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ORIGINAL ARTICLE

Normal ora: diversity and functions


Lynne V. McFarland

From the Department of Medicinal Chemistry, University of Washington, and Biocodex, Inc. Seattle, WA,
USA

Correspondence to: Lynne V. McFarland, Ph.D., Biocodex, Inc., 1910 Fairview Avenue E, Suite # 208, Seattle,
WA 98102, USA. Fax: 206-323-2968; E-mail: lvmcfarl@u.washington.edu

Microbial Ecology in Health and Disease 2000; 12: 193 207

Recent research into the therapeutic use of living organisms has focused attention on the impact of various disruptive factors (antibiotics,
surgery, immunosuppression) and their impact on the hosts normal ora. This review covers what is meant by normal ora, how the
microecology differs by the niche in the body, type of diet, age and health status. In addition, the functions and tools used to investigate
normal ora will be explored. The functions of the normal ora include digestion of substrates, production of vitamins, stimulation of
cell maturation, stimulation of the immune system, aid in intestinal transit and colonization resistance. A variety of factors can disrupt
the normal ora including age, diet, stress, illness and exposure to antibiotics. Research involving microecologic populations is difcult
due to the challenge of unraveling the complex dynamics within a usually inaccessible niche, but progress is being made.

INTRODUCTION predominant types of ora present within body niches and


In the past, the role that microorganisms played in the shared functional traits.
normal functioning of the body was not appreciated. In The adult human body contains 101 4 cells, of which only
the early 1900s when Dr. Metchnikoff was credited with 10% compose the body proper and 90% are accounted for
the discovery of the importance of intestinal ora, other by members of the microora (8). The predominating
physicians felt that the colon was totally unnecessary and types of species in humans differ according to the body
often surgically removed them from their patients (1). The niche (oral cavity, skin, vagina, stomach, ileum, colon or
colon was described as a poisonous cess-pit infecting the urinary tract), as shown in Fig. 1 (3, 9 13). Normal ora
body with rheumatism, tuberculosis, cancer and other found in the oral cavity has been found to vary by the area
diseases. Today, we know that normal ora is a dynamic sampled (tooth enamel, tongue, gingivital surface, saliva)
and complex mixture of microbes that have diverse func- and the state of periodontal health (14, 15). The oral cavity
tions including digestion of essential nutrients, maturation contains a wide mixture of microbes, which are mainly
of intestinal physiology, stimulation of immune system, anaerobic bacteria. Gagliardi et al. sampled normal ora
systemic effects on blood lipids and the inhibition of in the healthy esophagus during upper endoscopy proce-
harmful bacteria. Current research techniques allow better dures in 30 patients and the predominant ora was found
evaluation of the specic bacterial and fungal microbes to be Streptococcus viridans (16). Lactobacilli and alpha-
within various body sites. This paper revisits some popular hemolytic Streptococcus species are frequently isolated on
conceptions about normal ora and updates them with tonsils of healthy children (15, 17). Lactobacillus species
regard to recent scientic ndings. that have the ability to adhere to mannose-containing
receptors, such as L. plantarum, have a distinct advantage
in surviving in the oral cavity (18). Results from different
WHAT IS NORMAL FLORA? studies proling predominant ora may be difcult to
Although the term normal ora is commonly used, it is compare as subject age, sampling techniques (washing of
really a misnomer. Microbial ora has spatial and tempo- the surface, aspirates or biopsies), diet, sampled location
ral complexity that differs by individual, body niche, age, and microbiological assay techniques may produce signi-
geographic location, health status, diet and type of host (2, cantly different results. Fewer bacteria exist in the stomach
3). Even within the same individual, the composition of (usually below 103 :g due to acidic lumen). Helicobacter
the microbial ora can vary according to changes in diet, pylori has been found in patients with peptic ulcers and
stress, sexual behavior, medication, hormonal changes and gastric neoplasia, but is also found in 60% of healthy
other host-related factors (3 7). With this caveat in mind, hosts, which casts suspicion that this microbe is always a
the eld of normal ora can be examined for common cause for gastric disease (19 21). The concentration of

Taylor & Francis 2000. ISSN 0891-060 X Microbial Ecology in Health and Disease
194 L. V. McFarland

microbes increases as progression is made down the intesti- Vaginal ora has been shown to change over the menstrual
nal tract: small intestine ( 104 : ml contents), to 106 107 : cycle (4), sexual activity and hygiene habits (7, 24), and use
ml at the ileocecal region and 1011 101 2 :g in the colon. of intravaginal microbicides (such as nonoxynol-4) (25).
The intestinal microora consists of 1011 organisms:gram However, studies show most healthy women (52 78%)
of feces with over 500 different species, ranging in concen- have transient changes in vaginal ora (4, 7, 24). Some
trations from 102 1011 :ml luminal contents (22). Although more recent prospective studies have shown only a minor-
the variety of organisms is complex, generally there are ity (22 26%) of healthy women had a lactobacilli-predom-
more anaerobic microbes than aerobes (9). The develop- inant ora (5, 24). Thus, the characterization of vaginal
ment of new techniques and genetic probes has allowed ora is open to debate and requires additional prospective
better characterization of the types of organisms that studies in well-dened populations of healthy women.
comprise the normal intestinal ora. Franks et al. devel- The process of the development of normal ora starts at
oped six 16S rRNA-targeted oligonucleotide probes that birth. It is thought that colonization begins during parturi-
can detect at least 66% of the anaerobic fecal ora in tion when the neonates intestine is seeded with mostly
humans (23). When these probes were used to characterize Gram-positive facultative anaerobes from the vaginal mi-
the ora in nine healthy human volunteers, Bacteroides croora during delivery (22, 26, 27). Whether the vaginal
species accounted for 20% of the total fecal population, ora in the last trimester is similar to vaginal ora when
Clostridium coccides and Eubacterium rectale accounted the woman is not pregnant is not known. However, Kar-
for 29%, Gram-positive bacteria accounted for 12% and voven et al. documented that the vaginal ora collected
Bidobacterium species accounted for 3% of the fecal ora. from mothers after delivery was the same as ora found in
These probes may prove very valuable in the characteriza- the stools of neonates (27). Neonates born by caesarian
tion of the microecologic proles, but more research is section usually acquire their rst microbes from the envi-
needed (as discussed later). ronment of the hospital nursery (26). Neonates are quickly
Previous studies have reported that ora in the healthy colonized by facultative anaerobes (E. coli and Streptococ-
vagina is typically a mixture of aerobic Lactobacillus spe- cus), reaching concentrations of 108 to 1010 :g feces within
cies, including L. jensenii, L. acidophilus or L. rhamnosus 1 2 days (9, 26). Previous studies reported that anaerobic
(9). Two strains of lactobacilli (L. crispatus and L. jensenii ) ora do not become established until the second month of
protect vaginal surfaces by producing H2 O2 , which inhibits life (9). The hypothesis for this observation was that when
the colonization of pathogenic anaerobes and mycoplas- the newly seeded facultative microbes grew and produced
mas associated with bacterial vaginosis, Neisseria gonor- a more anaerobic environment, this established an anaero-
rhoeae or other sexually transmitted diseases (5, 13). bic environment suitable for anaerobes (9). However, these

Fig. 1. Predominant ora in different


niches of the human body. Compiled
from references: (3), (9 13).
Normal ora description 195

Table I FUNCTIONS OF THE NORMAL FLORA


Microbial-associated characteristics (MAC) relating to different Digestion
functions of the normal intestinal microecology
The functions of the normal ora have been called mi-
1. Digestion of metabolizable substrates croora-associated characteristics (MAC) by several re-
2. Colonization resistance searchers (22, 36 38). These MAC (Table I) include
3. Production of vitamins
digestion of metabolizable substrates, colonization resis-
4. Development of attachment sites
5. Induces development of the immune system tance, vitamin production, mucosal cell development, im-
6. Production of exogenous enzymes mune system stimulation and intestinal transit regulation
7. Stimulation of intestinal transit (36 49). An important role of the intestinal ora is the
8. Maturation and turn-over of intestinal cells digestion of metabolizable substrates. A major source of
nutrients is the upper intestinal tract and the available
Compiled from references: (22), (36 49). substrates may include dietary bers, starches, oligosac-
charides, sugars, some lipids and proteins. Another source
conclusions were based on standard culturing techniques
of nutrients is within the colon itself and includes endoge-
(9, 28, 29). Harmsen et al. compared newer techniques
nous mucins, sloughed epithelial and enterocyte tissues,
(FISH, 16S rRNA probes) with culturing techniques and
bacterial debris, bile acids and cholesterol. The types of
found high counts were found by culturing only after 8 9
metabolizable substrates are key in determining the type
days, but FISH detected anaerobes by the second day (30).
of ora present in the colon. The main products of bacte-
In addition, another study indicated that neonates can
rial digestion of non-absorbed dietary carbohydrates are
acquire strict anaerobes, such as C. difcile, within the rst
short chain fatty acids (SCFAs). The SCFAs produced in
2 days of stay on a neonatal ward (31). Therefore, the
the largest quantities by the normal ora include acetic,
theory that colonization of the neonatal intestine by anaer-
propionic and butyric acids (50). Acetic and propionic
obes is dependent upon facultative bacteria producing an
acids are rapidly absorbed and are a major source of
anaerobic environment may be erroneous.
energy, in addition to stimulating salt and water absorp-
The type of diet largely inuences the types of ora in
tion (22, 51). Butyric acid has several functions including
pre-weaning infants. Several studies have reported that
the maintenance of the integrity of the colonic epithelial
infants who are breast-fed have higher concentrations of
bidobacteria as compared to formula-fed infants (28, 29, layer, as a chief energy source for these cells and regulat-
32, 33). Breast milk contains low protein content and high ing cell growth and differentiation (22, 51). Treem et al.
levels of oligosaccharides and glycoproteins, which are studied fecal SCFA in patients with Inammatory Bowel
considered to be growth factors for bidobacteria (26). Disease (IBD) (52). Fecal homogenates from 10 patients
Formula-fed infants have a more complex microbiota con- with IBD and 10 age-matched controls were compared as
sisting of Bidobacterium, Bacteroides, Clostridium and to the ability of the fecal homogenat e to produce SCFA.
Streptococcus species (22, 28). However, other researchers Patients with IBD produced less total SCFA, less acetate
have not found a signicant difference in breast-fed and acid and less propionic acid. Since normal ora is respon-
formula-fed infants (34). sible for the production of SCFA, this study may indi-
Historically, the characterization of neonatal fecal ora rectly link normal ora disruption with IBD. However,
has relied on culturing techniques, which are not able to Treem et al. did not characterize the ora responsible, nor
detect non-culturable species and may not be able to compare the microbial proles of patients with IBD and
distinguish different microbial populations. More recent controls. In some rare instances, the role for a member of
techniques using 16S rRNA probes, which are able to the normal ora has been identied for a specic func-
amplify the bacterial genes and are followed by sequence tion. Oxalobacter formigenes is a normal anaerobe in the
analysis, are more sensitive than the traditional culturing colon responsible for regulating the breakdown of oxalic
techniques (23). Harmsen et al. used 16S rRNA probes acid and it has been demonstrated that patients with
and conrmed previous ndings that breast-fed infants recurrent calcium oxalate kidney stone formation prob-
have predominant populations of bidobacteria in their lems do not harbor this useful bacterium (53). Normal
stools and formula-fed neonates had a more complex ora are also involved in the conversion of primary bile
mixture of organisms (32). By 2 years of age, children have salts. The identication of the genes encoding conjugated
a similar complexity and range of microbes as adults (35). bile salt hydrolases has been identied in a strain of L.
Differences in breast-fed and formula-fed microbial pro- johnsonii (54). Usually the role for a specic member of
les found by different studies may be due to sampling the normal ora is not limited to one function, rather the
techniques, time of collection, assay method, hospital prac- role is usually to interact with other members of the ora
tices or by the buffering capacity of different formulas for more complex functions (such as colonization resis-
(34). tance).
196 L. V. McFarland

Colonization resistance the gut. In germ-free mice, S. exneri is present at 109 :g,
Colonization resistance is the rst line of defense against but if the mice are pre-colonized with a mixture of anaero-
bic bacteria, S. exneri is held to 105 :g and when E. coli
invasion by exogenous, pathogeni c organisms or indige-
was added to the mixture, the levels dropped to 103 :g (59).
nous opportunistic organisms and the normal ora is
Romond et al. showed that bidobacteria, given to gnoto-
responsible for this formidable task (8, 55). Even though
biotic mice, prevented the colonization by E. coli (60).
the focus here is the intestinal tract, it should be remem-
The role of colonization resistance has been well studied
bered that colonization resistance plays an important role
in the case of the resistance to C. difcile. C. difcile
at other body sites (oral, skin, vagina, etc.). Colonization
readily colonizes neonatal animals and human babies at
resistance is a dynamic phenomenon that may differ dra-
the time when there is scarce ora established, but is
matically by microbial species, type of host, diet and other
cleared from the intestines once a more mature microecol-
host factors. Colonization resistance has been found to be
ogy develops (55). In the adult host, colonization resis-
an extremely effective natural barrier against such patho-
tance adequately prevents infection by C. difcile unless
gens as C. difcile, Salmonella, Shigella, Pseudomonas, the microbial barrier is disrupted (61). Once the ora is
pathogenic E. coli strains, Candida albicans and others (8, disrupted, C. difcile colonizes the intestines, produces two
56 58). major toxins and may cause overt disease including di-
Autochthonous ora (indigenous species that normally arrhea, colitis, pseudomembranou s colitis or toxic mega-
inhabit a given ecologic niche) may become disrupted and colon (62, 63). If protective ora is seeded back into the
allochthonous species (not normally present) are then able intestines (either by fecal infusion of normal stools or
to colonize the site. Colonization with allochthonous bac- selected biotherapeutic organisms), colonization of C.
teria may or may not result in disease, as the colonizing difcile may be prevented and the disease does not develop
organism may not be a pathogenic species. Early evidence (64).
for colonization resistance arose from the observation that, The mechanism of colonization resistance is dynamic
in germ-free animals (animals with no ora), animals were and complex (Table II). Bacteria comprising the normal
extremely sensitive to colonization with pathogens and ora are capable of producing substances and antimicro-
subsequently developed disease at higher rates than ani- bial peptides that are inhibitory against colonizing bacteria
mals with intestinal ora (57). This increased susceptibility (40, 65). Bacteriocins are a group of anti-bacterial proteins
could be corrected if fecal ora or mixtures of bacteria and produced by intestinal ora that have a broad inhibitory
yeasts were administered (38). Germ-free animals and ani- spectrum including many Gram-positive and Gram-nega-
mals that have been given certain antibiotics are able to be tive bacteria. Lactobacilli bacteria have been shown to
colonized by exogenous bacteria at doses 1000 to 100 000 produce a bacteriocin called reuterin, which is inhibitory in
fold less than in animals with normal ora present (11). vitro for Salmonella, Shigella, Clostridium and Listeria
Freter and Abrams found the concentration of Shigella species (66). Although an intriguing in vitro nding, it has
exneri depends upon the degree of normal ora present in not been shown that these bacteriocins reach concentra-
tions inhibitory to pathogens in the intestinal lumen, thus
Table II the clinical signicance of bacteriocins is not known.
Mechanisms of action for colonization resistance Normal ora may also produce other metabolic end-
products that are inhibitory to other microbes. Most nota-
Mechanism Factor Factors active ble is hydrogen peroxide (H2 O2 ) produced under anaerobic
against
conditions by several strains of normal ora (66). The
Production of Bacteriocins Salmonella, presence of H2 O2 results in peroxidation of lipid mem-
inhibitory Shigella branes, increased bacterial membrane permeability, de-
substances struction of bacterial nuclear acids in bacterial strains that
Toxic metabolic Hydrogen peroxide Chlamydia, do not possess catalase. The vaginal tract is usually pre-
endproducts Gardnerella
dominantly colonized with lactobacilli (Fig. 1) and H2 O2
Adverse microenvi- Acidic endproducts, S. aureus, E. coli
roments short chain fatty producing Lactobacillus strains have been found in 75% of
acids vagina samples from healthy women. Vaginal colonization
Nutrient or sub- Monomeric glucose Clostridium with Lactobacillus strains that produce H2 O2 has been
strate depletion difcile shown to be protective of infections caused by Chlamydia
Attachment Non-toxigenic enterotoxigenic
trachomatis, Gardnerella vaginalis, Ureaplasma urealyticum
interference strains of E. coli E. coli
Immune system Secretory IgA rotavirus, and the development of bacterial vaginosis (13). In women
stimulation C. difcile with bacterial vaginosis, these strains of H2 O2 producing
lactobacilli are absent and, instead, high concentrations of
Compiled from references: (11), (13), (41), (55), (59), (60), (63), Gardnerella vaginalis and anaerobes are present (66). An-
(67), (68), (75), (80). other protective mechanism is the production of a low pH
Normal ora description 197

environment, which may be inhibitory for certain patho- Survival in various body niches necessitates the attach-
gens. The production of acids as an end product of ment to receptor sites, especially in the colonic lumen
carbohydrate metabolism is common in many species of where peristalsis sweeps unattached microbes and undi-
the normal ora and is inhibitory against Gram-positive gested debris away. Competition for attachment sites is a
and Gram-negative bacteria. Several pathogens, including successful mechanism to inhibit colonization of pathogenic
Staphylococcus aureus, Salmonella, E. coli, and Bacillus microbes. Colonization with non-enterotoxin producing E.
cereus, are inhibited by acids produced by normal ora coli has been reported to prevent the subsequent infection
such as lactobacilli and bidobacteria. A common end with enterotoxin strains of E. coli in several mouse and pig
product of microbial fermentation is short chain fatty animal models (40). Fuller reviews several studies in ani-
acids (SCFA). The presence of these SCFA have been mals showing positive interference with attachment of
shown to be inhibitory to nonindigenous bacteria (40). normal ora when an non-indigenous microbe is ingested
Rolfe showed in a hamster model of C. difcile disease (71). A variety of host factors are also involved with
that SCFA levels inhibited C. difcile growth (40). As colonization resistance including secretory IgA levels, peri-
newborn hamsters age, they start to produce high levels of staltic movement , production of mucus, epithelial or ente-
acetic, butyric and propionic acids by day 16 19. Growth rocyte turnover (40).
of C. difcile was signicantly reduced when levels of these An area of intense research is to determine which mem-
SCFA increased (40). If normal ora are disrupted, de- ber or members of the numerous species of microbes
creased levels of SCFA result and pathogenic microbes present in the intestine are responsible for colonization
may take advantage of this decrease and reproduce to resistance. Most studies support the role of anaerobic ora
levels that induce disease. To date however, the identica- as the major microbes responsible for colonization resis-
tance (11, 56). Hazenberg et al. found a mixture of human
tion of which specic SCFA or mixtures of SCFAs are
anaerobic ora restored colonization resistance against P.
responsible for the inhibition of pathogens has not been
aeruginosa in the germ-free mice model (57). Giuliano et
demonstrated.
al. showed cefoxitin increased the numbers of fecal enter-
Competition for nutrients may be another mechanism
obacteriaceae, enterococci and yeasts in human volunteers
for colonization resistance. As it is extremely difcult to
(72). The increase in all three groups of these aerobic
assess the levels of specic nutrients in the interior of the
organisms may be due to the suppression of selected
colon, most of the research on nutrient depletion has been
anaerobic bacteria by cefoxitin. Leonard et al. reported
done using continuous ow culture techniques. Wilson et
that ceftriaxone decreased anaerobic ora in mice and
al. inoculated normal ora from a mouse into a continu-
human volunteers and colonization resistance was also
ous ow culture and found one or more of the ora
depressed in ceftriaxone treated animals (73). Clindamycin
competed more successfully for monomeric glucose, N-
is effective against anaerobes and has been shown to
acetylglucosamine and sialic acid, resulting in signicantly
impair colonization resistance (58). Some antibiotics that
reduced levels of C. difcile (67). Sweeney et al. found that
are effective against anaerobes, but are only present at low
even small numbers of an ingested E. coli strain F-18 could levels in the gut (tinidazole, cephradine), do not impair
supplant established ora, as this strain utilized an avail- colonization resistance (58). Specic members of the nor-
able nutrient (gluconate) more efciently than the other mal ora have also been studied including Bidobacteria
microbes present in the system (68). However, continuous longum, peptostreptococci and Clostridium cocleatum.
ow cultures are extremely dependent on culturing and Romond et al. tested Bidobacterium longum in germ-free
incubation parameters and the applicability of these results mice model (60). Pre-colonization of germ-free mice with
is unclear. E. coli delayed colonization by B. longum for one month,
Normal ora may also produce extracellular enzymes compared to a colonization time of only 24 hours in the
that are inhibitory or interfere with pathogen attachment. totally germ-free animals. Herias et al. observed that pep-
A yeast (Saccharomyces boulardii ) has been shown to tostreptococci reduced translocation of E. coli in germ-free
produce a protease that destroys toxin A and toxin B rat model by priming the immune system (74). Boureau et
receptor sites in rabbit ileal models for C. difcile disease al. showed that a normal member of the mouse intestinal
(69). The toxins of C. difcile act by inactivating Rho ora, C. cocleatum, inhibited the growth of pathogenic C.
proteins that keep the cytoskeleton of the intestinal entero- difcile (75). C. cocleatum was found to produce a variety
cyte intact, thereby distorting the cellular morphology of glycosidases that attacked intestinal oligosaccharides.
leading to uid loss and diarrhea (70). Rho proteins are As the peptide core of the mucin layer in the intestine is
also involved in yeast budding processes (reproduction), protected by oligosaccharides, this bacteria may interfere
thus this yeast may produce the protease to protect itself with the attachment of C. difcile but further study is
against soil Clostridia that may produce similar toxins to needed. Bourlioux et al. found a Ruminococcus species that
C. difcile, but in the human, the protease may coinciden- prevented C. perfringens colonization in a gnotobiotic
tally protect the host against infection with C. difcile. animal model (76). Although these studies have identied
198 L. V. McFarland

potential candidates for species which may be responsible on mucin required 5 weeks before it was restored to
for colonization resistance; it is more likely that a complex pre-antibiotic levels, which may indicate the length of time
mixture of many microbes is responsible. that it takes for normal ora to recover from antibiotic
A note of caution must be exercised with these animal exposure. The most abundant intestinal species (Bac-
studies, as the behavior of microbial species has been teroides) does not possess the glycosidases necessary for
found to differ depending upon which animal is studied mucin degradation and only 1% of normal ora species
(including human). Wong et al. evaluated the mouse as a can degrade mucin. Hoskins et al. identied ve strains of
model for studying microbial ecology interactions that mucin oligosaccharide chain-degrading bacteria from
may occur in humans (77). Human strains fed to mice healthy humans, of which three were Ruminococcus strains
usually survived, with the exception of Bacillus and Lacto- and two were Bidobacterium strains (83). Mucin-degrad-
bacillus species. They also found that in the mouse, human ing bacteria represent a distinct subset of normal ora with
organisms only produced 25% of the expected organic a specic function.
acids, which may suggest a change in metabolism when
human strains colonize the mouse. Resistance to a chal- Stimulation of the immune system
lenge strain (Salmonella ) was preserved, indicating that the Normal ora also induces the maturation of the gut-asso-
strains maintain their ability to produce colonization resis- ciated lymphoid system (GALT). The intestinal ora pro-
tance (77). vides an array of antigenic stimulants to the GALT cells,
affecting both local and systemic levels (22). Gnotobiotic
Production of vitamins mice have been shown to have fewer intraepithelial
Intestinal ora are also involved in the production of lymphocytes, plasma cells and Peyers patches than mice
vitamins including panthothenic acid (B5), biotin (vitamin with intact intestinal ora (78). When gnotobiotic mice are
H), pyridoxine (vitamin B6) and menaquinone (Vitamin immunized, the only local immune response is secretory
K2) (66, 78, 79). Without the intestinal ora, these vita- IgA, however, mice with intact intestinal ora also respond
mins would not be produced or in some cases, not broken with IgM and IgG (40, 41). Intestinal ora may also be
down into an absorbable form. Intestinal ora can usually involved in the development of tolerance to antigens (42).
provide the daily minimum requirement for many of the Herias et al. gave germ-free rats E. coli alone or a mixture
vitamins in humans. Microbes that are added to foods of E. coli, Lactobacillus acidophilus and a strain of an
(such as fermented diary products) may also increase the obligate anaerobe (Peptostreptococcus) and observed two
dietary levels of these vitamins (66). Vitamins B12, niacin, effects (74). The peptostreptococci reduced translocation
riboavin and thiamin are also made by intestinal ora, rates of E. coli and increased serum anti-E. coli antibodies.
but are not absorbed in the colon. The value of micro- It may be that peptostreptococci act as an immune system
bially supplemented foods is that the Vitamin B12 is primer to other bacterial antigens, thereby leading to a
ingested and it can be absorbed in the small intestine and decrease in translocation. Further evidence that normal
utilized. ora may act as an immune primer was found in a study
using BALB:c mice. Pulverer et al. found that normal ora
Attachment releases low molecular weight substances that interact with
Attachment to the intestinal mucosa is an important sur- MALT (mucosa associated lymphoid tissue) and these
vival trait for organisms in the intestinal tract. The pres- substances appear to be essential for an adequate immune
ence of intestinal ora has been shown to stimulate the response (43). Antibiotic decontamination in a mouse
production of epithelial glycoconjugates, which may be model resulted in a decreased immune response. Thus,
receptors for some pathogenic bacteria (22). Umesaki et al. normal ora may have an important role as an immune
documented that the presence of a strict anaerobe, B. system primer.
thetaiotaomicron and a segmented lamentous bacterium
were associated with fucosylated glycoconjugates in the Intestinal transit
small intestinal tract (80). Colonic mucins are a class of The presence of intestinal ora has been shown to stimu-
high molecular weight glycoproteins, which are secreted by late peristalsis or otherwise involve the enteric nervous
the mucosa and exocrine glands. Mucin is present in the system (44, 45, 78). In gnotobiotic mice models, the small
lumen and functions as a lubricant, a modulator of water intestine has thinner walls and is smaller than in mice with
and electrolyte absorption, may aid in attachment of mi- intestinal ora (78). Husebye et al. studied germ-free rats
crobes and protects the mucosa from injury (81). A large (who have enlarged caeca) and found a slower progress of
number of oligosaccharide side-chains of mucin glyco- chyme through the intestinal tract when compared to rats
proteins aids in the stability of the mucin layer. Carlstedt- with a normal microbiota (46). He found that the mecha-
Duke et al. found antibiotics including bacitracin, nism may be through the enteric nervous system, rather
clindamycin and vancomycin resulted in altered mucin than stimulating motility by a direct action on intestinal
degradation in healthy human volunteers (82). The effect smooth muscle. This may explain why colonic bacteria are
Normal ora description 199

Table III
Geographical differences in intestinal microora in humans

Microorganisms or groups of English:mixed Western Ugandan vegetaria n Americans: Japanese:vege- Japanese:mixed


microorganisms diet (London) diet (in London) mixed Western tarian diet Western diet
diet

Total anaerobes 10.1 9.3 10.2 9.9 11.5


Total aerobes 8.0 8.2 7.5 9.49.8 9.6
Facultative anaerobes 7.2 4.8
Enterococci 5.7 7.0 5.5 8.4 8.4
Bacteroides 9.7 8.2 9.8 10.1 11.1
Bidobacteria 9.9 9.3 10.0 8.2 9.5
Lactobacilli 6.0 7.2 7.3 5.7 4.0
Clostridia 4.45.0 4.04.6 4.4 5.19.7 9.5
Yeasts 1.3 3.1

Indicates a signicant difference between groups as reported in the original publication (Adapted from reference 3).

able to inuence transit through the small intestine in ora in the elderly, but whether this variation is due to age
gnotobiotic mice. Pothoulakis et al. found that one intesti- or medical exposures, or due to illnesses was unclear (87,
nal response to C. difcile involved events relating to the 88).
neural cascade (84). The role of normal ora on the enteric
nervous system requires further study and may have Geography
promise for therapeutic intervention by medications that There are numerous studies which report differences in
inhibit the neural response. normal ora depending upon geographical location. Benno
et al. compared the fecal ora in elderly Japanese living in
Colonic physiology and maturation rural areas or urban centers (89). On the whole, the diversity
It has been shown that the presence of intestinal ora and counts of fecal ora were similar. Urban Japanese were
stimulates the maturation and turnover rates in colonic found to have signicantly less Bidobacterium adolescentis,
epithelial cells. The surface layer of the mucosa in the but signicantly more total anaerobic bacteria, bacilli and
intestines is replaced every 2 3 days, which allows basal Clostridium spp. than rural Japanese. Benno et al. at-
stem cells to migrate up the crypt, presenting differentiated tributed these slight changes to a different diet (high ber)
mature cells that are involved in nutrient absorption and in the rural population, but did not support this hypothesis
mucin secretion. In germ-free mice (gnotobiotic), the rate of with data on dietary patterns. Sepp et al. also cultured
cell turnover was found to be signicantly slower than mice neonates at 1 week born in Estonia and Sweden (90).
with established microora (78, 80). Bry et al. also found Estonian neonates had signicantly higher counts of
that normal ora induced enterocyte cell turnover (85). staphylococci (coagulase negative), enterococci and enter-
obacteri compared to Swedish neonates. Different feeding
habits and differences in antibiotic use in these two coun-
FACTORS THAT INFLUENCE NORMAL FLORA
tries was proposed to explain these differences, but further
Age study is needed.
Early infancy (B 2 years old) is a time of ux in the As shown in Table III, the composition of normal ora
composition of the normal intestinal ora, as this is the time reported in geographical populations are not due to genetic
that the microecology is becoming established. The diet in differences as originally thought, but to differences in diet
pre-weaned babies is largely inuenced by the diet (breast (3). For example, when intestinal ora is compared for
or formula fed) as previously discussed. Mitsuoka et al. English people living in London and eating a mixed western
characterized the Lactobacillus ora in four age ranges: diet against Ugandans living in London and eating vegetar-
infants B 7 months, children 4 6 years, adults 20 64 years ian diets, the English had more bdobacteria and bac-
and elderly 65 86 years old (86). Infants were found to teroides but less enterococci, lactobacilli and yeasts than
usually be colonized by three types of resident lactobacilli, Ugandans. Vegetarian diets are associated with fewer
and in the older ages both number of different species and anaerobes and higher counts of facultative and aerobic
transient nature increased. Normal ora in older adults may microbes (3). Generally, one individual has a fairly constant
also differ from younger adults. Postmenopausal women prole of microbes that they can consider as normal ora
have been found to have increased numbers of fungi, unless the person is exposed to antibiotics or other factors
clostridia and lactobacilli compared to women who are that disrupt the ora, but inter-individual differences of
pre-menopausal (3). Other studies have shown variation of normal ora may vary considerably.
200 L. V. McFarland

The comparison of differences in normal ora for di- Stress


verse geographi c populations is complicated (and may be Stress is frequently cited as a major inuence on intestinal
completely obscured) by vast differences in diet, culturing
function, but the evidence for its impact on normal ora is
techniques, degree of sophisticated technology and differ-
limited. A frequently quoted reference to document the
ent study populations. What is needed are studies which
impact that daily stress has on normal ora may be
follow similar populations (same age range and sex), using
misleading (99). This study was done with postoperative
identical microbiologic techniques and thoroughly docu-
patients, so that the disruption of the normal ora may
menting dietary constituents, in order to compare differ-
have been due to antibiotics or medications associated
ences seen in normal ora of different countries.
with the surgery itself and not due to stress (99). Studies
Diet linking stress and the Irritable Bowel Syndrome (IBS)
often do not provide data on normal ora (100). Bochkov
Diet has been shown to change the ora in several animal
et al. studied the fecal ora of cosmonauts during training
studies, but there is little direct evidence from human
and space ight and concluded that stress affected the
studies other than studies with calcium, sugars or ber
ability of the normal ora to mount effective colonization
manipulation (40). Dietary calcium precipitates cytotoxic
resistance (101). Stress may decrease hydrochloric acid
substances such as bile acids resulting in less cytolysis. A
production in the stomach allowing for the growth of
decreased luminal cytotoxicity may help to reinforce en-
dogenous ora. Bouvee-Oudenhoven et al. found when coliforms and bacteroides (102). There is no direct evi-
calcium supplemented diets were given to rats Salmonella dence that stress causes a signicant shift in normal ora
enteritidis colonization was reduced as was translocation populations.
(91). Fructo-oligosaccharides (FOS), found in bananas,
onions, asparagus and artichokes, are fermented mostly by Antibiotics
Bidobacterium species. Increased ingestion of FOS at
The effect of antibiotics on intestinal ora has been well
doses of 4 g:d was found to increase levels of bidobacte-
studied, mainly because of the frequent occurrence of
ria in the intestines in human volunteers, but resulted in
disease associated with antibiotic use. Antibiotics are often
excessive atus at doses over 20 g:day (92, 93). Budding-
used to treat a specic illness without considering how the
ton et al. also studied dietary fructo-oligosaccharides in 12
antibiotic will impact the normal ora (11). A common use
healthy volunteers (39). FOS was found to increase num-
of antibiotics is to decontaminate the intestines in prepara-
bers of total anaerobes and bidobacteria in this small
tion for intestinal surgery. As it was observed that the
study. Sucrose was found to increase Bacteroides species
etiology of some post-surgical infections was of intestinal
and inulin was found to increase mostly bidobacteria
origin, it was thought that decontaminating the intestines
(92). Different types of ber have found to result in altered
with antibiotics might reduce the risk. However logical this
levels of bidobacteria, lactobacilli and fungi in rats and
tactic appeared it has not been productive, largely due to
pigs (94 96). Some types of fermentable bers may sup-
underestimating both the protective role of normal colonic
port the growth of normal ora, yielding SCFA and
decreased colonic pH, which can act to inhibit the growth ora and the profound disruptive impact of broad-spec-
of certain pathogens , such as C. difcile (95, 97). Tea trum antibiotics. Tetteroo et al. studied selective gut de-
polyphenols given to pigs resulted in increased levels of contamination and found patients experienced rebound
lactobacilli and decreased levels of bacteroides (98). colonization with potentially pathogenic organisms after
Evidence that diet inuences normal ora in adults is surgery (103). Of 135 patients studied, 20 patients had
sparse, but numerous studies have been done in young rebound colonization with a nosocomial aerobic pathogen.
infants. Previous studies have shown that breast-fed in- Lingnau et al. studied 357 patients with trauma who had
fants have been found to harbor mostly bidobacteria. In topical and gut decontamination using two mixtures of
contrast, formula-fed infants are colonized with a wider antibiotics and compared them to trauma patients who
variety of species, namely enterobacteria, bacteroides and were not given mixtures of antibiotics (104). Treatment
clostridia (40). A recent study by Heavey and Rowland with the antibiotics did reduce colonic bacteria, but had no
reported that high levels of bidobacteria were seen in impact on the incidence of expected post-surgical infec-
infants fed formula, but only for formulas that had a low tions (pneumonia, sepsis or organ failure). Unfortunately,
buffering capacity (34). Rubaltelli et al. studied the effect the identication of the inhibited microbes was not per-
of an adapted formula (high maltose) on the intestinal formed. A follow-up study showed that gut decontamina-
colonization of bidobacteria compared to breast-fed in- tion did result in higher rates of oxacillin-resistant
fants (33). Breast-fed babies were again found to have Staphylococcus aureus (105). Terg et al. studied four differ-
more bidobacteria (48%) than formula-fed (15%) infants, ent dose regimens for ciprooxacin in 29 patients with
even as early as the fourth day of life. Unfortunately, cirrhosis (106) but found no statistical differences by dose.
controlled studies that document how well-dened diets Most studies of antibiotics and their impact on ora
can change normal ora have not been done. have been directed at either the antibiotics potential to kill
Normal ora description 201

pathogenic bacteria (and not normal ora) or the inuence treatment with either three of these antibiotics resulted in
antibiotics have on the development of antibiotic resis- increased numbers of C. albicans in the feces. Unfortu-
tance. However, there are a few studies on the impact nately, he did not study other types of normal ora. Van
antibiotics have on normal ora. A summary of antibiotics de Leur et al. suggested that before giving neutropenic
by the impact on the normal ora is given in Table IV. patients ciprooxacin to decontaminate the colon and
Thijm et al. studied mice treated with ampicillin or epicillin reduce disease, the effect of low dose ciprooxacin should
or cephradine and observed the rates in which resistant be studied when the ora has been disturbed (by another
strains of E. coli colonized as the measure of colonization antibiotic) in healthy subjects (113). Therefore, he then
resistance (107). Both penicillins resulted in a loss of treated ve healthy volunteers with ciprooxacin for 20
colonization resistance to E. coli. Oral treatment with days and then ciprooxacin combined with clindamycin
cephradine did not inuence normal ora or effect colo- for 14 days. Fecal samples were analyzed for Gram-nega-
nization resistance. No direct measures on specic strains tive bacilli, enterococci, yeasts and antibiotic levels. Not
of normal ora were done. surprisingly, the addition of a second antibiotic was found
Most of the studies of normal ora and antibiotics have to dramatically increase the acquisition of Gram-negative
been done using healthy volunteers. Barza et al. studied 20 bacilli. When low-dose ciprooxacin was used alone, no
healthy subjects, of whom 16 were given 4 antibiotics increases in Gram-negative bacilli were noted. Edlund et
intravenously and 4 were given no antibiotics (108). Only al. studied 20 healthy human volunteers who received
those subjects exposed to antibiotics (7:16) were later cefuroxime axetil (500 mg:d for 1 week) and ten of those
found to be colonized with Gram-negative bacilli. Van who additionally received vancomycin (500 mg:d) for the
Nispen et al. studied trovaoxacin on the effect on normal following 7 days (114). Cefuroxime resulted in rapid de-
fecal ora in 19 healthy male subjects (109). In comparison crease of anaerobic Bidobacterium and Lactobacillus spe-
with the seven men who received placebo, the 12 who cies, but increased the numbers of three species of
received trovaloxacin had signicantly reduced rates of Enterococcus. Vancomycin resulted in a rapid decline of
enterobacteriaceae, but there were no differences in Gram- Enterococcus faecium, E. faecalis and E. durans, Bac-
positive cocci, anaerobes or yeast populations. Vollaard et teroides and Clostridium species and a signicant increase
al. studied the effect of amoxycillin, erythromycin and in vancomycin-resistant Enterococcus gallinarum and E.
roxithromycin on the colonization resistance in healthy casseliavus. Van der Auwera et al. also documented that
volunteers (110). Amoxycillin decreased colonization resis- 64% of human volunteers given vancomycin carried van-
tance (evidenced by increased numbers of enterobacteri- comycin-resistant enterococci as part of their intestinal
aceae and yeasts). Vollaard followed this study with ora (115).
another in 6 healthy volunteers when he dened impair- These studies show clearly that antibiotics may disrupt
ment of colonization resistance by signicant increase in normal intestinal ora and may predispose patients to
the numbers of fecal yeasts, Gram-negative bacteria or by disease from opportunistic pathogens. Recovery of the
increased colonization by a challenge strain (111). colonization resistance brought on by antibiotic exposure
Thomakos et al. studied the effect of cefamandole, ce- may take weeks to months. Hashimoto et al. exposed rats
furoxime and cefoxitin on Candida albicans colonization in to 6 days of broad-spectrum antibiotics and found that, by
28 surgical patients (112). Thomakos found that 10 days the seventh day, fecal anaerobic levels decreased, bile acids

Table IV
Inuence of antibiotics on normal ora from human volunteer studies

Antibiotic tested Daily dose Number of volunteers Effect Reference

Clindamycin 800 mg bid 10 anaerobes (136)


Erythromycin 500, 1000 mg, tid 12 anaerobes, (137)
aerobic GNB,
No effect on enterococci or GPB
Ciprooxacin 50, 100, 200 mg 10 No signicant effect by dose (138)
bid
Cefoxitin, cefoxitin or cefamandole na 28 in yeasts (112)
Ciprooxacin 20 mg 5 GNB (113)
Ciprooxacin (and clindamycin) (300mg) 5 Cipro resistant GNB
Trovaoxacin 200 mg bid 12 E. coli (109)

in surgical patients.
Abbreviations: GNBGram-negative bacilli, GPBGram-positive bacilli, nadata not available, bid twice a day, tid three times
a day.
202 L. V. McFarland

decreased and the cecum enlarged in size (116). After required, but these assays still cannot identify unknown
antibiotics were stopped, fecal microbes recovered to their species if there is no known biomarker. Corthier et al. also
initial counts within a week, but the restoration of bile used luciferase gene biosensors to detect Lactococcus lactis
acid and cholesterol metabolism did not return to baseline (124). Serotyping and plasma proles have been helpful to
until 3 weeks later. Unfortunately, no other measures of differentiate different strains of similar bacterial and fun-
colonization resistance were made. Larson and Borriello gal species. The most practical use for these techniques
found that the susceptibility to C. difcile in hamsters may be in the tracking of outbreaks. However, these
exposed to clindamycin was prolonged (74 days) compared techniques cannot quantitate levels of different microbes
to a relatively short period of susceptibility (2 days) if the nor provide any functional measurements.
hamsters were given ampicillin (117). Although many an-
tibiotics have their activity tested against pathogenic or- 16 S ribosomal RNA typing
ganisms or on selected populations of normal ora, few Cumulative databases allow placement of unknown species
studies have measured their impact directly on coloniza- using 16S ribosomal RNA ngerprint techniques (125).
tion resistance. This technique is applicable for both culturable and non-
culturable microbes and is becoming increasing popular as
TOOLS AND MODELS a research tool. Use of polymerase chain reaction tech-
Selective culture techniques nologies are increasing, but it is costly. Dore et al. used
16S rRNA targeted oligonucleotide probe to detect and
Routine biochemical assays can identify specic species quantify Bacteroides species in human fecal samples (126).
and these traditional methods are standard and well- Franks et al. used 16S rRNA-target oligonucleotide probes
known (118). However, the results will only quantitate to quantify predominant groups of anaerobic bacteria in
culturable bacteria and cannot identify unknown types of human fecal samples (23). These probes were able to detect
microbes nor test microbial interactions or protective func- at least two-thirds of the anaerobic fecal ora.
tions. Routine microbiological culturing will underestimate Several researchers report good agreement between 16S
both the number and diversity of ora and in addition, rDNA probe identication and culturing techniques (32,
frozen samples may result in less sensitive results (119). 127, 128). But other researchers have noted DNA probes
can detect more anaerobes than by culturing techniques
Continuous ow cultures
(30, 129). Several advantages of the newer probe tech-
In an attempt to control some of the interactive factors niques are that frozen stools may be used, and non-cul-
present in microecologies within various body sites, re- tivable species may be detected. Standard culturing has
liance upon continuous ow cultures has become estab- advantages of being inexpensive and widely available.
lished. Although the results may depend more on culture When 16S rDNA probes are used to characterize the
specications than the microbial interactions, this tech- species composition of a population, several sources of
nique is popular. Bernhardt et al. studied the growth of C. bias may arise. Bacterial strains may react differently to
albicans using a continuous ow culture (120). This the processing required by this technique. Bacteria will be
method exhibited a colonization resistance effect on C. detected more frequently if they are susceptible to lysing,
albicans. Macfarlane et al. used a three-stage compound amplication and permeability (129).
continuous culture system to study normal ora (121).
Stage 1 models for the proximal colon and Stages 2 and 3 Functioning analysis
model the distal colon. Metabolism, bacterial interactions The above probes all share the disadvantage of not guar-
and changes in the diet and environmental conditions may anteeing that the species identied by the probe may not
be studied using this system. Kontula et al. used SHIME be the microbe responsible for a specic microbial associ-
(simulator of human intestinal microbial ecosystem) to ated function. Also, as colonization resistance results from
study ora changes and observed changes in fatty acids the interactions of many microbes, the above probes do
and gas production and enterococci numbers (122). Con- not measure these interactions. Measuring the function of
tinuous ow cultures have the advantage of being able to the target organ as a whole corrects for this disadvantage.
tightly control various factors (nutrient concentration, Collinder et al. and others have suggested using MACs
types of species, etc.) but whether the results can be (Microora-Associated Characteristics) to study coloniza-
extrapolated to the intact intestinal microecosystem is tion resistance (36 38, 130). MACs include: degradation
uncertain. of mucin, conversion of cholesterol to coprostanol, biliru-
bin to urobilinogens (to study hepatic-intestinal interac-
Serotyping, plasmid proles, antibiotic resistance patterns tions), inactivation of tryptic activity (to study
Monoclonal antibodies have been used to enumerate Bac- pancreatic-intestinal interactions), degradation of b-as-
teriodes species present in human fecal ora samples (123). partylglycine (found in disturbed ora) and production of
These techniques do not require culturing, so less work is SCFA. Several types of indicators of functions can be
Normal ora description 203

measured, including bacterial enzymes and bacterial differ for the distal and proximal segments of the intestine.
metabolite levels. The metabolite levels include NH3 levels Marteau et al. overcame this difculty by sampling jejunal
(breakdown product of protein and urea), phenols and ora directly by using a lumen tube with a proximal
cresols (amino acid catabolism), and the breath hydrogen occluding balloon (135). This allows sampling of the ora
test (118, 131). at the desired location within the intestine. However, this
technique is invasive and not as simple as culturing feces,
Animal models but is well tolerated by subjects. The use of selective media
Gnotobiotic models have been used to test normal ora and polymerase chain reactions (PCR) probes of 16S
and disruptive factors (132, 133). Wilson et al. used gnoto- rDNA have enhanced the detection of fastidious organ-
biotic mice to test the ability of hamster ora for the isms and should provide valuable assistance to future
protection for C. difcile and E. coli (134). Segmented studies of normal ora.
lamentous bacteria restored diffuse GALT in gnotobiotic
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