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

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.