MICROBIAL ECOLOGY IN HEALTH AND DISEASE
VOL.
1: 101-108 (1988)
Diversity Analysis of the Human Intestinal Flora:
A Simple Method Based on Bacterial Morphotypes
F. BAQUERO*,A. FERNANDEZ-JORGE,M. F. VICENTE, J. I. ALOS and M. REIG.
Servicio de Microbiologiu, HospituI Ram& y Cajal. Carr. Colmenar Km 9J.28034 Madrid, Spain.
Received 10 October 1987; revised 11 January 1988
A preserved diversity is a distinctive feature of the health of any ecosystem. A reasonable estimation of the diversity of
the human intestinal microflora can be easily obtained without the need of complicated methods of culture and
speciation. From a study of 100 healthy human adults, 40 different bacterial morphotypes were recognised from colour
prints obtained by microphotography of Gram-stained smears of faeces. On average each individual harbours 15.5
morphotypes (morphological diversity index, i.e. MDI = 15.5/40=0.38). The number of morphotypes (or the MDI)
increases in formula-fed newborns in contrast with breast-fed babies. In a semi-continuousculture system mimicking
the microbial community of the human large intestine, the MDI sharply decreases when a broad spectrum antibiotic
(imipenem)is added to the system. The results obtained in these two models of rise and fall of diversity as indicated by
the MDI, suggests the potential usefulness of this simple approach for the study of bacterial diversity and stability in a
variety of clinical circumstancesin large human populations.
KEY
WORDS-Diversity analysis; Bacterial morphotypes; Human intestinal flora.
INTRODUCTION
It has been statistically calculated that more than
400 bacterial species are permanent members of the
human intestinal microbial ecosystem. l 9 Many of
them are extremely difficult to cultivate. In practice,
and taking advantage of the more sophisticated
techniques of culture and identification, only about
20 separate species are generally recovered from
each i n d i ~i d ua l .~,'
In many studies on the effect of
antibiotics on the 'intestinal flora', only six to eight
predominant species or groups of organisms are, on
average, identified by culture. Because of the complexity of the faecal flora and associated problems
of isolation and identification of bacteria, only a
limited number of individuals, generally less than
20,6 are investigated in this type of study. At present,
reliable, practical and cheap methods based on
bacterial cultures which are able to describe the
general variations of the human intestinal microflora are unavailable. As there is a need for this type
of information, alternative approaches must be
investigated.
A preserved high degree of diversity is probably
the most descriptive feature of the health of a given
e c o ~ y s t e m . 'The
~ ~ ~basis
~ of the diversity of the
*Author to whom correspondence should be addressed
089 1 ~ 6 0 X / 8 8 / 0 2 0 1 0 1 ~$05.00
8
0 1988 by John Wiley & Sons, Ltd.
intestinal flora remains virtually unknown. Our
group, based on the postulates of Freitas and
Frederickson,* suggested the value of a negative
feed-back based on low molecular weight inhibitors,
like m i c r ~ c i n s , ~ ~where
~ * " the same product could
produce hetero-inhibition and, to a certain extent,
auto-inhibition.
Any disturbance of a previously integrated ecosystem will probably be reflected in a loss of the
diversity of its members. Certainly in ecological
terms diversity means stability;27 in practice, ecosystems presenting low diversity are highly fragile
to external influences, including colonisation by
external invaders. The ideal method to determine
the actual diversity of the intestinal flora would be
the precise enumeration of each of the different
microbial types present, a virtually impossible task.
Nevertheless, in many studies of environmental
microbial ecology, determination of the bacterial
diversity within natural communities has been
performed disregarding any accurate species identification. l o Diversity can probably be evaluated in
practice without a precise knowledge of the species
structure.
Miscroscopic examination of the colonic intestinal content or faeces in normal human adults shows
a great diversity of bacterial morphological types.
102
The apparent complexity of all these types has
hitherto prevented the establishment of an objective
and systematical study of their prevalence and significance. Of course, early studies in the first decades
of microbiology’ ‘discovered the presence of several
‘microscopic patterns’ in stained smears of human
faeces, but there are to our knowledge no systematic
catalogues of faecal bacterial morphotypes. In
this study we present a simple approach for the
establishment of such a catalogue as a tool for
further analysis of microbial diversity of the human
intestinal flora in different clinical situations. The
applicability of such analysis to this type of situation
is illustrated by preliminary studies on newborns
and in an in-vitro semi-continuous culture system of
the human large intestine microbial community.
MATERIALS AND METHODS
The diversity of bacterial morphotypes in faecal
specimens was studied on apparently normal
samples obtained from 100 healthy human adults
ranging in age from 20 to 30 years (most of them
students or members of the laboratory). Forty-five
samples from four selected newborn babies, two of
them hospitalised, were also studied sequentially
over 15 days. In each case, a portion of about 1 g
was carefully homogenised with 7 ml of sterile
phosphate-buffered saline (PBS). Smears were prepared with the homogeneous suspensions on clean
glass slides marked with circles of 1 cm2, air dried,
gently heat-fixed and stained by the standard Gram
procedure. l6 Four photographs were taken, from
separate parts of the smear from at least two different slides, in a Nikon-Fluophot photomicroscop
(Nippon Kogaku K.K.), using Kodacolor film.
Colour prints were enlarged to a size of 175 x
125 mm. At least 1,500 bacteria were photographed
by this method in each sample. All apparently different morphological types of bacteria were cut from
the developed photographs, carefully classified by
their morphology and Gram reaction and stuck on
individual record cards.
Morphological types were selected according to
the folIowing criteria: 1. Gram reaction; 2. rod,
coccal or yeast form; 3. length (very short, short,
medium or large); 4. width (wide, thin, filamentous);
5. shape and arrangement of bacterial cells; and
6 . regular or irregular staining. The work was
independently repeated by at least two different
investigators. A catalogue, including in most cases
several typical images of each recognisable type, was
F. BAQUERO ET AL.
used for further comparison and identification of
morphotypes in new samples.
The same type of analysis was performed in
sequential samples obtained from a semi-continuous
culture system mimicking the human faecal
microbial community, based on the model of Miller
and Wolin.” Two 250 ml sterile glass vessels for an
environmental incubator shaker (Ecologen G24,
New Brunswick Scientific Co. Edison, N.J.) were
filled with 175 ml of a sterile nutrient suspension of
fibrous food (lettuce, celery and carrots) with the
addition of sodium deoxycholate, urea, haemin,
acid-hydrolised casein, vitamins and salts, at a final
pH of 7.2. Culture was started with 75 ml of a recent
suspension of fresh human faeces representing a
high morphological diversity index (MDI =0.45see below-), in sterile PBS, in a ratio of 1:2 v/v.
Both vessels were flushed with nitrogen, closed to
maintain anaerobiosis, and the system was slowly
rotated (50 rpm/min) and incubated at 37°C.
Samples of a third of the total volume of each vessel
were removed at 12 h intervals during the first 48 h
and then every 24 h, and substituted by fresh nutrient
suspension, which was always checked for absence of
bacterial forms. Samples were diluted and cultured
for anaerobes in Brucella agar and Bacteroides-bileesculin agarz5 and for aerobes in McConkey
agar (OXOID) and Columbia-sheep blood agar
(OXOID) with and without the addition of 40 pg/ml
of nalidixic acid. After 48 h of incubation (in a
Forma Scientific Anaerobic Chamber), the colonies
were enumerated and identified. In only one of the
vessels, imipenem (provided by Merck, Sharp and
Dohme de Espaiia) was added at the time of each
medium replacement, to obtain a final concentration
of 25pg/ml, to detect the influence of a large
spectrum inhibitor on bacterial diversity.
RESULTS
Detectable morphotypes and the morphological
diversity index (MDI)
All different morphological types in every set of
photographs corresponding to each sample were
selected according to the following criteria: 1. Gram
reaction; 2. rod, coccal or yeast form; 3. length (very
short, short, medium or large); 4. width (wide, thin,
filamentous); 5. shape and arrangement of bacterial
cells; and 6. regular or irregular staining. The initial
number of distinguishable morphotypes in the first
five cases examined (14 morphotypes) progressively
increased when the number of cases studied reached
15 (28 morphotypes), 25 (35 morphotypes), 35 (38
103
DIVERSITY A N D BACTERIAL MORPHOTYPES
morphotypes) and finally45 cases (40 morphotypes).
In over 45 cases, a non-classifiable morphotype was
only rarely found. These 40 morphotypes were
retained in this way as a comprehensive catalogue
for further classification. Possibly a higher number
would be unpractical for comparisons by eye.
Surprisingly, the results obtained by three different
people working independently on the same photographs were highly consistent in the recognition of
morphotypes. After examination of 100 cases, the
catalogue proposed in Table 1 and Fig. 1 was
eventually selected. Among Gram-positive organisms, 30 morphotypes were selected, 21 corresponding to rods and nine to coccal forms. The number of
recognisable morphotypes among Gram-negative
organisms is unfortunately smaller, and includes ten
types of rods and one coccal type. The distribution
of such morphotypes among the 100 studied cases
is also shown in Table 1. In general, the average
number of different morphotypes per case was 15.5
(s.d. 2.71). Considering the comprehensive list of 40
detectable types, a mean ‘morphological diversity
index’ (MDI) of Spanish healthy young adults of
15.5/40(0.38) can be established. This type of index
serves as an indication of the bacterial diversity in
the intestinal microbial ecosystem.
Rise and fall of bacterial diversity: two models
The evolution of the number of morphotypes
found in two breast-fed normal newborns in comparison with two bottle-fed hospitalised newborns
during a period corresponding to their first two
weeks of life is shown in Fig. 2. Interestingly, both
groups of newborns can be differentiated as early as
the fourth day by the different number of faecal
morphotypes, the number obtained in the bottle-fed
group being higher.
In contrast, the faecal semi-continuous culture
system with an original high diversity reduces its
MDI, apparently as a consequence of the presence
of inhibitory levels of the antimicrobial agent
imipenem, whereas the number of morphotypes
were rather well preserved in the non-treated vessel
(Fig. 3). As the corresponding bacterial estimations
demonstrate a fairly good maintenance of the
original proportions in the original faecal sample,
including the major anaerobic groups, it can be
suggested that the observed MDIs over the observation period parallel the real diversity of the
system. On the contrary, a drop in MDIs in the
imipenem-treated vessel coincides rather well with
the general disturbances in the colony counts of the
major bacterial groups. Pseudomonas aeruginosa,
an unidentified aeromonas-like organism and
Klebsiella pneumoniae appeared after the third day
of imipenem therapy (only P.aeruginosa is shown in
Fig. 3).
DISCUSSION
Most bacterial organisms belonging to the intestinal
flora have fastidious growth requirements, require
selective or differential media for isolation and are
highly susceptible to the conditions of handling
and transport of the specimen. Moreover, their
speciation is difficult, and sometimes impossible
because of the bias caused by the selective exclusion
of some species in the community. All these facts
have in practice limited the establishment of good
studies on microbial diversity in natural environments, based on conventional methods of culture
and identification. The proposed simple method
based on the detection of morphotypes also has
obvious limitations. Firstly, there is the problem
of pleomorphism in this type of approach. l 2
Certainly within each Gram-type group, the same
bacterial organism could appear in different forms.
For instance morphotypes 5 and 6 could be interpreted as the vegetative and replicative form of a
single organism. On the contrary, quite different
types of bacteria may present exactly the same
microscopic morphology. On some occasions, a
given morphotype could be easily related with a
characteristic group of organisms, such as morphotype 9, which suggests Clostridium cochleatum or
Clostridium spiroforme, or morphotype 10, suggesting Actinomyces-Bijidobacterium, or morphotype
29, indicative of Veillonella. The analysis of
morphotypesesentially servesto predict the presence
of any abnormal diversity.
Whether the limited information (reduced to the
consideration of about ten characters) which is
attainable by simple microscopic examination is
really sufficient for the establishment of any kind of
classification into groups for studies on diversity
must also be considered. In fact a very limited
number of tests, less than ten, have been successfully
applied to generate clusters of bacterial organisms
related at supraspecificlevels for examining bacterial
diversity in environmental studies.lO”* On the
other hand, bacterial morphology remains a useful taxonomical tool, particularly for anaerobic
bacteria. 3*24
The neonatal gut is a sterile habitat which is
immediately colonised after birth under natural
104
F. BAQUERO ET AL.
I
6
2
7
22
23
24
3
8
25
4
9
26
5
--
27
20
29
**
*
30
10
II
II
31
4
36
12
32
37
13
33
38
14
=
\
I
i
34
J
35
40
t
15
Figure 1. Representative forms of bacterial morphotypes in human faecal samples. 1 to 21, Gram-positive rods; 22 to 28, Grampositive cocci; 29, Gram-negativecocci; 30, yeast-likeforms; 31 to 40, Gram-negativerods. See description in Table 1
105
DIVERSITY AND BACTERIAL MORPHOTYPES
Table 1. Catalogue and percentage of recovery of bacterial morphotypes in human faecal samples
~~
Morphotype
Length
Width
Shape
long
medium
medium or short
short
medium
long
short
long
any length
wide
wide
wide
wide
medium
medium
medium
medium
any width
10
any length
any width
11
12
13
14
short or very short
short
very short
any length
medium
thin
thin
thin
15
16
17
long or medium
long or medium
short
medium or short
wide
wide
wide
thin
long or medium
any width
Gram-positive rods
I
2
3
4
5
6
19
20
21
22
23
24
25
26
21
28
-
-
large
medium
medium
small
small
minute
small
round or oval
round
elongate, tapered ends
elongate
round
round or elongate
round
straight
straight
straight square ends
straight
straight
curved
curved
branched
semicircular, circular or
spiral
branched in X,V,Y forms.
Irregular staining.
straight
straight
curved
straight or angled;
beaded or barred
non-swelling round spore
swelling round spore
round spore
terminal swelling round
spore
oval spore
oval free spore
round free spore
pairs or chains
pairs or chains
pairs or chains
pairs or chains
pairs or chains
pairs or chains
irregular masses
Gram-negative cocci
29
any size
round
pairs
7
8
9
18
Yeast-like
30
Gram-negative rods
31
32
33
34
35
36
31
38
39
40
~~
Percentage
of recovery
19
39
24
50
32
38
25
20
5
22
50
37
6
14
9
10
5
40
5
18
31
32
54
57
38
22
22
32
6
5
short
medium
medium or short
very short near round
short
medium
long
medium
very short
very short
medium
medium
medium
medium or large
thin
thin
filament
filament
filament
thin
straight, bipolar staining
straight
curved, tapered end
cocobacillary
straight
curved or spiral
straight or angled
straight
straight
straight
26
32
14
40
20
45
20
26
24
25
106
F. BAQUERO ET AL.
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D A Y S
Figure 2. Increase in the number of morphotypes during the neonatal colonisation process: a and b (-),
breast-fed newborns; c and d (- - -), formula-fed hospitalised newborns
circumstances by different microbes. It is known that
diet profoundly influences the process of bacterial
colonisation in the infant intestine. Bijidobacteriurn
species predominate in the faecal flora of infants fed
on human milk whereas Gram-negative anaerobes
and facultative organisms are the most common
inhabitants in the colon of infants fed on a diet based
on cows milk.23,26The observed rise in the number
of morphotypes may correlate with the well-known
increase of bacterial diversity with this type of diet.
It would be of interest to detect a sudden increase in
MDI at the time of weaning in the breast-fed babies.
Obviously the build-up of an integrated microbial
system in the human intestine must be related to an
increase in bacterial diversity.
Our results obtained in the faecal semi-continous
culture system suggest that the establishment of
predictions of the effect of antibiotics on the human
intestinal flora could be done through the MDI
determination of faecal specimens. Preliminary
work in progress suggests that the MDI is significantly decreased in subjects treated with
metronidazole (mean MDI =0-28),oral gentamycin
plus vancomycin (MDI = 0.16), and to a much lesser
extent with oral ampicillin (MDI = 0.35). Obviously
. the study of the effect of new drugs on the human
intestinal flora could also be done by determining the
MDI under in-vitro conditions, as described above.
Of note is the appearance in the system of
some opportunistic pathogens, such as Pseudu-
rnonas after the third day of imipenem therapy,..
which could be related with the simultaneous
drop of the 'protective diversity'. It can be conceived that a low MDI in the faecal flora of
an hospitalised patient treated with antibiotics
may be a sign of a high risk of colonisation by
unusual and potentially dangerous opportunistic
pathogens.
The results obtained in the semi-continous culture
system, in which the damage to the ecosystem
corresponds to a drop in the MDI, have precedents in
observations obtained in artificial sewage digesters
mimicking the rumen, where several causes of
derangement of the system also result in large
variations in morphological types.'* It is certain
that new developments in the identification of
organisms, in faecal smears, like specific fluorescence antibodies, already used by some authors,'
or the application of computerised image processing
analysis to the automatic recognition of bacterial
morphotypes, may soon enlarge the possibilities of
the MDI analysis of diversity. Obviously the same
type of approach can be used for other bacterial
communities associated with humans and other
animals, such as the vaginal flora, urethral flora,
throat flora, and possibly periodontal flora and the
flora of dental plaque.
The potential usefulness of the proposed technique is based on its simplicity and accessibility,and
the possibility of its application to large numbers
107
DIVERSITY AND BACTERIAL MORPHOTYPES
- 0.45
- 0.35
- 0.25
- 0.15
- 0.05
I
I
1
0
I
2
/
I
I
I
I
3
4
5
6
I
M. D . 1
D A Y S
- 0.45
- 0.35
- 0.25
-0.15
- 0.05
M. D. I.
D A Y S
Figure 3. Bacterial colony forming units (c.f.u.) and morphological diversity index (MDI) in
a semi-continuous culture system of human faeces: a: imipenem (25 pg/ml)-treated vessel;
b: control vessel. A--.-A
E. coli; 0-0
group D streptococci; 0-0
Bucteroides
frugils; A - .-A Gram-positiveanaerobicbacteria; 0--0
Gram-negativebacteriaother
than B.frugilis; * - . . . -* Pseudomonus ueruginosa; W = = = = W evolution of the MDI
of specimens without the need for preservation or
prolonged transport of samples. As diversity most
likely correlates with colonisation resistance, and
there is evidenceto suggest that the danger of enteric
infection, for example due to organisms such as
salmonella or shigella is greater in circumstances of
low
some kind of analysis of the actual
susceptibility of subjects at risk could be conducted
by MDI determination. In such groups, diversity
may be reduced by the use of antimicrobials, by
chronic or endemic infections, and also because of
the type and amount of food. Preliminary experiments in our laboratory suggest a very important
drop in bacterial diversity measured by the MDI
following enteral nutrition (F. Baquero, A. Sastre,
unpublished observations), as expected by the
results obtained in previous studies.l4 Finally it may
be possible to apply the MDI determination of bacterial diversity to a variety of clinical circumstances,
such as nutritional problems, infections with
I08
opportunistic pathogens, cross-infection risk, etc.,
either in hospitalised patients or in the community.
ACKNOWLEDGEMENTS
We are grateful to Professor Ian Phillips for encouragement during the preparation of the manuscript. One of us
(A.F-J.) was supported by a grant of the Instituto Berna
de Espaiia, SA.
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