16S RRNA Hybridization Probes For The Major Groups Of Intestinal ...

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F. Schut
168 rRNA Hybridization Probes for the Major Groups of Intestinal
Bacteria: Development and in situ Application
Abstract
'Whenever a man gets the idea that he is going to
work out the bacteriology of the intestinal tract of
any mammal, the time has come to have him
quietly removed to same suitable institution'. Jordan, In: Gorbach et al., 1967.
Investigations into the role of the gut microflora in pathogenesis are continuously frustrated by technicallimitations. We are currently developing 16S
rRNA targeted oligonucleotide probes for the enumeration of various
phylogenetic groups of intestinal bacteria. Preliminary results show that
bifidobacteria are grossly overestimated by cultural methods, and th at members
of the Bacteroides vulgatus cluster are underestimated. Microscopic image
analysis in combination with 16S rRNA hybridization probes has proven to be
a very powerful technique in ecological studies of intestinal flora.
The intestinal microflora
The richest and most complex part of the human intestinal microflora resides in
the colon. N ormal populations exceed 10 11 per gram of stool and consist of a
mixed culture of an estimated 100-400 species of bacteria (Moore and
Holdeman, 1974). The initial inoculum is derived from the mother at the time
of birth and although the climax flora alters as the subject ages (Mitsuoka,
1992) it is fairly constant in composition with Bacteroides and Eubacterium spp.
dominating in adults. The stability of the gut flora can be appreciated when
bearing in mind that food and drinks are in actuality minor components of the
intestinal contents. Saliva (1.0 litre/day), gastric (1.5 ljd) and pancreatic (1.2ljd)
secretions, bile (1.0 l/d) and secretions of the glands in sm all and large intestine
(2.0 ljd) form the bulk of the estimated 7 litres of fluid that enters the intestinal
tract daily (Guy ton, 1986). These highly concentrated endogenous secretia
ensure a constant intestinal environment thus enabling the digestive enzymes to
operate under controlled conditions.
The composition of the diet, and the administration of specific food supF. Schut 69
plements such as fibre, has been suggested to influence the composition and
activity of the flora (Benno et al., 1989; Mitsuoka, 1992). However, direct
evidence is difficult to obtain due to statistical inaccuracy of the cultural technique employed (Gorbach et al., 1967; Finegold et al., 1974), a high inter-subject
variability (Minelli et al., 1993), the relatively low numbers of subjects th at can
be investigated (Drasar et al., 1986), and the difficulty in determining causative
relationships from observed correlations (e.g. Benno et al., 1989).
Over 95% of the bacteria encountered in faeces are non-sporulating, strict
anaerobes and pre-reduced, anaerobically sterilized (PRAS) media or anaerobic
chambers are essential for cultivation of these organisms (Holdeman et al.,
1977). Although it is relatively easy to obtain a total viabie count, counting of
individually identifiabie bacterial species is laborious and time consuming.
Viabie counts of the various aerobic and facultatively anaerobic species can be
obtained by using selective media. However, such media are not available for
most of the strict anaerobes. Since some 3~O species account for 99% of the
flora, several hundred isolates from each sample should be identified for reliable
statistics and end-product analysis of fermentation in pure cultures is essential
for reliable identification. As a result, studies on population dynamics of the
intestinal flora are of ten limited in the number of subjects or the number of
species investigated and, as a consequence, the observed changes in composition
are often not statistically significant.
Clinical relevance: intra-abdominal sepsis as a case
Due to the advances in medical technology and the significant elevation in live
expectancy of the severely ill, an increasing number of patients in hospitals have
compromised immune systems. Subjects undergoing major surgery (e.g. transplantations ), chemotherapy as a treatment for cancer, and those with major
trauma form the majority of such patients. Concurrently, the type of infection
has changed from predominantly pathogenic to potentially pathogenic, or
opportunistic. Furthermore, antibiotic resistance among nosocomial infections is
becoming a world-wide threat and treatment may soon become ineffective. It is
expected th at alternative strategies for infection prevention will arise from
knowledge on the natura 1 physiological con trol of potential pathogens.
However, up till now such studies have rarely been performed.
Although probably not as important on a world-wide scale as colon cancer,
intra-abdominal infections are in this respect of significant interest due to the
presumed involvement of normal host immunity, gut flora, and antibiotics.
Almost all bacteria isolated from intra-abdominal infections are indigenous to
the intestinal tract. Wh ether they have escaped from the intestine by 'naturally'
occurring translocation or after perforation of the gut wall is the subject of
debate (Sedman et al., 1994), but the excessive accumulation of opportunistic
microbes on the mucosal surface is believed to be very hazardous in this context
(McClean et al., 1994). The predominance of certain isolates does not always
70 16S rR NA hybridization pro bes
correlate with the use of antibiotics (Baron et al., 1992; Sawyer et al., 1992). It
is probably through changes in the structure of the mucous colonizing community as a whole that certain opportunists gain a foothold (Kennedy and Volz,
1985; Van der Waaij, 1989). Antibiotics greatly affect the community structure
and for both Candida and enterococcal intra-abdominal infections antimicrobial
therapy has already been identified as a major risk factor (Kujath et al., 1990;
Boulanger et al., 1991).
So, although immediate antimicrobial treatment during peritonitis is essential,
especially in the case of Candida infections, the increased use of antimicrobial
agents in immunocompromised hosts will promote changes in the normal
intestinal flora and relatively harmless organisms can suddenly give ri se to
serious complications (McClean et al., 1994).
The human intestinal flora is responsible for a number of other health disorders. Through the production of carcinogens, some species of the intestinal flora
are believed to play a role in the development of colon cancer. Crohn's disease,
rheumatoid arthritis and chronic diarrhoea, are all believed to be related to
intestinal bacteria or their products. Furthermore, there are indications that
graft versus host reactions after transplantation are also influenced by the gut
flora. The development of fundamental insight in the interaction between the
healthy host and its gut flora is of primary importance to the understanding of
the pathogenesis of these diseases.
The use of 16S rRNA probes for enumeration
The above clinical ex am pIes serve to illustrate the need for detailed and rapid
community-Ievel screening of the composition of the intestinal flora. Only when
changes in the composition are accurately described can relationships with
pathogenesis be recognized. The possibi1ity of using 16S rRNA hybridization
probes for this purpose deserves investigation.
Each living cell contains ribosomes for protein synthesis. Several tens of
thousands of ribosomes are present in growing cells. Because of evolutionary
changes, the sequence of the nucleotides in the rRNA molecules is unique in
each bacterial species. Therefore, the bacterial ribosome is uniquely suited as a
phylogenetic (evolutionary) or taxonomic marker (Olsen et al., 1986; Pace et al.,
1986). Synthetic deoxyoligonucleotide probes can be constructed which
hybridize specifically to a certain sequence of nucleotides in the rRNA
molecules. The specificity of the oligonucleotide pro bes can be adjusted to fit any
taxonomic level, from kingdom to subspecies. If a fluorescent molecule is
attached to the probe, individual bacterial cells can be identified by using an
epifluorescence microscope. Since the 16S rRNA sequence of over 2,000 bacterial
species is currently known (Larsen et al., 1993), most bacterial groups can be
included in searches for taxon specific 16S rRNA sequences.
Presently, species and group-specific hybridization pro bes can be developed
on a rational basis, the essence of which lies in the identification of a unique
F. Schut 71
sequence of approximately 20 nucleotides in the sequence of the target
organism( s). Shorter stretches (10 bases) harbour the danger th at the same
sequence occurs at other (unforeseen) regions in one of the three ribosomal
RNA's. Long sequences (50 bases) have the disadvantage that the mismatches necessary for discrimination between target and non-target cells - do not result
in a significant decrease of the dissociation temperature of the hybrid complex.
This dissociation temperature can be estimated from the relationship
T ct = 81.5 + 16.610g(Na +) + 0.41(%G + C)-820jprobelength
(Stahl and Amann, 1991).
The 16S rRNA contains various 'conserved' regions essential for the biochemical
function of the ribosome. Such regions are used as annealing sites for primers
used in PCR amplification and sequencing of the 16S (Lane, 1991). Other
regions are hypervariabie by nature and can serve as target regions for species
and subspecies-specific hybridization probes. Sequences of intermediate
specificity can also be found and these can be used to distinguish higher
taxonomic groups.
During an extensive study, using cultural methods of enumeration, some 200
species of bacteria have been isolated from human faecal samples (Finegold et
al., 1974). Approximately 30 of these are numerically important and represent 10
distinct genera. Genus specific pro bes could thus prove appropriate in describing
community structures at large. However, the natural classification system for
bacteria, based on phenetic characteristics, is known not to comply fully with
genetically based methods: not all taxonomic genera are monophyletic on the
basis of their 16S rRNA sequence. Eubacterium spp., for example, are clustered
with several Clostridium and Peptostreptococcus spp. and peptostreptococci
themselves are spread over several distinct phylogenetic clusters (fig. 1). Within
these phylogenetic clusters, however, conserved regions can be identified and
16S rRNA probes designed for such regions can be used to identify the majority
of the population to a well defined taxonomic level. Only a dozen pro bes is thus
required for the enumeration of the various phylogenetic groups of the gut flora
(fig. 1).
Although many copies of the rib os ome should ensure sufficient signal, reality
is of ten far from ideal. The accessibility of the target region, the number of
fluorochromes attached to the probe, the hybridization tempera tu re, the
metabolic status of the cells and the level of autofluorescence of non-target cells
are important factors in obtaining sufficient specific fluorescent signal. Optical
aid in the form of CCD-camera's is often required for small and slowly growing
cells with little rRNA. Image analysis can be very helpful in discriminating
between non-fading autofluorescence and rapidly fading fluorochrome
Fig. 1. (opposite page) Unrooted distance tree obtained after Kimura-2 parameter analysis and
neighbour joining of 200 nucleotides of the 16S rRNA sequence of some bacterial species isolated
from human faecal samples. Analysis was performed using MEGA software. The assignment of the
twelve large phylogenetic clusters is supported by findings of the RDP project (Larsen et al.,
1993). Fluorescent oligonucleotide pro bes have been deve10ped for all clusters assigned.
72 165 rRNA hybridization probes
F. Schut 73
Fig. 2. Fluorescence lifetime imaging of walled mycoplasmas subgr. 11 in human faecal flora using
16S rRNA hybridization probes. For a period of 8 sec FITC fluorescence is recorded (panel A).
Subsequently, a DAPI image is recorded under UV illumination (panel B). Af ter 20 sec of UV
illumination during which FITC fluorescence but not autofluorescence fades significantly, a second
FITC image is recorded (panel C). Graphical subtraction of the image in panel C from that in
panel A produces the frame in panel D with c1early distinguishable hybridized cells.
fluorescenee (fig. 2). Using such fluorescenee lifetime imaging techniques, only a
few positively hybridized cells can be distinguished among several thousand nontarget cells in a microscope field (manuscript in preparation). This is neeessary
to enumerate those bacterial clusters that form a minority of the population,
(e.g. streptococci).
Wbole eell in situ bybridization of faeces
We have developed a relatively simple protocol for the enumeration of
hybridized eells in faecal samples (fig. 3): A known quantity of homogenized
faeees is resuspended in phosphate buffered saline (PBS). For cultivation,
appropriate dilutions are made and eells are inoculated on the surfaee of selective and non-selective agar media. Plates are incubated anaerobically at 37°C.
For microscopic enumeration and probe hybridization, eells are fixed for a mini74 165 rR NA hybridization pro bes
1 gr
Faeces
9ml
PBS
Formaldehyde PBS Ethanol
1 h
Fig. 3. Protocol for the hybridization and enumeration of faecal bacteria.
mum of 2 hrs in 4% paraformaldehyde. Debris is separated from bacterial cells
by low-spin centrifugation. Cells are pelleted, washed in PBS and suspended in
gradually increasing concentrations of ethanol (to 75%). After pelleting, the cells
are resuspended in hybridization butTer and FITC-Iabelled probe is added.
Hybridization is carried out at a temperature 2°C below the dissociation temperature of the probe for a minimum of 2 hrs. After hybridization, cells are
stained with 4',6-diamidino-2-phenylindole (DAPI), filtered onto a O.2,um poresize polycarbonate filter and washed with hybridization butTer at hybridization
temperatures. The filter is mounted on a micro scope slide and viewed under an
epifluorescence micro scope equipped with the appropriate excitation and emission filters for FITC and DAPI. An image analysis system (Wilkinson, 1995) is
used when fluorescence signals of target cells are too low to allow proper
enumeration by eye.
Laboratory trials
We investigated the population of bifidobacteria in the faecal samples of ten
volunteers. During this study, cultural methods were compared with the 16S
hybridization method. A genus-specific Bifidobacterium pro be targeted for position 164 (Bif164) was used (Langendijk et al. , submitted). The mean number of
culturable bifidobacteria (± S.E.) from all samples was 2.45 (± 1.40)x109 per
gram of wet faeces. The mean number of total culturabie anaerobes on BBA
from all samples was 3.87 (± 1.73)xlO lO per gram of wet faeces. On average,
bifidobacteria accounted for 6.9% ± 3.3% of the total culturable population. For
F. Schut 75
all ten individuals investigated, the total counts of faecal bacteria as obtained
on non-selective anaerobic brucella blood agar were significantly lower than
the total microscopic counts on membrane filters with DAPI as a nuclear
stain.
The membrane filter technique was also used to enumerate the number of bacteria that hybridized with probe Bif164. In theory, all bifidobacteria present in
faeces, including those that are nonculturable on agar media, should be detected
when using this probe hybridization technique. In analogy to the discrepancy
between cultural counts and DAPI total counts, we expected the cultural counts
of bifidobacteria to be considerably below probe hybridization counts. By contrast, the number of culturabie bifidobacteria enumerated on BIF agar (Sutter
et al., 1985) was not significantly different from the microscopic count using the
Bifidobacterium probe Bif164 (av. 2.38xl09 per g wet faeces). Based on DAPI
total counts, Bifidobacterium spp., on average, accounted for 0.8% ± 0.4% of the
total population. This implies th at nearly all bifidobacteria in human faeces are
culturable, possibly as a result of their oxygen tolerance and th at the contribution of bifidobacteria to the total intestinal microflora is largely overestimated
when using cultural methods as sole method of enumeration. Such overestimations can approach an order of magnitude.
In a second study, we have enumerated members of the Bacteroides vulgatus
subgroup (Bacteroides fragilis group). Cultural methods of enumeration result in
counts half of those obtained when using a subgroup-specific hybridization
probe. Currently we are developing and testing other phylogenetic group-specific
pro bes for detection of major subpopulations of bacteria in human faeces.
Included are probes specific for streptococci, the Clostridium coccoides subgroup,
Bacteroides distasonis (species-specific), and subgroup 11 of the walled
mycoplasmas (Larsen et al., 1993) (see also fig. 2). Together these pro bes 'cover'
around 50% of the total microscopic count in faecal samples.
Conclusions and prospeets
Phylogenetic group-specific 16S rRNA hybridization probes can successfully be
used for the enumeration of the major subpopulations of bacteria in human
faeces. This method has several advantages to the classic cultural method for
enumeration. The most important one is the relative ease and speed of the
technique. With FITC-Iabelled 16S probes several samples can be processed per
day. Once large-scale dynamics in composition of major subpopulations have
been established species-specific pro bes can be used to elucidate the more
detailed dynamics within such phylogenetic clusters. Before this can be achieved,
the sensitivity of the technique is limited to threshold population densities of 1
positive cell per 500 negative cells, i.e. around 0.2% of the population. Several
species of the intestinal flora, specifically the aerobic and facultatively anaerobic
potentially pathogenic bacteria, drop well below this level. This is wh ere the
cultural technique is superior.
76 16S rRNA hybridization probes
The use of flow cytometers, in which several tens of thousands of cells can be
analyzed within minutes, will prove useful in the future. Flow cytometry in combination with 16S pro be hybridization dramatically increases the number of
samples th at can be analyzed per study. In preliminary trials we have
enumerated six phylogenetic clusters of intestinal bacteria in a single sample,
some with good precision wh en compared with microscopic counts, in less than
a day. The hybridization time being the rate limiting step. Although several
problems related to signal to noise ratio still have to be overcome, 16S rRNA
hybridization has al ready proven to be a very powerful technique in ecological
studies of intestinal flora. Investigations into the dynamics of the community
structure will help to fill the gaps in knowledge on the interaction between the
host immunity and gut microflora. Such knowledge is crucial to the understanding of gut flora related pathogenesis.
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78 165 rRNA hybridization pro bes
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F. Schut
Department of Medical Microbiology
University of Groningen
Oostersingel 59
9713 EZ Groningen
The Netherlands
Tel: + 31 50633507
Fax: + 31 50633528
E-mail: f.schut@med.rug.nl
F. Schut 79

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