Hedgehogs In Your Garden

"…there were many very little living animalcules, very prettily a-moving. The biggest sort … had a very strong and swift motion, and shot through the water (or spittle) like a pike does through the water. The second sort … oft-times spun round like a top … and these were far more in number."
– Antonie van Leeuwenhoek (1632 – 1723)
 

by Christoph
 

(Click to enlarge )
Figure 1. Spatio-tempo­ral mo­del of oral bacterial co­lo­nization, show­ing re­cog­ni­tion of salivary pellicle recep­tors by initial co­lo­ni­zing bacteria and coag­gregations between initial co­lo­ni­zers, fusobacteria and late co­lo­ni­zers of the tooth surface. Salivary pellicle: layer of proteins and glycoproteins of salivary origin that permanently coats the sur­faces of oral tissues. Source

If someone told you that your mouth harbors roughly 700 different bacterial species you might think "that's gross!" but you'd probably just say "Ooh!". Wait! Please, keep your mouth open for a minute and look into a mir­ror. Consider the size of bacteria, so for them your mouth is a huge garden. And now take a look at the 'ge­og­ra­phy' of your mouth-ly garden: there's the tongue (the fruit trees) with the tongue dorsum (the berry bushes), the keratinized gingiva and teeth covered by plaque (the ve­getable patches, with weeds, and hiding a hedgehog or two). Then there's the buccal mucosa (the flower beds), the hard palate and the palatine tonsils (a potato field), and the throat (the compost heap). And, finally, there's the saliva (a well). These were the locations chosen for sampling the human oral microbiome (HOM) (you may close your mouth now! ). You would not expect all 700 different bacteria to be present in equal numbers and at all locations. But who's where?, and how many of them? One location has attracted dentists and microbiologists for a long time: the dental plaque or tartar (calculus in medicinese), which forms regularly on the tooth surfaces close to the gum (gingiva). You're probably familiar with the re­mo­val of excess plaque that is part of dental prophylaxis, done to prevent gingivitis and periodontitis

 

Figure 2. Methacrylate-embedded section show­ing corn­cob structures, hybridized with probes for Cory­ne­bac­terium, Strep­to­coccus, Por­phy­ro­mo­nas, and Haemo­philus/Aggregatibacter (no scale given). Source

Microbiologists have for long tried to study the spatio-temporal organization of the biofilm that is the dental plaque. For example, by pairwise co-cultivation of se­lec­ted species on artificial ena­mel-ty­pe surfaces, they en­countered an intriguing network of reciprocal in­ter­ac­tions, metabolically and locally, of some 22 bacterial spe­cies/strains from 8 different bacterial phyla (Figure 1). What is still missing, though, is the 'micro-biogeography' of individual plaque-forming consortia, the 3D-re­sol­ved arrangement of their members. Knowing this would be crucial for understanding the localized physiology within the oral cavity. And should, in turn, give clues on the when & why the ecology de­­viates towards periodontitis or other disease states.

 

Figure 3. Corncob structures formed in plaque. Co­ryne­bacterium cells (magenta) are visible as long filaments, with cocci (green) bound to the tips of the filaments. Partially disrupted plaque was hybridized with a probe for Co­ry­ne­bac­te­ri­um and a universal bacterial probe. Image was acquired using a Zeiss Axio­Imager 63/Plan-Apo­chro­mat 1.4 N.A. ob­jec­tive and Apo­tome struc­tured illu­mination (Scale bar: 20 μm). Source

A good deal of this 'micro-biogeography' has now been resolved in a study by Jessica Mark Welch, Gary Borisy, and their coworkers. Here's how they did it. They asked volunteers to refrain from dental hygiene for 48 h and then collected their plaque, which they either mounted directly onto microscope slides or embedded in me­tha­cry­la­te for thin-sectioning. "Staining" of these samples for microscopy was done by an intricate variation of the FISH technique (fluorescence in situ hybridization) called CLA­SI-FISH: a large number of oligonucleotides (DNA probes) specific for individual taxa (from phylum down to genus or species) are coupled to different fluorophores and then hybridized to the samples. Following the hy­bri­di­za­tion procedure, "reading" of the samples is done by scan­ning with a confocal laser microscope for the individual channels (=wavelengths) responding to the fluorophores. The CLASI acronym stands for com­bi­na­to­ri­al labeling and spectral imaging, a procedure that includes as the last step software-based linear channel un­mixing (Note that the colors in the pictures are not 'true' colors but manually as­signed after unmixing according to the fluorophores' specific channel ). With oligonucleotide se­quen­ces based on their previous study they designed probes for 4 phyla, 2 classes, 3 families, and 15 genera. With these probes they ex­pected to detect 96 – 98% of the cells in a healthy su­pra­gin­gi­val plaque microbiome. Examples of specific structures involving distinct taxa detected in plaque samples are shown in Figures 2 + 3.
 

Figure 4. Interpretation of hedgehog structures. See text for details. Crevicular fluid: fluid that seeps through the gingival epithelium. Source

Among the various distinct structures in their plaque sam­ples, Mark Welch et al. found one prominent consortium composed of 9 taxa, which they termed 'hedgehog'. They found this grouping in every volunteer sampled re­peat­ed­ly and in 80% of individuals sampled only once. These hedgehog structures were prevalent among some vol­un­teers, but the fraction of plaque consisting of hedgehogs was highly variable from sample to sample, even within a single volunteer. Hedgehogs were found on the exposed buccal side of teeth and also at the gingival margin. Some samples contained multiple hedgehog structures ad­ja­cent to one another; other samples lacked hedgehogs com­ple­tely. The authors summarize their impressive pic­ture gallery of partial hedgehogs from thin sections and intact hedgehogs from whole mounts in the following in­terpretation: Corynebacterium filaments bind to an ex­is­ting supragingival biofilm containing Streptococcus and Actinomyces (Figure 4). At the distal tips of the Co­ry­ne­bac­te­ri­um filaments, corncob structures form in which the filaments are surrounded by cocci, Streptococcus, and rods, Porphyromonas, in direct contact with the Corynebacterium fi­la­ment as well as Haemophilus/Aggregatibacter in contact with Strep­to­coc­cus. Clusters of Neis­se­ri­a­ce­ae also occupy the periphery of the hedgehog. The Streptococcus cells create a mi­cro­en­vi­ron­ment rich in CO₂, lactate, and acetate, containing peroxide, and low in oxygen. Elongated filaments of Fusobacterium and Leptotrichia proliferate in this low-oxygen, high-CO₂ environment in an an­nulus just proximal to the corncob-containing peripheral shell of the hedgehog and aligned along the orientation of neighboring Corynebacterium filaments. The CO₂-requiring Capnocytophaga also proliferates abundantly in and around this annulus. The base of the hedgehog is dominated by Corynebacterium filaments and thinly populated by additional rods, filaments, and/or cocci.
 

Metagenomic studies – like those of the human oral mi­crobiome – have seen a stunning increase in numbers over the last decade: from a handful in 2003 to more than 900 in 2015 alone (PubMed search with keywords "metagenomic, bacteria"). Mainly these studies have caused this unsettling feeling most microbiologists share today: drowning in numbers, i.e., in microbial diversity. Most metagenomic studies employ the "deep se­quen­cing" approach (sequencing all DNA in a sample down to clones that represent less than 1% of the pool), which allows gene assembly from short sequence reads. Thus, we know that gene diversity is real. Virtual remain for now are, in most ca­ses, the organisms that carry these genes. Either they resist cultivation or a reconstruction of their complete genomes has not yet been achieved. (Ge­nome reconstructions from short reads require sub­stan­tial efforts even for the typically small-sized genomes of bacteria or archaea. ) A puzzling finding in many metagenomic studies is that sampling of highly similar habitats – or re-sampling a habitat at later time points again – led to the detection of many of the same taxa yet at highly va­ri­able abun­dan­ces. To account for this fluctuation, the apparent lack of a consistently abundant "core" mi­cro­bi­ome in a given habitat, it has been proposed that it is the genes and their functions that are con­ser­ved within a microbiome, distributed among various organisms whose identities are largely ir­re­le­vant. Enter the hedgehogs into this 'statistical garden'. Mark Welch et al. found that hedgehog structures were highly consistent in species/strain composition and structure across 22 healthy volunteers suggesting a pivotal role of individual bacterial species/strains for the dy­na­mics of this consortium. Turning the above argument upside-down they conclude: "...that an un­der­standing of the ecology and physiology of the organisms in the consortium will provide an or­ga­nizing principle for understanding and interpreting metagenomic and metatranscriptomic da­ta." And, beware, hedgehogs are not the only plaque consortia that these authors detected: one variety, termed 'cauliflower', contained clusters of Lautropia (Betaproteobacteria) at the center surrounded by bunches of Streptococcus (Firmicutes), Haemophilus/Aggregatibacter (Gam­ma­pro­te­o­bac­teria), and Veillonella (Firmicutes). But for now, I'm done with gardening...
 

Figure 5. DnaA tree. DnaA sequences were ob­tained from the NCBI protein database. Align­ment of DnaA domains 3 + 4 (gap open=10; gap extension=1.0) and tree building (Neighbor Joi­ning, Jukes-Cantor, 1,000 replicates) were done with CLC SequenceViewer 7.7.1 software. Boots­trap values are shown at the branch points. Red dots indicate C. durum and C. matruchotii DnaA, respectively. By the author

Appendix 1.  Mark Welch et al. found that in all their hedge­­hog structures two distinct corynebacteria provide the filamentous scaffold for the entire consortium, either C. matruchotii or, less often, C. durum. They had earlier observed during cataloging the oral microbiomes of 148 individuals that these two species – out of altogether 72(!) different Actinobacteria in the human oral microbiome database (HOMD) – are preferentially found in supra- and subgingival plaque but rarely in samples from other oral locations. You might guess that these two co­ry­ne­bac­te­ria, which thrive in an apparently identical habitat, are closely related. Probably they're not. A crude (be­cau­se incomplete ) phylogenetic tree for one of their house­keeping proteins, DnaA, reveals that they belong to dif­fe­rent clades of the (taxonomic) genus Corynebacterium within the large phylum Actinobacteria. While C. durum belongs to one clade together with the workhorse of biotechnology, C. glutamicum, the other, C. matruchotii, belongs to a different clade that also includes the pathogen C. diphtheriae (Figure 5). Their DnaAs are less homologous to each other (51% identity) than those of E. coli MG1655 and Pseu­do­mo­nas aeruginosa PAO1 (63% identity), which are 'third-degree cousins' (colloquially ) in the phylum Gam­ma­pro­te­o­bac­teria (Figure 5). Both corynebacteria interact with bacteria from eight different taxa during the formation of dental plaque of the hedgehog type, which raises the question of whether they share a common set of genes for this purpose despite being more distantly related. This is not yet known. Also, it is unknown whether hedgehog consortia built on C. durum or C. ma­tru­cho­tii scaffolds show preferences for distinct species, or strains, of their bacterial interaction partners from any of the other taxa. Preliminary results suggest that this may be the case at least for the fusobacteria. But taken together, the combinatorial possibilities are daunting, no fast-forward here for the curious.
 

Figure 6. van Leeuwenhoek's drawing of bac­te­ria from dental plaque (slightly com­pressed). See text for details. Source

Appendix 2.  I chose the introductory quote of A. van Leeuwenhoek as a reference to the first known ob­ser­va­tion of bacteria in dental plaque, but there's more to it. The full text of his Letter No.76 [39] to the Royal Society from September 17th, 1683, begins with a lengthy de­scription of his habitual way of cleaning his teeth before continuing: "... Yet all this does not make my teeth so clean but that I can see, looking at them in a magnifying glass that something will stick or grow between some of the molars and teeth, a little white matter, about as thick as batter. Observing it I judged that, although I could not see anything moving in it, there were yet living ani­mal­cu­les in it. I then mixed it several times with pure rain-wa­ter, in which there were no animalcules, and also with saliva that I took from my mouth after eli­mi­na­ting the air-bubbles lest these should stir the spittle. I then again and again saw to my great as­to­nishment, that there were many very small living animalcules in the said matter, which moved very prettily. The big sort had the shape of fig. A (see Figure 6); these had a very strong and swift motion, and shot through the water or spittle like a pike through the water. These were mostly few in num­ber. The second sort had the shape of fig. B. These often spun round like a top and eve­ry now and then took a course like that shown between C and D. These were far more in num­ber. I could not make out the shape of the third sort, for at one time they seemed to be long and round while at another time they appeared to be round. These were so small that I could see them no bigger than fig. E and therewithal they went forward so rapidly and whirled about among one another so densely that one might imagine to see a big swarm of gnats or flies flying about together. These last at times appeared to me so numerous that I judged that I saw several thou­sands of them in a quantity of water or spittle (mixed with the aforesaid matter ) no bigger than a sand-grain, although there were quite nine parts of water or spittle to one part of the matter taken from between my front-teeth and grinders." (a lightly modernized translation from the original Dutch ). His description of the experiment – equivalent to the 'Material and Methods' section in contemporary scientific papers – is so precise that it could be easily reproduced today by students in an undergraduate lab course, who, by the way, could also make their own Leuwenhoek-style magnifying glass for such an experiment. Much of science is about reproducibility, so it's good to be reminded of that, by someone who lived 300 years ago after all.