杏吧原创

THE SCENE could be mistaken for a piece of modern art. In the foreground
stands a forest of slightly limp tubular balloons. Beyond it, a buffalo herd of
tightly packed spheres fades into a distant horizon. On the skyline are
elaborately shaped edifices: corn-on-the-cobs, bottle brushes, rosettes and
palisades.

But this isn鈥檛 fantasy land. The images are of something real鈥攎undane
even鈥攁nd close to all of us. These are the microbes that live in a human
mouth, as seen through an electron microscope. And much-maligned microbes they
are. For decades, dental researchers have been waging all-out war on these tiny
interlopers. The makers of toothpaste and mouthwash have treated them as if they
were an army of comic-book villains. The Evil Plaquemen, as one television
commercial puts it.

Great white sharks

In your mouth, there is a jungle made up of hundreds of species of fungi,
protozoans, viruses, intracellular parasites and鈥攁bove all鈥攂acteria.
Most are permanent residents, and many of them are found nowhere else, not even
in the mouths of other mammals. 鈥淲hat we are seeing is a very elaborate
ecosystem,鈥� says George Bowden a microbiologist at the University of
Manitoba.

Perhaps this shouldn鈥檛 come as too much of a surprise. Kept at a balmy 36
掳C, with constant moisture and a steady influx of nutrients, the mouth is a
year-round tropical paradise. Add to this a busy procession of incoming
flights鈥攆ingers, food, pencils, you name it鈥攅ach loaded with
microbial passengers, and we鈥檙e talking Mardi Gras. Every square millimetre of
cheek tissue, every fold beneath the gum line, every crevice in the tongue is
swarming with occupants. Even a well-brushed tooth hosts billions of
bacteria.

Most estimates of the number of species that count as fully paid-up members
of the oral community range from 200 to 500, of which some 50 have been named.
There are simple bacterial spheres, rods or twisted threads that anchor
themselves in place and advance only by dividing. Then there are others that
glide or creep about, driven by filament-like propellers. Up to 50 times larger
than the bacteria are beasts such as Trichomonas tenax, a great white
shark of a protozoan that uses its flagellum to whiplash its way through saliva,
or the oral jungle鈥檚 sabre-toothed tiger, Entamoeba gingivalis, a
predatory amoeba that lurks around tooth surfaces and gum pockets, searching for
food particles or hunting down hapless bacteria.

鈥淚t鈥檚 quite a fascinating thing to look at, especially the samples you take
from beneath the gums,鈥� says Sigmund Socransky, an oral ecologist at the Forsyth
Dental Center in Boston. 鈥淚t鈥檚 literally a seething, swimming mass.鈥�

Despite this richness of species, most of the interest over the years has
focused on just one, Streptococcus mutans. This nondescript,
sphere-shaped bacterium, which is found only on human teeth, has been known
since the 1920s. Its habits of chowing down on sugar and secreting lactic acid
were discovered in 1956 to be the cause of tooth decay. Since then, researchers
have toiled long and hard in an effort to develop an antimicrobial agent or
vaccine that could destroy it for good. 鈥淭he Swedes in particular went after it
with everything short of nuclear war,鈥� says Page Caufield, an oral
microbiologist at the University of Alabama at Birmingham. 鈥淭hey were the ones
who coined the phrase, `a clean tooth never decays鈥�.鈥�

Swarming masses

Efforts to look in detail at the entire mouth bug community began with
studies by a team at the Royal Dental College in Aarhus, Denmark, in 1966. They
found 11 volunteers who were prepared to have removable plates fitted to their
teeth, and to abandon their toothbrushes for three weeks. By removing the plates
after various lengths of time and examining them under a microscope, the
researchers were able to observe the growth of the bacterial colonies commonly
known as plaque. What they saw was ecological succession鈥攅ach community of
organisms successively replacing its predecessor, and then being replaced in its
turn, until equilibrium was reached and a climax community established.

Human plaque, a type of biofilm
(see 鈥淪lime City鈥�, New 杏吧原创, 31 August, 1996, p 32),
develops like this. Eight hours after brushing, a tooth is
swarming with spherical bacteria such as Streptococcus mitis and
Streptococcus oralis, along with a few rod-shaped organisms such as
Actinomyces naeslundii. After a day, the teeth have shrouded themselves in
a blanket of these early colonisers, and the first longer rods and thread-like
species such as Fusobacterium nucleatum, a sulphur producer linked to
bad breath, begin to appear on top. The next few days see the development of
complex structures made up of bacteria of different species. For example, those
microscopic corn-on-the-cobs are in fact hundreds of round Streptococcus
sanguis crowded along the elongated bodies of Corynebacterium
matruchotii. When oral hygiene has really gone to pot, more choosy species
such as the corkscrew-shaped spirochete Treponema denticola join the
party.

As the community develops, local conditions such as pH change,
encouraging new organisms to dominate until conditions change again and others
take over. After three weeks, this kaleidoscope of activity gives way to the
relative calm of the climax stage鈥攖he mouth鈥檚 version of an old-growth
forest. The base is now a dense undergrowth of bacteria up to 20 cells鈥� or
15 micrometres鈥攄eep; the canopy is a seething mesh of bacteria and other
microorganisms, sometimes more than a tenth of a millimetre
thick鈥攕ubstantial enough to be visible to the naked eye.

Kissing couples

At the Centre for Applied Microbiology and Research at Porton Down in
Wiltshire, Philip Marsh and his team are discovering how group living helps oral
microbes adapt to the mouth鈥檚 changing environment. The tool they use is called
the multispecies chemostat, an apparatus that houses discs closely resembling
tooth enamel, which it keeps under conditions mimicking those found in the
mouth. Researchers used to believe that bacteria unable to tolerate oxygen, such
as Porphyromonas gingivalis which rots gums, will only flourish on an
unbrushed tooth coated with lots of plaque to block out the gas. But when Marsh
introduced 10 species of bacteria, several of them oxygen-hating anaerobes, into
an oxygen-rich chemostat, they all grew almost immediately. At first, Marsh
assumed that the survival of the anaerobes was made possible by the extremely
rapid growth of the one aerobic species, Neisseria subflava, using up
all the oxygen. But when he removed N. subflava and ran the test again,
he got the same result. The facultative anaerobes鈥攂acteria that consume
oxygen when it鈥檚 around, but also grow quite happily without it鈥攕eemed
perfectly capable of detoxing the environment for the oxygen-haters by
themselves. The microorganisms also formed tight 鈥減ackages鈥�, both on the discs
and in the surrounding liquid, that may have helped the oxygen-haters to cope.
鈥淲e assume the aerobic organisms are on the outside of these structurally
organised packages,鈥� Marsh says, 鈥渁nd that they very rapidly consume the oxygen,
so over a few cell depths it鈥檚 anaerobic.鈥�

Discovering how communities of oral flora develop is all very well, but how
do the organisms get there in the first place? To find some of the answers,
Caufield is using DNA fingerprinting to track bacteria as they move from host to
host. His analysis of saliva collected from infants and their mothers over a
five-year period reveals key details about the traffic of oral microbes. All but
eight of 46 children Caufield studied became infected with S. mutans
during a 鈥渨indow of infectivity鈥� between 19 and 31 months of age, a period that
corresponds to the eruption of primary molars. The eight children who didn鈥檛 get
infected during this period remained free of S. mutans throughout the
study, suggesting that the organism had missed its opportunity to fill that
niche.

Last year, Caufield reported that S. sanguis has an even tighter
window of opportunity: every one of 45 children were infected during a
three-month period around nine months of age. As he makes his way through
additional species, Caufield expects similar patterns to emerge. 鈥淭hese bacteria
come in at very specific times in the development of the infant,鈥� he says. 鈥淎nd
the acquisition is orderly and in sequence.鈥� That鈥檚 because the exact conditions
that enable each micro-organism to get a foothold鈥攁 certain amount of
virgin tooth surface, for example, or the presence of another
species鈥攈appen at distinct times in a child鈥檚 life.

Caufield has also discovered that the source for these bugs is the children鈥檚
own mothers. After scrutinising the DNA from individual bacterial cells, he has
concluded that pretty much every one of us has our own strain of S.
mutans. Only mothers and their children share the same strain. When
Caufield re-examined some of the samples from his original study, he found that
24 out of 34 children had been infected by a strain from their mother; the rest
carried a strain of unknown origin. 鈥淲e think what you get early on is what you
keep for the rest of your life, and this sets the pattern for health and
诲颈蝉别补蝉别.鈥�

Intimate contact

Another surprise is that husbands and wives don鈥檛 swap S. mutans
strains, although other bacteria, such as Actinobacillus
actinomycetemcomitans鈥攐ne of the agents implicated in gum
disease鈥攎ay be passed between kissing couples. Caufield has fingerprinted
bacteria from members of over 300 families, and has yet to find a S.
mutans match between the two parents. People appear to be remarkably
effective at fending off foreign strains of the bug. 鈥淚f you take a teenager or
an adult and try to put S. mutans into their mouth, it won鈥檛 go,鈥� he
says, referring to trials in the 1970s on student volunteers. An established
bacterial colony, like a troop of territorial monkeys, refuses to yield its neck
of the woods.

There鈥檚 more: none of the DNA fingerprints has so far shown a match between a
child and its father, not even amongst the 10 who failed to inherit maternal
strains of S. mutans. Nor was there a father-child match reported from
a recent study of 11 families in Sweden, where fathers spend more time with
their babies because of mandated paternity leave.

What鈥檚 going on? Caufield hasn鈥檛 ruled out the straightforward possibility
that mothers have more intimate contact with their babies than even the most
devoted of fathers. But he thinks it more likely that the immune system is
involved. A child receives maternal antibodies while in the uterus, and from
breast milk, to help it deal with the many unaccustomed threats it faces during
the months after birth. These antibodies might well be tolerant of Mum鈥檚
lifetime microbial companions, Caufield reasons, leaving her child open to those
particular strains. Perhaps the mother also passes on an acquired reaction
against her husband鈥檚 germs, which would explain the absence of transfer from
him.

But what about those 10 children who didn鈥檛 get S. mutans from their
mothers? Caufield admits that if his theory is true, the transfer rate should
have been higher. But he points out that many of these children were bottle-fed,
and that this, coupled with exposure to antibiotics, may represent an unnatural
situation. Last year Yihong Li, a molecular epidemiologist who is also at the
University of Alabama, studied the mouth bugs of young children in a Chinese
village where virtually all infants are breast-fed and few people are exposed to
modern drugs. Just as Caufield predicted, Li found an almost 90 per cent
transfer rate between mother and child.

In this respect as in many others, your mother鈥檚 influence is likely to be
with you for years鈥攑erhaps even for a lifetime. Caufield was recently sent
a saliva sample from a Swedish woman whose bacteria had been used in experiments
during the 1970s. A comparison of DNA from these bacteria with material from the
fresh sample showed that the woman had been carrying the same strain of S.
mutans for over 25 years.

Oral bacteria might even make a useful tool for studying evolution, says
Caufield. Palaeontologists already use random mutations in mitochondrial DNA,
which is passed from mother to child without any contribution from the father,
to estimate how closely related different groups of people are to each other and
hence where modern humans originated. Given that bacterial DNA is thought to
undergo more frequent mutations than mitochondrial DNA, oral microbes might give
a more accurate measure of the timing of genetic changes, he says. 鈥淪.
mutans has probably been around for millions of years, so it should reflect
the changes that went on in the human genome during that time.鈥� In the past few
years, Caufield has analysed a fragment of DNA in S. mutans collected
from humans in Asia, Africa and Europe, and found greater sequence diversity
among Africans. This finding mirrors the diversity in mitochondrial DNA that is
taken as evidence that humans have been in Africa longest and so originated
there鈥攖he 鈥淥ut of Africa鈥� theory of human origins.

Wipe out

The growing knowledge about the ins and outs of oral ecology could have more
prosaic uses, too. For one thing, it could pave the way to a new approach to
fighting oral diseases鈥攐ne that replaces the 鈥渂last `em鈥� mentality of the
past with more subtle attacks behind the enemy鈥檚 lines.

One promising strategy uses a strain of human S. mutans that
naturally produces a chemical that kills rival S. mutans strains. Jeff
Hillman, a microbiologist at the University of Florida in Gainesville, has
genetically manipulated organisms belonging to this fratricidal strain to
produce a superbug that still wipes out its fellows but has lost the ability to
produce tooth-rotting acids. Last October, he infected young rats with the
superbug and then put them on a high-sugar diet. Unlike rats carrying normal
human S. mutans that were fed the same diet, the teeth of animals given
the replacement strain remained cavity-free. What鈥檚 more, Hillman鈥檚 superbug
doesn鈥檛 seem to upset the oral ecosystem.

Hillman has already shown that the undoctored version of the killer strain of
S. mutans has the unusual ability to oust a person鈥檚 natural strain,
presumably by killing it off and settling into its niche. In these experiments,
a group of volunteers at the Forsyth Dental Center had their teeth
professionally scraped, and then spent three minutes with a toothbrush and
dental floss coated with the aggressive S. mutans. A year later, the
killer bacteria were still very much around.

Hillman is now seeking permission to run clinical trials using the
genetically altered, non-rotting superbug. His goal is to use these bugs as an
inoculant that would do for cavities what vaccines have done for polio. 鈥淚t
could be used by everybody,鈥� he says, 鈥渁nd it should be relatively
inexpensive鈥攁 one-time treatment that could prevent tooth decay for a
濒颈蹿别迟颈尘别.鈥�

This way of fighting oral diseases could even have big advantages over the
scorched earth approach of conventional oral hygiene. Some of the organisms in
your mouth do a useful job, stopping nastier incoming germs gaining the foothold
they need to wreak havoc. For example, one consequence of blasting the oral
ecosystem with heavy doses of antibiotics is thrush, a fungal infection that has
been linked to certain oral cancers.

The resident microbes also produce a battery of chemicals that are lethal to
some of our worst foodborne and airborne enemies. One of the latest examples to
emerge was reported last year by researchers at the University of Aberdeen. They
showed that bacteria growing naturally on rats鈥� tongues produce nitrite, which
reacts with stomach acid to create conditions that in the lab kill off
salmonella, Escherichia coli and other unwanted organisms. Closely
related microbes on the human tongue are thought to be doing the same.

All of which represents a remarkable turnabout in our attitude towards the
bugs that live in our mouths. 鈥淒ay after day, year after year, these organisms
are doing us no harm,鈥� says Socransky. 鈥淚n many ways they鈥檙e actually benefiting
us on a continuous basis.鈥� The image of the Evil Plaquemen may still rule, but
the day of Benevolent Biofilm is at hand.

* * *

Perfect harmony

EVERY one of us carries round something like a hundred trillion
bacteria鈥攖hey live in your mouth, your anus, the crease at the side of
your nose, behind your ears, in your stomach and intestines, and in every other
crack and crevice of the human body. How do we survive?

Certainly these germs are capable of doing us in. When they get where they
shouldn鈥檛鈥攚hen a bite wound from a fellow human injects oral bacteria into
the blood, for instance鈥攖hey can be deadly. 鈥淭hese bacteria,鈥� says cell
biologist Brian Henderson, 鈥渁re like little sticks of dynamite.鈥� Yet they rarely
explode.

Perhaps more mystifying is why they are not eliminated by the ever-vigilant
immune system, even though their surfaces are loaded with molecules that under
other circumstances trigger a hearty immune response. Henderson, who works at
University College London, has a theory that as in any stable relationship the
secret is communication.

In this case, it is chemical crosstalk that is keeping us and our house
guests at peace. One of the key go-betweens are those cytokines which activate
the immune system in response to infection and damp it down when the danger has
passed. Henderson鈥檚 idea is that bacteria manipulate this activity with chemical
signals of their own鈥攑roteins that he and fellow University College
microbiologist Michael Wilson are calling 鈥渂acteriokines鈥�.

Viruses have already set a precedent for this sort of behaviour: some can
subdue their host鈥檚 immune response by manufacturing copies of its
anti-inflammatory cytokines. 鈥淚f you knock the gene that makes these molecules out of
the viruses, animals infected with them often die,鈥� says Henderson. The
undoctored virus doesn鈥檛 appear to adversely affect the animal鈥檚 health.

So far, Henderson and others have only identified bacteriokines that trigger
the release of inflammatory cytokines from human cells in a test tube. The human
bacteria that produce those bacteriokines cause no problem under natural
conditions, suggesting, says Henderson, that the bacteria are also sending out
signals that keep the immune response in check.

In Germany, researchers have other evidence for crosstalk between microbe and
host. Genetically altered mice that are unable to produce the cytokine
interleukin-2 suffer a violent immune response that leads to an often fatal
intestinal inflammation. Similar mice reared in a germ-free environment are
healthy, suggesting that interleukin-2 somehow helps bacteria suppress the mouse
immune response.