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REVIEWS

Microbiota restoration: natural and


supplemented recovery of human
microbial communities
Gregor Reid*‡, Jessica A. Younes§, Henny C. Van der Mei§, Gregory B. Gloor||,
Rob Knight¶# and Henk J. Busscher§
Abstract | In a healthy host, a balance exists between members of the microbiota, such that
potential pathogenic and non-pathogenic organisms can be found in apparent harmony.
During infection, this balance can become disturbed, leading to often dramatic changes in
the composition of the microbiota. For most bacterial infections, nonspecific antibiotics are
used, killing the non-pathogenic members of the microbiota as well as the pathogens and
leading to a substantial delay in the restoration of a healthy microbiota. However, in some
cases, infections can self-resolve without the intervention of antibiotics. In this Review, we
explore the mechanisms underlying microbiota restoration following insult (antibiotic or
otherwise) to the skin, oral cavity, and gastrointestinal and urogenital tracts, highlighting
recovery by natural processes and after probiotic administration.

*Human Microbiology and Humans live in symbiosis with a diverse community of communities that were more similar to the general skin
Probiotics, F2‑116, Lawson microorganisms, the composition of which has evolved surface communities of the mothers and were domi-
Health Research Institute,
268 Grosvenor Street, London,
to carry out many specific tasks that benefit the host, as nated by Staphylococcus spp., Corynebacterium spp., and
Ontario N6A 4V2, Canada. well as to survive and thrive in sites that provide these Propionibacterium spp. (FIG. 1). Furthermore, the compo-

Departments of Microbiology microorganisms with a suitable nutrient-filled habitat. sition of the microbiota can differ between infants that
& Immunology, and Surgery, These symbionts can be mutualists (benefiting them- are fed with either breast milk or formula2. However, it
The University of Western
selves and the host), commensals (benefiting themselves seems that after weaning there is a degree of matura-
Ontario.
§
Department of BioMedical but not the host) or pathogens (benefiting themselves by tion and the gross differences between the two groups
Engineering, University harming the host). The goal of identifying specific in terms of the microbial populations of their faecal
Medical Center Groningen roles for these 100 trillion symbionts, the gene pool of microbiota disappear 3. Indeed, a study of the gut micro-
and University of Groningen, which far exceeds that of their host, is currently elusive, biota of twins has identified a ‘core microbiome’ with a
Antonius Deusinglaan 1,
9713 AV Groningen,
but the composition of this microbiota is now being wide array of microbial genes that are shared between
The Netherlands. deciphered. individuals, although with substantial diversity in bacte-
||
Department of Biochemistry, As far as can be determined, the body is sterile before rial lineages4. This functional homogeneity in the infant
The University of Western birth. However, during and following birth the body microbiota is surprising, given the differences in infants’
Ontario, London, Ontario
becomes colonized by a vast array of microorganisms primary colonizing microorganisms, nutrient intake and
N6A 3K7, Canada.

Department of Chemistry that originate from the mother’s birth canal, from the macro-environments.
and Biochemistry, University living environment and from handling by other individ- The extent to which such differences in the infant
of Colorado at Boulder, uals. A recent study of babies delivered vaginally and by microbiota influence health later in life is also of great
Boulder, Colorado 80309,
Caesarean section showed that, despite the highly differ- interest. For instance, it is known that bacterial strains
USA.
#
Howard Hughes Medical
entiated maternal communities, the neonates’ microbi- can prime the immune system and alter susceptibility
Institute, Boulder, ota were undifferentiated across multiple body habitats, to respiratory ailments and metabolic syndromes5–7,
Colorado 80309, USA. regardless of delivery mode1. Vaginally delivered babies and that strains embedded in the microbiota, such as
Correspondence to G.R. acquired bacterial communities resembling their own Clostridium spp., uropathogenic Escherichia coli or per-
e‑mail: gregor@uwo.ca
doi:10.1038/nrmicro2473
mother’s vaginal microbiota, dominated by Lactobacillus haps sulphate-reducing bacteria, might later be asso-
Published online spp., Prevotella spp. or Sneathia spp., whereas infants ciated with disease8–10. The crucial part played by the
29 November 2010 delivered by Caesarean section harboured bacterial microbiota in human health has only recently become

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Vaginal delivery C-section delivery Vaginal delivery C-section delivery

Acinetobacter Lactobacillus Propionibacterineae


Bacillales Micrococcineae Sneathia
Coriobacterineae Neisseria Staphylococcus
Corynebacterineae Pasteurellaceae Streptococcus
Haemophilus Prevotella Other

Figure 1 | Taxonomic distribution of microorganisms in mother and baby. Immediately after birth, humans become
Nature Reviews
colonized by a range of microorganisms, particularly on the skin, oronasopharyngeal area, gastrointestinal | Microbiology
tract and
urogenital tract. The host and its microbiome have developed mechanisms to not only protect against infection, but also
restore microbial homeostasis, even without pharmaceutical therapy. The genus-level distributions are shown for a sample
of mothers and their babies, grouped according to the delivery method of the babies (vaginal or by Caesarian section
(C-section)). The data shown are averaged for each group. Data from REF. 1.

widely recognized, and this realization has come about members of the Actinobacteria (Corynebacterium spp.,
mainly because of studies that have led to, and stemmed Propionibacterium spp., Microbacterium spp. and
from, the Human microbiome Project 11 (BOX 1). Micrococcus spp.), Firmicutes (Staphylococcus spp.
and Clostridium spp.), Proteobacteria (Pseudomonas
The human microbiota spp., Janthinobacterium spp., Serratia spp., Halomonas spp.,
The most comprehensive analysis of the human Stenotrophomonas spp., Delftia spp. and Comamonas
microbiota to date looked at 27 distinct sites on the spp.) and Bacteriodetes (Sphingobacterium spp. and
body and revealed the presence of 22 bacterial phyla, Chryseobacterium spp.)13,14 — the distributions are col-
with most sequences (92.3%) related to just four lectively much more similar than those found in the gut
phyla: Actinobacteria (36.6%), Firmicutes (34.3%), or vagina. In fact, the variations in microbial-community
Proteobacteria (11.9%) and Bacteroidetes (9.5%) 12. constituents between body sites are much greater than
The sites included the oral cavity, gut and skin (three of those between different free-living communities (for
Microbiota the four areas discussed in this review). each habitat example, in soil and water)12. Indeed, bacterial dis-
The microorganisms that harboured a characteristic microbiota and a set of abun- tributions differ between the tongue and faeces far
inhabit a region of the body. dant taxa that were stable between individuals and over more than between faecal samples from different indi-
time (FIG. 1), although the rare taxa varied immensely. viduals15, and samples from the same individual taken
Sebaceous
Pertaining to sebum, the
Despite the differences in the abundance of phyla three months apart, although dissimilar, resemble each
substance secreted by glands between sites — such as sebaceous, moist and dry skin other more than they resemble samples from different
of the skin. sites which are colonized by differing abundances of individuals.

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Box 1 | The Human Microbiome Project niche to another that causes problems for the host. For
example, uropathogenic E. coli strains emerge from but
The goals of the Human Microbiome Project (HMP) are: to take advantage of new, do not infect the gut en route to infecting the bladder.
high-throughput technologies to characterize the human microbiome more fully by The switch from homeostasis to a disease state can occur
studying samples from at least 300 individuals with no known health problems
through various means, including physical insult to the
(sampled at 15–18 body sites over two to three visits); to determine whether there are
mucosa, disruption of the indigenous microbiota by
associations between changes in the microbiome and health or disease by studying
several different medical conditions; and to provide a standardized data resource and the use of antimicrobials, expression of specific virulence
new technological approaches to enable such studies to be undertaken broadly in the factors and access to the site by excessive numbers of
scientific community113. pathogens.
With 24% (375) of projects complete, over 1,500 bacterial species have been
identified and 178 genomes have been sequenced from more than 285 individuals. Microorganisms at wound sites. The main func-
Almost half of the organisms identified are members of the Firmicutes (46%), followed tions of the skin are to provide mechanical strength,
by Actinobacteria (20%), Proteobacteria (16%) and Bacteroidetes (12%). More than regulate water and salt loss and protect the body from
29,693 unique proteins have been identified, and the functions of many of these environmental damage, including that caused by
proteins await characterization.
microorganisms. The outermost layer of the skin, the
stratum corneum, consists of corneocytes surrounded
by lipid regions containing long-chain ceramides, free
studies of the vaginal microbiota show a different fatty acids and cholesterol25. Interspersed among the
Dysbiosis pattern; at this site the bacterial type16 and abundance17 keratinocytes in the epithelium are specialized antigen-
An imbalance in the bacterial in the microbial community can change rapidly in a presenting langerhans cells (of the dendritic cell family),
population at a naturally matter of weeks and sometimes days. In addition, the which serve as sentinels of the immune system by sam-
occurring site of colonization, vaginal microbiota differs between individuals, even pling antigens that pass through the stratum corneum.
often resulting in health
problems.
those who are reportedly healthy 18. In ecological terms, These langerhans cells then migrate to draining lymph
a more diverse microbiota with adequate nutrients nodes and relay antigenic information to the adaptive
Eubiosis would be expected to equate to a more stable ecosys- immune system26. Diseased skin is often character-
The state of having a healthy tem19, although relationships between diversity and sta- ized by a reduced barrier function and an altered lipid
population of bacteria
bility have long been debated in the ecological literature. composition and organization25. Although some sta-
naturally occurring in a body
site. The vagina provides a marked contrast: here, increased phylococci, such as Staphylococcus epidermidis, are con-
diversity, especially decreased dominance of lactobacilli, sistently found on the skin surface, other species, such
Probiotic equates with an aberrant microbiota and infection17. as Staphylococcus aureus, may colonize intact skin only
A live microorganism that, transiently. However, when the skin surface is penetrated,
when administered in
adequate amounts, confer a
Natural and artificially aided restoration both species can colonize the damaged area to exploit
health benefit on the host. The past 60 years have seen the introduction of anti- the nutrient-rich bloodstream27. staphylococci cause a
biotics to treat bacterial infections. However, both before range of cutaneous and systemic infections, including
Homeostasis and during this era, countless individuals have recovered impetigo, furuncles, subcutaneous abscesses, staphylococ-
A condition of equilibrium
naturally from dysbiosis and infection through mecha- cal scalded skin syndrome, toxic shock syndrome and neo-
or stability in the internal
environment of the body, nisms that are not yet fully understood. with dwindling natal toxic shock syndrome-like exanthematous disease.
which is maintained by antibiotic options and growing bacterial resistance, it is The virulence factors produced by S. aureus can disrupt
adjusting physiological timely to examine the mechanisms of natural restoration the epithelial barrier, inhibit opsonization by antibody
processes to counteract of eubiosis, with a view to developing new methods of and complement, interfere with neutrophil chemotaxis,
external changes.
preventing and curing disease. Furthermore, the recog- cytolyse neutrophils and inactivate antimicrobial pep-
Corneocyte nition that antibiotics also eradicate non-pathogens and tides28. In a recent study of microbial shifts with skin
A dead keratin-filled squamous disrupt the natural microbiota for some time after their wounding in a diabetic mouse, there was a selective
cell of the stratum corneum. administration20,21 has been one reason for the growing shift noted in the colonizing bacteria after wounding,
interest in administering beneficial microorganisms accompanied by transcriptional changes indicative of
Keratinocyte
An epithelial cell that produces (probiotics) to aid in recovery from infection22–24. In this mobilized defence and immune responses29. This shift
keratin in the process of review, we examine how indigenous microorganisms was towards an increased abundance of Firmicutes spp.
differentiating into the and probiotics can help the host overcome infection and (including Staphylococcus spp.) and occurred alongside
corneocytes of the stratum restore the microbiota to a state associated with health. a prolonged immune response.
corneum.
Four body sites — the skin, oronasopharyngeal cavity, skin can be damaged by burns owing to fire, heat,
Impetigo gastrointestinal tract and vaginal tract — are discussed cold, electricity, radiation or caustic chemicals. The
A contagious bacterial skin (FIG. 2 ), as the complex microbial communities that extent of the burn is measured by the depth to which the
infection characterized by the occupy these sites carry out a broad range of func- skin layers are damaged. Partial-thickness burns (first-
eruption of superficial pustules
tions that are indispensable for human health, while and second-degree burns) usually heal well because new
and the formation of thick
yellow crusts. being constantly challenged by potentially pathogenic skin can grow upward from the dermis. However, when
organisms. the dermis is destroyed (a full thickness or third-degree
Furuncle burn), skin growth is inhibited and deep scarring devel-
A localized suppurative Perturbation of the human microbiota ops. In the case of heat causing partial-thickness dam-
staphylococcal skin infection
originating in a gland or hair
The ability of microorganisms to selectively colonize a age, some of the microorganisms on the skin are likely
follicle and characterized by niche reflects their evolutionary adaptation. In many to survive and, during handling of the patient, other
pain, redness and swelling. instances, it is the migration of an organism from one microorganisms will also contaminate the site, providing

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Gastrointestinal tract non-pathogenic microorganisms in this process, further


• Dietary sources studies are warranted to examine sequential changes in
• Environmental sources the skin microbiota after insult and during healing.

Urogenital tract Recovery following antibiotic therapy. In the vagina the


• Migration from the skin and
lower gastrointestinal tract microbial profile can change quite rapidly and exten-
• Vaginal birth canal sively from one in which members of the Firmicutes,
• Environmental sources especially lactobacilli, dominate to one with a high abun-
dance of Bacteroidetes and Actinobacteria members and
an aberrant condition termed bacterial vaginosis (BV)
(FIG. 3). BV occurs in an estimated one-third of women
Oronasopharyngeal cavity at any given time, with rates being over 70% in sex work-
• Dietary sources ers and HIV-infected individuals34–36. In about one-third
• Migration from the skin
• Environmental sources of cases, BV is accompanied by vaginal discomfort and
homogeneous, malodorous vaginal discharge. It is diag-
nosed most commonly by one of two methods. The
Amsel criteria requires three out of the following four
Skin parameters: a homogeneous, milky discharge; a vaginal
• Vaginal birth canal pH of greater than 4.5; the presence of ‘clue cells’ on
• Environmental sources
microscopic examination of a vaginal smear; and a posi-
Figure 2 | Known sites of natural microbiota restoration. The main areas discussed in tive amine or ‘whiff ’ test 37. The nugent scoring system
this Review that are colonized by bacteria (from the sourcesNature Reviews
indicated) Microbiology
and |for which involves performing a Gram stain on a vaginal smear
there is some evidence for natural restoration after pathogen insult. and enumerating lactobacilli versus Gram-negative
rods and other bacterial morphotypes38. Irrespective of
the symptoms present, BV has been associated with an
the bacteria with access to the blood. Burn infections increased susceptibility to preterm labour (perhaps due
are common, with S. aureus being the most common to inflammatory processes)39, sexually transmitted infec-
pathogen, followed by E. coli, Pseudomonas aeruginosa, tions (perhaps owing to a damaged epithelial layer or
coagulase-negative staphylococci and a range of aerobic altered expression of protective compounds like elafin)40,
and anaerobic pathogens30,31. During the initial 48 hours including HIV infection41, and pelvic inflammatory
following injury, Gram-positive bacteria heavily colo- disease caused by pathogens42.
nize the burn wound. By 5–7 days, a shift occurs, with when treated with the nitroimidazole antibiotic
Gram-negative opportunists, armed with invasive and metronidazole, the composition of the microbiota
virulent properties, superseding the initial colonizers32. recovers, becoming close to (but not necessarily exactly
Antibiotics are often administered locally with a view to the same as) the healthy, pre-infection state, even up to
reducing the pathogen count and preventing infection. 25 weeks later (FIG. 3). notably, the lactobacilli abundance
The ability of the non-pathogenic indigenous micro- increases with recovery, coinciding with a decrease in
biota to outcompete the pathogens and restore bacterial members of the phyla Proteobacteria, Bacteroidetes and
Toxic shock syndrome and immune homeostasis (defined in this case as main- Actinobacteria, perhaps because lactobacilli are resistant
A rare, life-threatening taining the stability that is associated with health of the to metronidazole.
complication of bacterial
infection that has been most
host) to burn wound sites has not been studied, to our In terms of organisms and host site, it is intriguing that
often associated with the use knowledge. But, given the competitive nature of indig- Prevotella spp. can persist in the mouth in some patients
of superabsorbent tampons enous organisms and the availability of progeny that sur- without any major pathological adversity, whereas in oth-
and occasionally linked with vive at the burn site and that are present on adjacent skin, ers they cause destructive periodontitis, but in the vagina
the use of contraceptive
it is reasonable to suggest that the indigenous microbiota they become part of an aberrant microbiota that causes
sponges. Often resulting
from toxins produced by may help prevent infection in many instances, assuming BV43. such a change in mucosal site exposes Prevotella
staphylococci, but also from that immune defences are intact. spp. to a new community of microorganisms, some of
those produced by group A The process of wound healing necessitates repair which may activate dormant virulence factors or provide
Streptococcus spp. of the skin barrier and revascularization, as well as the a substrate for the production of amines that are respon-
Biofilm
return of normal immune function. It is not uncom- sible for malodour 44. Although Prevotella spp. coloniza-
A microbial community that mon for wounds to become persistently infected with tion and an increase in bacterial diversity are key features
is enveloped by extracellular Corynebacterium spp., Peptoniphilus spp., Staphylococcus of BV, the exact trigger (or triggers) for the dysbiotic
polymer matrices produced by spp., Serratia marcescens, Prevotella spp., P. aeruginosa state has yet to be identified. However, when Prevotella
the microorganisms and that
and members of the order Bacteroidales31. The per- spp. drop in abundance or disappear altogether and
adheres to a surface.
sistence of these infections can have several causes, Lactobacillus crispatus or Lactobacillus iners increases,
Bacterial vaginosis including the ability of the organisms to form biofilms33. the host no longer has BV17. In some women this proc-
An aberrant condition in the However, in most otherwise healthy people, skin repair ess can occur naturally, without treatment. Alternatively,
vagina that is characterized by mechanisms allow the reparation to be completed, probiotic lactobacilli can be administered to aid the res-
pH >4.5, malodour and
depletion of normal
and the indigenous skin microbiota are subsequently toration process. The cure rate for antibiotic treatment
microbiota, particularly restored to the surface. To better understand the natu- of BV in otherwise healthy women would be expected to
lactobacilli. ral restoration process and determine the role, if any, of be higher than that for HIV-positive women, but it still

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Order Class Phylum phylum level abundance of microorganisms in the gut51 —


for example, by microbiome transfer 52 — demonstrates
the power of microorganisms to influence health and
disease. In addition, the fact that different microbiota
compositions can be associated with a healthy state17 sug-
gests that microbiome functionality, rather than precise
species abundance, may be key to maintaining health. By
understanding how indigenous and probiotic organisms
respond to different conditions in vivo, it should become
possible to understand the mechanisms by which
pathogens are displaced and a return to homeostasis
occurs.

Mechanisms of restoration
restoration of a healthy microbiota is driven by multi-
ple species and requires substantial reorganization after
N BV R N BV R N BV R insult. There are numerous mechanisms by which the
microbiota can return to normal, as depicted in FIG. 4.
rem rem rem
Fusobacteriales Fusobacteria Fusobacteria Co-aggregation. The phenomenon of co-aggregation
Lactobacillales Bacilli Firmicutes is the assembly of microbial communities into distinct,
Clostridiales Clostridia Bacteroidetes interlinked structures53. Following disruption of the oral
Bacteroidales Bacteroidia Actinobacteria microbiota by antibiotic administration or mechani-
Bifidobacteriales Actinobacteria cal cleaning, the reorganization of a non-pathogenic
Coriobacteriales biofilm with specific subsets of species derived from
the planktonic phase is a key component in the return
Figure 3 | changes in the vaginal microbiome before and after bacterial vaginosis. to homeostasis. A problem arises if the antibiotic has
Vaginal samples from HIV-positive individuals in Mwanza, Tanzania, were processed by
Nature Reviews | Microbiology
depleted the levels of non-pathogenic species to the
Illumina16. One group (n = 60) was regarded as normal (N), or healthy, at day 0 of recruitment, extent that recovery of their normal levels is delayed and
with a Nugent score of ≤3 at screening. A second group had bacterial vaginosis (BV) so the new biofilm that forms is essentially made up of
on recruitment, with a Nugent score of ≥7. Individuals with BV were then treated pathogenic species.
with metronidazole, and those who subsequently returned to health at any time in In the vagina, L. iners survives antibiotic therapy for
the 25 weeks following treatment were designated as restored (R). The plots show the
BV, and it is possible that this and other lactobacilli aggre-
proportion of the microbiota in each clade at the level of order, class and phylum; the
remainder (rem) groups contain all groups of organisms that made up less than 0.5% of
gate with BV constituents as a key step to the restoration
the total in any of the three patient groups. Data from REF. 17. of homeostasis. If lactobacilli are depleted and planktonic
drug-resistant pathogens are in the vicinity, a recurrence
of BV is more likely. A genomic study of L. iners has
varies from 27% to 63%, depending on the cohort and identified potential surface adhesins that could bind other
regimen45,46. By contrast, intra-vaginal use of probiotic bacteria54, but differential binding of non-pathogens
lactobacilli, or oral antibiotic plus oral lactobacilli, gives versus pathogens has not been investigated.
a cure rate of around 88%22,47. Interestingly, in a study Pathogens that inhibit interleukin-8 (Il-8), disrupt
following individuals for two months, BV self-resolved epithelial cell homeostasis or block access of other spe-
partially or completely in 31% of individuals48. cies to Toll-like receptors (Tlrs) can more easily infect
the host 55. Potentially, non-pathogens could compete
Recovery of the intestinal microbiota. Another exam- with pathogens and enhance host defences, or the patho-
ple associated with a shift towards an increased diver- genic biofilm could reach a state in which pH levels or
sity in the microbiota comes from coeliac disease, an certain metabolites stress the cloistered structure and
autoimmune disorder of the small intestine in which lead to its disintegration or detachment 56. The admin-
dietary gluten ingestion leads to chronic enteropathy. istration of probiotic lactobacilli may simply accelerate
In a study of the dominant duodenal microbiota of these pH and metabolic changes57.
children with coeliac disease, differences were noted
not between individuals but between active and remis- Biosurfactant production. There is evidence from two
sion states, with Bacteroides vulgatus and E. coli being body sites that biosurfactants — surface-active com-
Planktonic
detected more often in patients than in controls49 and pounds that are synthesized by microorganisms — may
Referring to microscopic
organisms that are suspended a potential decrease in the abundance of B. vulgatus have a role in the restoration and maintenance of micro-
in a liquid (for instance, media and E. coli seen in individuals in remission. Another bial homeostasis. In the oral cavity, which is home to an
or a buffer). study suggested that a return to health coincides with a unexpected microbial diversity 58,59, regular tooth brush-
change from a microbiota dominated by Lactobacillus ing and use of antimicrobial mouth rinses disrupt the
Adhesin
An external bacterial product
curvatus, Leuconostoc mesenteroides and Leuconostoc microbiome, whereas dietary components, particularly
that enables adhesion to and carnosum to one dominated by Lactobacillus casei and sugars, increase bacterial growth. Despite these insults,
colonization of a host. Bifidobacterium adolescentis 50. The ability to alter the the oral cavity in most individuals maintains microbial

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a Co-aggregation b Biosurfactant production c Bacteriocin and d Signalling effects


H2O2 production

H2O2

H2O2
Bacteriocins

e Competitive exclusion f Immunomodulation g Modulation of tight junctions

Alkaline
phosphatase

Defensins and Lipopolysaccharide


lysozyme

NF-κB-dependent Macrophage
Lactobacilli Gram negative pathogens
antimicrobial genes
Non-pathogenic cocci Gram positive pathogens
Bifidobacteria

Figure 4 | Possible mechanisms contributing to restoration of the microbiota. a | Co-aggregation of non-pathogens


Nature Reviews | Microbiology
and pathogens interferes with the ability of the pathogenic species to infect the host. b | Biosurfactants produced by
lactobacilli help prevent the adhesion of pathogens to mucosal surfaces. c | In addition to host immunoglobulin defences,
it has been suggested that bacteriocins and hydrogen peroxide produced by lactobacilli can inhibit or kill pathogens.
d | Signalling between bacteria can lead to downregulation of toxin production in pathogens. e | By competing for
nutrients and surface receptors, non-pathogenic bacteria can competitively exclude pathogens from host surfaces.
f | The regulation of immune responses by the microbiota can result in: the production of host factors such as antimicrobial
peptides (for example, defensins), lactoferrin and lysozyme, which can kill pathogens; the production of alkaline
phosphatases, which bind to lipopolysaccharide and negate its toxicity; and the deregulation of nuclear factor-κB
(NF-κB) signalling in host epithelia (whether this is influenced by lactobacilli, as indicated, remains to be confirmed).
g | Upregulation of tight junction proteins might help to limit the damage that is caused to epithelia by inflammatory
processes or pathogens. The damage disrupts the cell linkages and induces a host response, shown here as a macrophage,
which further induces local damage. The lactobacilli-coated surface retains an intact junction.

stability for long periods of time. one reason for this alteration in surface tension repels hydrophobic patho-
seems to be the presence of indigenous non-pathogens gens65. The biosurfactants therefore provide a means
such as Streptococcus mitis, which produces biosurfactant of restoring the lactobacilli-dominated microbiota and
molecules that can substantially reduce the presence displacing the pathogenic species.
of pathogenic species such as Streptococcus mutans 60.
when adsorbed onto surfaces, these rhamnolipid-rich Bacteriocin and hydrogen peroxide production.
compounds can prevent S. mutans adhesion61 through Following contact with pathogens, the ability to produce
weakening the adhesion forces between the pathogen substances that kill or inhibit their growth could be an
and the enamel, but other, early colonizing members of important factor in restoring homeostasis. Bacteriocins
the oral microbiota are not blocked, suggesting selective are molecules that are synthesized by ribosomes and
activity against cariogenic strains. have a narrow killing spectrum; their modes of action
Vaginal lactobacilli produce biosurfactants that are include interfering with cell wall structure and bio-
made up of a mixture of proteins, lipids and carbohy- synthesis, forming pores in the target bacterial mem-
Rhamnolipid drates and that help to displace dense mixed cultures brane and permeabilizing membranes66. However, the
A biosurfactant produced by of uropathogenic E. coli, Enterococcus faecalis and organisms that produce bacteriocins are not simply
Pseudomonas spp. Gardnerella vaginalis 62–64. These effects occur even indiscriminate killing machines. rather, they probably
Cariogenic
with only a few lactobacilli, suggesting that the secreted sense the bacterial dynamics of their niche and pro-
Producing or promoting the low-molecular-mass biosurfactants spread out over duce bacteriocins to retain a competitive position. This
development of dental caries. the surface of the vaginal mucosa and the subsequent is illustrated by Lactobacillus acidophilus str. la-5, in

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which bacteriocin expression is controlled by an auto- one possible mechanism for these effects is that host
induction mechanism involving the secreted peptide and/or bacterial signalling factors induce a downregu-
IP-1800: bacteriocin production increases when the lation in the expression of virulence factors, forcing the
organism senses its target 67. The bacteriocin-producing pathogens to retreat deeper into the bladder epithelium76
strains make immunity proteins, such as the product of or back to the vagina. For uropathogenic E. coli, this
the associated abortive infection (abi) gene (skkI; locus process might involve a two-component response system
lsA0564_c) found in Lactobacillus sakei, to protect the composed of a histidine kinase activated by extracellular
organisms from their own bacteriocins, and this is cru- signals in the host environment and a response regula-
cial for the survival of the organism and retention of the tor that, in turn, modulates the expression of genes that
microbiome structure68. induce cell invasion77. whether clinical recovery from
several studies have strongly suggested that bacte- the infection is influenced by indigenous bacteria in the
riocins could have a role in restoring homeostasis. An vagina is debatable, but anti-virulence signalling mol-
S. mutans strain in which the lactate dehydrogenase ecules have been identified in some strains of lactobacilli
gene is deleted (making it entirely deficient in lactic acid that are known to be present in the vagina and intestine, as
production) produces the lantibiotic mutacin 1140, a well as in some probiotics. one example of such a strain is
peptide antibiotic, which gives the mutant a strong selec- Lactobacillus reuteri str. rC-14, which produces signalling
tive advantage over most other strains of S. mutans 69. molecules that inhibit the expression of toxic shock toxin
experiments in gnotobiotic rats have shown that this 1 in multiple strains of S. aureus and interferes with the
strain is genetically stable and less cariogenic than wild- P2 and P3 promoters of the staphylococcal global regula-
type S. mutans and that it possesses strong colonization tory system agr 78. Presumably, the ability to repress toxins
properties, such that a single application can result in in vivo could reduce host damage and inflammation (and,
its permanent colonization and displacement of indig- thus, symptomatology), but whether it would help restore
enous, disease-causing S. mutans strains. mutacin 1140 microbiota homeostasis remains to be seen.
was shown to remove lipid II from the target’s septum
and to block cell wall synthesis. Competitive exclusion. In order for indigenous bacteria to
In arguably the most elegant in vivo verification to compete with transient pathogens, the commensals must
date of how a bacteriocin functions in the gut, Corr have attributes that strengthen their ability to colonize the
et al.70 showed that the bacteriocin Abp118 produced host. For instance, Bacteroides fragilis produces multiple
by Lactobacillus salivarius str. uCC118 was essential for capsular polysaccharides that are essential for coloni-
protecting the host against infection by invasive Listeria zation of the gut 79. These polysaccharides not only aid
monocytogenes. This does not prove that the bacteriocin in persistence but also function in immune regulation,
restores homeostasis, but it does show one mechanism by helping to exclude pathogens and restore homeostasis.
which pathogenesis can be interfered with in the gut. Arguably, the most extreme assessment of competi-
Hydrogen peroxide (H2o2), a powerful oxidizing agent tive exclusion of pathogens comes from whole-stool
produced by lactobacilli, can kill pathogens in the vagina implantation to treat individuals with chronic gut
through the production of free radicals71. lactobacilli infections. In this process, a sample of homogenized
seem to protect themselves from toxic accumulations of faeces from a healthy relative or friend is instilled
H2o2 through the production of Fe3+-activated extracel- through a nasogastric tube into the recipient’s stom-
lular peroxidase72. H2o2 induces nuclear translocation ach, through enema infusion or through nasoduodenal
of peroxisome proliferator-activated receptor-γ and tubes80,81. For a cohort of 159 individuals, the overall
enhances transcriptional activity in gut epithelial cells, reported success rate for restoring normal homeostasis
directly modulating epithelial cell responsiveness to to the gut microbiota was 91%82. In one patient who
inflammatory stimuli73. The use of H2o2 as an antibac- was infused with her husband’s microbiota through a
terial agent and host signal transduction molecule makes colonoscope, there was a rapid and prolonged change
its contribution to homeostasis twofold. in the bacterial composition of her gut microbiota, with
Bacteroides spp. coming to dominate, Clostridium dif­
Signalling effects. In the 1960s, several studies on uri- ficile disappearing and defecation frequency returning
nary tract infections (uTIs) showed that the infections to normal83. This was a particularly interesting case, as
resolved without treatment in a proportion of women. it showed that colonization with exogenous bacteria is
more recently, in a study of 1,143 women in northern possible, an end result that has so far eluded probiot-
sweden who had symptoms suggestive of a uTI, 28% ics. The implantation seems to re-establish resistance to
of the 288 individuals treated with a placebo for 7 days colonization, whereby the dense population of infused
showed a spontaneous curing of symptoms after the microorganisms blocks the pathogens from causing a
first week, and 37% were cured after 5–7 weeks74. After relapse of enteric disease.
evaluating dropouts, the overall spontaneous cure rate This treatment seems to competitively exclude C. dif­
of symptoms and bacteriuria was calculated as 24%. In ficile and prevent potentially fatal symptomatic relapse.
another study of 50 women, the median time for spon- The recipients had received extensive antibiotic treat-
Free radical taneous remission was reported to be 4 weeks with pla- ments during their C. difficile management programme
An atom or molecule that has
at least one unpaired electron
cebo treatment, which is substantially less time than the and just before faecal transplant, so much of their indig-
and is therefore unstable and 7 months for remission of those who required concurrent enous microbiota would have been eradicated. In the
highly reactive. antibiotics75. future, it may be possible to create a healthy microbiota

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REVIEWS

in a chemostat or to generate other complex multistage For example, the capacity to form biofilms, to use capsules,
continuous cultures that simulate the large bowel, and to to produce adhesins and toxins, and to induce inflam-
use these as inocula rather than human stool. mation are regarded as pathogenic properties, but these
characteristics can apply to many non-pathogenic micro-
Immunomodulation. Antimicrobial factors such as organisms depending on the circumstances. Indeed, part
defensins, lysozyme and haemocidins can have a role of the reason that E. coli str. nissle 1917 is considered to
in the restoration of microbial equilibrium. The host’s be a probiotic and useful for treating some inflamma-
immune system must properly calibrate the responses to tory bowel disease cases is the fact that its capsule plays
pathogens and must differentiate indigenous and exog- a part in chemokine induction91. As Blaser and Falkow
enous non-pathogenic organisms84. It seems to do this argue, there is a need to increase our focus on the rules
through pattern recognition receptors, which mediate that govern the evolution of cooperation between
the detection of bacterial antigens and activate signal- mammalian and microbial cells in the human body90.
ling cascades that regulate the immune response. The In terms of ecology, equilibrium requires boundaries
homeobox-containing protein Caudal has been shown and penalties for transgressors, and the maintenance
to regulate the interaction between Drosophila melano­ of homeostasis requires inputs from multiple sources,
gaster and its indigenous microbiota by repressing including the indigenous microbiota. when in a state
nuclear factor-κB (nF-κB)-dependent genes encoding that is defined clinically as being healthy or devoid of
antimicrobial peptides, thereby maintaining homeosta- disease, the homeostatic equilibrium must permit the
sis85. These findings illustrate the fact that immune regu- co-evolution of competing organisms with sufficient
lation, which is to a large extent instigated or controlled elasticity to cope with invaders. studies are needed not
by microorganisms, can target pathogens and thus help only to understand these phenomena, but also to inves-
indigenous organisms to re-emerge. tigate aberrant conditions in which the equilibrium is
The microbiota can have a role in intestinal inflam- not functioning, has never been attained or is unable to
matory autoimmunity, but it has been suggested that be recalibrated.
disruption of the dialogue between the microorganisms
and the host can also lead to aberrations such as allergy, Modulation of tight junctions. The integrity of the epi-
rheumatoid arthritis and type I diabetes86. It is thought thelial lining in the mouth, gut and vagina is crucial
that disruption of the intestinal and nasopharyngeal to maintaining health. when this lining is disrupted
microbiota allows certain organisms to induce autoim- or breached, the microorganisms on the outer surface
munity and the onset of diseases through Tlr-mediated gain access to the tissue and bloodstream and induce
signalling. In terms of a protective role, polysaccharides disease. For example, HIV can decrease trans-epithelial
from the prominent human symbiont B. fragilis pro- resistance through disruption of tight junction proteins
tected animals from experimental colitis induced by (claudin 1 (ClDn1), ClDn2, ClDn4, the transmem-
Helicobacter hepaticus, a commensal bacterium that can brane protein occludin and the scaffold protein zonula
sometimes be pathogenic. The mechanism for this pro- occludens 1 (Zo1)) and thereby allow viral entry into
tection seems to involve suppressing pro-inflammatory the host 92. lactobacilli could potentially counter this
Il-17 production by intestinal immune cells and pro- effect, as they upregulate Zo1 and occludin93,94, as well
tecting the host from inflammatory disease by triggering as elafin (G.r., unpublished observations), a molecule
Il-10-producing CD4+ T cells87 that inhibit inflammation. associated with HIV resistance41. However, there is no
short-chain fatty acids produced by intestinal symbionts evidence to date that the sparse coverage of vaginal cells
also seem to be able to counter immune aberrations, as by indigenous lactobacilli or the presence of specific
illustrated by their binding to G protein-coupled recep- bacterial species can protect against HIV, and it remains
tor 43 (GPr43; also known as FFAr2) in models of to be determined whether probiotic application could
colitis, arthritis and asthma88. In relation to restoration confer any level of protection.
after skin injury, staphylococcal lipoteichoic acid can act In the intestine, various sensory pathways can influ-
selectively on keratinocytes through Tlr3, which inhibits ence homeostasis and recovery from, or prevention of,
the release of inflammatory cytokines from keratinocytes, inflammatory processes95. For example, Paneth cells,
and can also inhibit inflammation triggered through a the function of which can be regulated by the unfolded-
Tlr2-dependent mechanism after injury 89. protein response and by autophagy, have a crucial role
These examples illustrate some of the molecular in limiting barrier breaches of the epithelial lining: they
mechanisms used by mutualistic bacteria to help pre- can sense the microbiota and produce antimicrobial
vent disease and restore equilibrium. Intriguingly, the molecules through cell-autonomous myeloid differentia-
effects can be conferred by species that, under other tion primary response protein 88 (myD88)-dependent
conditions, may be pathogenic, such as B. fragilis. The activation of Tlrs95.
interface between the microbiota and the host’s immune
system is changing with dietary modifications and the Probiotics to aid restoration of the microbiota
widespread use of antimicrobials90. likewise, our cat- one rationale for the use of probiotics is to help restore
egorization of pathogens versus non-pathogens needs and maintain homeostasis. Although the commercial
to be carefully considered, as some so-called pathogenic field of probiotics is sadly still lined with too many
or virulence factors are used by mutualistic and perhaps undocumented products making unproven claims, the
even probiotic organisms to convey benefits to the host. pending implementation by regulatory agencies of some

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Table 1 | Probiotics that aid in preventing infection and hastening recovery


Probiotic Administration outcome and apparent mode of action ref.
Streptococcus A pilot study of three subjects: a 3-day regimen of The subjects’ levels of volatile sulphur compounds 1 week 114
salivarius str. K12 chlorhexidine mouth rinsing, followed by the use after treatment showed an 85% reduction in the S. salivarius
of lozenges containing either S. salivarius K12 or a str. K12 group and a 30% reduction in the placebo group;
placebo likely reasons are the displacement and killing of bacteria
that are implicated in halitosis
Bifidobacterium A double-blind, randomized crossover study on There was a statistically significant reduction of salivary 115
animalis 24 healthy adolescents: during periods 2 and 4 Streptococcus mutans after probiotic yogurt consumption
str. DN 173 010 (of 2 weeks each), the subjects ingested 200 g of (P < 0.05); the likely mode of action was competitive
probiotic yogurt or pasteurized control yogurt once exclusion
daily, and periods 1 and 3 were run-in and wash-out
periods of 1 and 6 weeks, respectively.
Lactobacillus 256 women were randomized at their first trimester Probiotic intervention reduced the frequency of gestational 116
rhamnosus str. GG of pregnancy, and an intervention group received diabetes to 13% compared with 36% in those receiving
and B. animalis subsp. intensive dietary counselling plus daily probiotic or the placebo and 34% in controls (P = 0.003); proposed
lactis str. Bb12 placebo capsules mechanisms involve improved immunomodulation and
energy harvest in the maternal gut
L. rhamnosus str. GG 6-month-old infants were fed daily supplements of The probiotic promoted phylogenetically clustered taxa 117
probiotic or placebo in a double-blind, randomized characteristic of communities that are more resistant to
trial perturbation and outgrowth of pathogens; it was suggested
that the promotion of a stable, even and functionally
redundant infant gut microbiome is a key mechanism for
preventing the development of allergic disease
Bifidobacterium A randomized, double-blind trial of 31 subjects Only L. johnsonii str. La1 was recovered, and its presence 118
longum str. BB536 undergoing elective colorectal resection for cancer: correlated with reduced intestinal pathogen load and a
and Lactobacillus patients received either probiotics or a placebo modulation of local immunity in favour of homeostasis
johnsonii str. La1 orally (two doses per day for the 3 days before the
operation and then post-operatively from day 2 to
day 4)
Bifidobacterium In a randomized, double-blind, placebo-controlled The cumulative incidence of eczema in the infants during 107
bifidum str. BGN4, trial, 112 pregnant women with a family history of the first 12 months was reduced significantly in infants
B. animalis subsp. allergic diseases received a once-daily probiotic whose mothers were given the probiotic (36.4% versus
lactis str. AD011 supplement or placebo, starting at 4–8 weeks before 62.9%; P = 0.029); the concept is that the probiotics helped
and Lactobacillus delivery and continuing until 6 months after delivery; create a more eubiotic state in the mother and newborn,
acidophilus infants were exclusively breast-fed during the first resulting in immune modulation and a lower risk of eczema
str. AD031 3 months and subsequently fed with breast milk or
cow’s milk formula from 4 to 6 months of age
L. rhamnosus 40 women diagnosed with bacterial vaginosis (BV) Follow-up at days 6, 15 and 30 showed a cure of BV in 47
str. GR-1 and on the basis of symptoms of discharge, fishy odour, significantly more probiotic-treated subjects (16, 17 and 18
Lactobacillus reuteri a sialidase-positive test and Nugent Gram stain out of 20, respectively) than in metronidazole-treated subjects
str. RC‑14 scoring were randomized to receive either two dried (9, 9 and 11 out of 20, respectively; P = 0.016 at day 6,
capsules of probiotic intravaginally each night for P = 0.002 at day 15 and P = 0.056 at day 30); the mode of
5 days or 0.75% metronidazole gel applied vaginally action is believed to be displacement of the BV pathogens
twice a day (in the morning and evening) and restoration of the lactobacilli-dominated microbiota

or all of the guidelines published on what constitutes a several studies have shown that probiotic bifido-
probiotic96 will hopefully focus attention on those strains bacteria and lactobacilli may help prevent or reduce the
that have been proved to confer health benefits (exempli- formation of caries99. This is somewhat counter-intuitive,
fied in TABLE 1). not all probiotic applications have been as both groups can actually be involved in deep carious
successful. For example, attempts at using probiotics to lesions. However, when given as probiotics, these organ-
treat Crohn’s disease met with failure, although this may isms displace or reduce S. mutans in the saliva and, by
not have been unexpected, given that there is only sparse failing to colonize, they do not induce acidic damage to
evidence to indicate that microbial imbalance is the pri- the teeth100,101.
mary cause of this disorder 97. In other cases, such as the The recent interest in the potential for probiotics
treatment of Helicobacter pylori infections, the inability to influence obesity through altering the gut microbi-
of probiotic organisms to colonize and displace deeply ome is fuelled by studies showing that a higher abun-
embedded pathogens is also not unexpected, given dance of certain species, such as Bacteroides spp. and
that it is difficult for probiotic organisms to propagate Staphylococcus spp., can be found in obese individu-
in the stomach and to dislodge the intracellular patho- als102. However, to date no interventions have been
gens. nevertheless, probiotic therapy has been shown to tested for obesity, although a randomized study of 256
reduce some of the side effects of pharmaceutical treat- pregnant women showed that ingestion of the probiot-
ment of H. pylori (such as nausea, vomiting, a metallic ics Lactobacillus rhamnosus str. GG and Bifidobacterium
taste in the mouth and headaches) and to potentially animalis subsp. lactis str. Bb12 reduced the frequency of
decrease pathogen density 98. gestational diabetes threefold103, potentially through the

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REVIEWS

alteration of energy uptake and modulation of plasma using L. rhamnosus str. GG in a prospective, double-
lipopolysaccharide concentrations. These findings sug- blind, randomly assigned trial of 131 children (aged
gest that innate immune pathways can regulate glucose 6–24 months)110 indicates that more studies are needed.
metabolism and insulin resistance in response to pro- The prevalence of an aberrant microbiota does not
biotic microorganisms. Furthermore, a combination necessarily translate into symptomatic infections in all
of L. rhamnosus str. GG and B. animalis subsp. lactis cases. This is certainly the situation with BV. It is likely
str. Bb12 improved glucose tolerance in non-obese preg- that one of the main reasons for a natural return to homeo-
nant women, suggesting that this approach is worthy of stasis is the ability of indigenous lactobacilli to ascend
consideration for obesity 104. from the rectum into the vagina and dislodge BV biofilms
Atopic dermatitis is a chronic inflammatory skin dis- using the mechanisms outlined in FIG. 4. several studies
ease, the hallmark of which is dry, red, itchy skin. The have provided conceptual proof of this. Ingested probiotic
prevalence of atopic dermatitis is ~18–25% in children105. L. rhamnosus str. Gr-1 and L. reuteri rC-14 have been
risk factors include sensitizations to ingested and inhaled shown to reach the rectum, ascend to the vagina, reduce
allergens. unlike microorganisms on healthy skin, the the ascent of yeast and bacterial pathogens and increase the
microorganisms colonizing the skin of atopic dermatitis number of women whose BV resolved48,111. This approach
patients — Staphylococcus spp., Corynebacterium spp. holds great promise for the ability to interfere with
and Candida spp.106 — are characterized by profound dysbiosis before symptomatic BV or uTI recurs.
polymorphisms. Atopic dermatitis is accompanied by
destructive changes to the skin and to the bacteria on In closing
the upper layers of the epidermis. It is possible that the Chau et al.112 speculated that probiotic species may have
maturation of the intestinal microbiota has a role in arisen by a selection process favouring peptidoglycan
the spontaneous resolution of the disease that occurs by that can downregulate the effects of the toxic virulence
age 2 years in many children. To test this theory, probiotic factors produced by pathogens. The proposal was that
lactobacilli and/or bifidobacteria have been adminis- pathogens might prefer to be commensal or mutualistic
tered to pregnant woman and newborns at high risk of and to live in an environment that is not pathologi-
atopic dermatitis. In one study of 68 infants who were cal to the host. This implies that there are triggers that
followed for a year, the prevalence of eczema was signifi- can sway the balance of the microbiome away from
cantly lower in the group given probiotics than in the homeostasis to pathogenesis and, similarly, factors
group given a placebo (18.2% versus 40.0%; P = 0.048)107. that return the balance from an infected state. many
In another study, children aged between 2 and 10 years such triggers of disease have been identified, but far
old who had atopic dermatitis and had received a L. sakei less is known about the triggers of a natural return to
Eczema supplement 108 showed significant clinical improvement, homeostasis. studies that provide insight into this area
Non-contagious inflammation suggesting that the effect may not be specific for probiotic will be challenging but may lead to new methods to
of the skin, characterized
chiefly by redness, itching and
strain. However, the failure of L. reuteri to remediate treat and prevent disease. An alternative challenge will
the outbreak of lesions that eczema109 contradicts this theory, and the lack of clini- arise if we let the current, often ineffective, arsenal of
may discharge serous fluid. cal effect on atopic dermatitis or asthma-related events therapies run out.

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