (PRAS), and the other non-growth dependent (ROSCO diagnostic), both corroborated that D. invisus, quote, [Did] “Not ferment arabinose, cellobiose, fructose, glucose, lactose, maltose, mannitol, mannose, melezitose, melibiose, rafﬁnose, rhamnose, ribose, salicin, sorbitol, sucrose, trehalose or xylose” (Downes et al 2003).
The ROSCO diagnostic test indicated that trace amounts of acetate and propionate were detected as end products of metabolism when grown within a peptone/yeast extract/glucose medium. Thus as an asaccharolytic bacterium, D. invisus doesn’t metabolise sugars (Downes et al 2003). Instead this bacterium is speculated to use other non-carbohydrate nutrients such as amino acids and peptides for its growth (Rôças and Siqueira 2006). This in part is reflected by its ‘preferred’ environmental niche within the oral cavity, which is deep within the root canals, were such nutrients during inflammation are abundant (Rôças and Siqueira 2006,).
Biochemically D. invisus is unreactive to common diagnostic tests such as the Rapid ID 32A identiﬁcation system, with a profile of 0000000000 from current known strains (Downes et al 2003). However in a way, this makes this species diagnostically distinct within its own genus as most species within display some form of reactivity, with the exception of D. invisus and D. propionicifaciens (Morotomi et al 2008) (see figure 3).
Figure 3: Table sourced from (Morotomi et al 2008) representing both G+C content within the Dialister genus and members respective API codes: 1- D. propionicifaciens, 2- D. propionicifaciens, 3- D. invisus, 4- D. micraerophilus and 5- D. pneumosintes. Note the lack of positive scores in D. invisus, conferring its general lack of reactivity for compound used in the Rapid ID 32A API test.
D. invisus is an obligate anaerobic bacterium. Thus it can only survive in conditions deprived of oxygen. Originally identified in the oral cavity, this bacterium is usually associated with periodontal and root canal based infections, thus it resides in low oxygen environment within the oral cavity.
It has also been identified in stool samples and within the gut microbiota. One such study identified three cases in which D. invisus, co-occurred among their subjects oral and gut microbiota (Franzosa et al 2014). In all of these cases the abundance of the species was low within the oral cavity but reached a high abundance within stool samples. A point of interest of the study was that although there was a high abundance of the organism DNA within the gut samples, very little species specific transcripts could be isolated from the same samples. It was concluded that this could indicate that while the species is present within the gut it may exhibit a reduced transcriptional activity. Other members within this study carried the bacterium exclusively in the oral cavity. It was thusly concluded that D. invisus can co-occur within the oral and gut microbiota with some form of movement between the two niches, but this association of high visible abundance may be atypical (Franzosa et al 2014).
The bacterium has only been associated and isolated from human hosts, probably due to the bias in focused sampling for the human microbiome. In the oral cavity its interactions with its host can be akin to a pathogenic. Within the gut microbiota however it may play a more passive or even beneficial role.
Irritable Bowel Syndrome links
A correlation with the abundance of D. invisus in the gut and a dysbiosis diagnosis of the IBS patient has recently been established. Namely that patients with gut dysbiosis (IBS) had low levels of D. invisus within their gut microbiota compared to controls and healthy relatives (Nagao-Kitamoto and Kamada 2017). Is was speculated since patients with IBS often have a disequilibrium with their levels of acetate and propionate, and since D. invisus is known to actively produce both of these compounds, perhaps this is the association between the bacterium and the disorder (Nagao-Kitamoto and Kamada 2017). Other studies have shown that the lack of D. invisus in conjunction with four other species prevalence’s, is a diagnostic indictor of IBS (Joossens et al 2010). This species has thus been used a case example to demonstrate that IBS patients have different microbiota compositions to healthy controls. This study also outlined that of its three predominate disproportioned species, including D. invisus, all suggest that the lack of butyrate producing capacity is a plausible component of the physiopatholigical explanation of IBS (Joossens et al 2010).
Studies show that D. invisus is highly prevalent in periodontal infections, either asymptomatic or persistent infections. From a 2005 study (Rôças and Siqueira Jr 2005) of 29 participants diagnosed with a periodontal infection, in 55% of cases D. invisus was isolated from regions of infection. It was inferred that this association and high prevalence of the bacterium is suggestive of a pathogenic role within the oral cavity (Rôças and Siqueira Jr 2005). However this pathogenic role may be isolated to the oral cavity, as some new studies have found an interesting correlation with the abundance of this species within the gut microbiota and IBS patients.
Application to biotechnology
Currently, as of September 2017, there is no published or known projects investigating bioengineering of this bacterium. Neither are any specific enzymes, compounds or drugs being manufactured from it, or specifically for it.
There may be some possible avenues of interest in the future with its antibiotic resistance capabilities within the oral cavity or potentially it’s identified CRISP arrays. If its role within the gut microbiota is characterised further, it may have a potential (untested) to act as part of treatment or management plan for IMS patients.
The current research for this species is split along two lines. Some research teams are focussing on the role D. invisus plays with IBM (Nagao-Kitamoto and Kamada 2017). While other groups are continuing to work on its pathogenic contributions within the oral cavity (Rôças and Siqueira. 2006). All these studies are aiming to help fully characterise the functional aspect D. invisus within the human microbiota.
1.  type strain] 2. [Olitsky, P.K. and Gates, F.L., 1921. Experimental studies of the nasopharyngeal secretions from influenza patients. Journal of Experimental Medicine, 33(2), pp.125-145.] 3. [Downes, J., Munson, M. and Wade, W.G., 2003. Dialister invisus sp. nov., isolated from the human oral cavity. International journal of systematic and evolutionary microbiology, 53(6), pp.1937-1940.] 4. Rôças, I.N. and Siqueira, J.F., 2012. Antibiotic resistance genes in anaerobic bacteria isolated from primary dental root canal infections. Anaerobe, 18(6), pp.576-580. 5. Al-Ahmad, A., Ameen, H., Pelz, K., Karygianni, L., Wittmer, A., Anderson, A.C., Spitzmüller, B. and Hellwig, E., 2014. Antibiotic resistance and capacity for biofilm formation of different bacteria isolated from endodontic infections associated with root-filled teeth. Journal of endodontics, 40(2), pp.223-230. 6. [https://doi.org/10.1016/j.femsle.2005.07.017 Rôças, I.N. and Siqueira Jr, J.F., 2005. Detection of novel oral species and phylotypes in symptomatic endodontic infections including abscesses. FEMS microbiology letters, 250(2), pp.279-285. 7. Nagao-Kitamoto, H. and Kamada, N., 2017. Host-microbial Cross-talk in Inflammatory Bowel Disease. Immune network, 17(1), pp.1-12. 8. Morotomi, Masami, Fumiko Nagai, Hiroshi Sakon, and Ryuichiro Tanaka. "Dialister succinatiphilus sp. nov. and Barnesiella intestinihominis sp. nov., isolated from human faeces." International journal of systematic and evolutionary microbiology 58, no. 12 (2008): 2716-2720. 9.  10.  11. [orgn 12. Isabela N. Rôças, José F. Siqueira, Characterization of Dialister Species in Infected Root Canals, In Journal of Endodontics, Volume 32, Issue 11, 2006, Pages 1057-1061, ISSN 0099-2399, https://doi.org/10.1016/j.joen.2006.04.010. 13. Franzosa, E.A., Morgan, X.C., Segata, N., Waldron, L., Reyes, J., Earl, A.M., Giannoukos, G., Boylan, M.R., Ciulla, D., Gevers, D. and Izard, J., 2014. Relating the metatranscriptome and metagenome of the human gut. Proceedings of the National Academy of Sciences, 111(22), pp.E2329-E2338.
This page is written by C. Stephenson for the MICR3004 course, Semester 2, 2017