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Biotech.Adv. Vol. 13, pp. 75-90,1995
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THE USE OF IMMUNOLOGICAL METHODS TO DETECT AND
IDENTIFY BACTERIA IN THE ENVIRONMENT
M. SCHLOTER, B. At~MUSand A. HARTMANN
GSF-Forschungszentrumfiir Umwelt und Gesundheit, ln.stitut fiir Boden6kologie,Neuherberg,
Postfach 1129, D-85758 Oberschleissheim, Germany
ABSTRACT
Immunological detection methods have become increasingly important in
microbial ecology for the tracking of specific microorganisms and for community
analysis. For a reliable application of these techniques, the monoclonal antibodies or
polyclonal antisera used have to fulfill several quality criteria. Cross reactivity, cellular
localization of the antigenic determinant, affinity characteristics and the expression of
the antigenic determinant at environmental conditions have to be determined.
Immunological methods can be used for the identification, quantification and
enrichment of specific bacteria in extracts as well as for the visualization of cells in situ.
The sensitivity of advanced immunological methods can be compared to PCR
techniques. Using image processing of epifluorescence micrographs or confocal laser
scanning microscopy, the immunofluorescence approach can now be applied to study
complex environmental samples.
KEYWORDS
monoclonal antibody, polyclonal antiserum, antigenic localization, cross reaction,
affinity characteristics, antigenic stability, agglutination techniques, ELISA, flow
cytometry, immunofluorescence, immuno-isolation
INTRODUCTION
It is the major goal of ecological studies in microbiology to understand the life of
specific bacteria in their habitats and their interaction with other biota or their chemical
and physical environment. For a long time microbial ecology was unable to function at
75
76
M. SCHLOTERet al.
this level, because methods for the selective identification of certain microbes in a
complex environment were not available. Classical microbiological techniques use
selective growth media for certain physiological groups of bacteria. However, the
detection at the species or strain level is often not possible using cultivation approaches
due to the lack of stringent growth criteria (57). In addition, it has been repeatedly
reported that bacteria may reach a viable but non culturable state under environmental
conditions (54, 58). Artificial transposon or plasmid located antibiotic resistance
markers made specific tracking of introduced bacteria possible (4). However, many
bacteria from water or soil samples show natural antibiotic resistance (30), which can
bias this approach. Grant et al. (25) cloned the lux CDABE operon from Vibrio fischeri
in Erwinia carotovora and it was possible to detect the inoculated microbes by
luminescence. However, a genetically engineered microorganism might show different
properties in the environment compared to the wild type (30). An alternative for the
detection of specific bacteria in the environment is the direct identification approach
using oligonucleotide probes and specific antibodies. The use of oligonucleotide probes
for the detection of microbes in complex ecosystems was recently reviewed by
Hartmann et al. (27). Immunological detection methods are based on the ability of
antibodies to recognize specific three-dimensional structures (e.g. parts of proteins or
polysaccharides) of biological macromolecules. These techniques nowadays play an
important role as diagnostic tools in medicine and food technology (35). Bohlool and
Schmidt (10) introduced the immunofluorescent approach for microbial ecology studies
using polyclonal antisera. With the invention of monoclonal antibodies (37), which
bind to a single, well defined antigenic determinant, the potentially improved detection
of bacteria became possible. In phytopathology, serological techniques became
increasingly important for the tracking of specific microorganisms and for community
analysis (64). In this review we discuss quality criteria for monoclonal antibodies and
polyclonal antisera and cover advanced methods for in situ localization of microbes as
well as recent developments for the selective identification, quantification and
enrichment of bacteria in environmental samples.
CHARACTERIZATION OF THE QUALITY OF ANTIBODIES
Antibodies are produced by immunization of animals. To obtain sufficiently
high densities of pure bacterial cultures, the microbes have to be isolated from the
environment and cultured in an adequate media. Therefore, "non-culturable" bacteria
cannot be used for the production of antibodies. Monoclonal antibodies are produced in
vitro by cell-culture techniques (37): B-lymphocytes of an immunized animal, e.g. a
mouse, are isolated and fused with myeloma-cells, which enable them to grow
continuously. One fused hybridoma-clone produces one type of antibody. Antisera, in
contrast, are "natural" mixtures of different antibodies, produced by immunized
IMMUNOLOGICALMETHODS AND BACTERIA
77
animals, due to the polyclonal nature of the immune response and to the mixture of
antigenic determinants generally used for immunization. After several weeks of
immunization, animals (e.g. rabbits) are bled and the serum is used for further
examinations (14).
For the evaluation of the quality of an antiserum or an monoclonal antibody,
four features must be taken into account:
Localization of the Antigenic Determinants
Depending on the location of the antigenic determinant, samples may have to be
pretreated before immunological detection is possible. For detection of the
enterobacterial common antigen (ECA), Obst et al., (52) pretreated the bacteria by
boiling water. If the antigenic determinant is localized directly on the cell surface,
detection with antibodies without further treatment is possible (66). Therefore,
antibodies which recognize cell surface structures are preferable for the use in complex
ecosystems.
The outer membrane of Gram-negative bacteria harbours many different
lipopolysaccharides and proteins. Furthermore, slimes which are rich in carbohydrates,
as well as protein capsules can form the outer envelope of bacteria. The use of outer
membrane proteins for the production of antibodies has in many bacterial families
demonstrated the conserved structure of certain membrane proteins (26, 49). In
addition some monoclonal antibodies are described which react strain-specifically with
outer membrane proteins (60, 67, 77). The outer part of lipopolysaccharides, the ospecific side chain, is a very variable region on the bacterial cell surface. The sequence
and connection of the sugar molecules determine the o-antigenic structure of a bacterial
strain (43). The high specificity of o-antigens has been used for serotyping of pathogenic,
Gram-negative bacteria (34) and some microbes isolated from natural ecosystems (3).
The lipid A part of lipopolysaccharides is an ubiquitous and is very conserved in
structure. Monoclonal antibodies, produced against the lipid A of E. coli and
Pseudomonas aeruginosa showed a high of cross reactivity with other bacterial families
(49, 61).
Cross Reaction
Polyclonal antisera contain a variety of antibodies against typical and less typical
structures of the target. The latter can lead to non-specific cross reactions. The degree of
cross-reactivity depends on the number of unspecific antibodies in the serum. If
polyclonal antisera are produced, usually subfractions of the microorganisms e.g. cell
wall extracts or purified components are used for immunization (20). Schr6der and
Kunz (68) described a method to remove unspecific compounds from polyclonal
78
M. SCHLOTERet al.
antisera by preabsorption steps. In contrast, monoclonal antibodies ensure less crossreactivity with non-target microorganisms, because a single antibody is directed against
one antigenic determinant only. Depending on the distribution of the particular
epitope, a monoclonal antibody can show strain- (67), species- (8) or family-specificity
(52). However, some cross-reaction of even strain specific monoclonal antibodies with
structures of the humic acids must be taken into account, because the organic fraction of
soils contains a large number of different antigenic determinants (30).
Affinity Characteristics
The interaction of antibodies with the target antigen is characterized by the
equilibrium which is reached between the initial molecules that entered into the
reaction and the complex. The equilibrium state is described by the binding constant kb
(21). The kb is therefore an important means of characterizing antibodies. Since
polyclonal antisera are a mixture of different antibodies, it is difficult to assign a single
kb for the whole serum. This is in contrast to monoclonal antibodies. The kb of a
monoclonal antibody with high affinity for the antigen can be about 10-l° M d, when
optimal temperature and medium conditions for the immunological reaction are used.
Stability of Antigenic Determinants
As detection with antibodies is based on the bacterial phenotype only the stable
expression of antigenic determinants makes the detection sensitive and reliable. If an
epitope is expressed only during certain stages of growth or u n d e r certain
environmental conditions, the target microorganism can not be detected (30). For
example, components of the flagella are not expressed under all environmental
conditions (48) especially if monoclonal antibodies are used. It is therefore important
that the recognized antigenic determinant is expressed constitutively.
IMMUNOLOGICAL TECHNIQUES FOR THE LOCALIZATION, IDENTIFICATION,
QUANTIFICATION AND ENRICHMENT OF BACTERIA IN COMPLEX ECOSYSTEMS
In situ Localization Techniques
Immunofluorescence techniques. Immunofluorescence staining (IF) was suggested for
the identification of bacteria by Kikumoto and Sakamoto (36), who identified
Enterobacter aroideae in host plant tissues and soil using epifluorescence microscopy.
The antibodies can be coupled directly with a fluorescence marker (23), or marked with
a secondary antibody coupled with a fluorescent dye (83). The use of the enhancer
IMMUNOLOGICAL METHODS AND BACTERIA
79
system streptavidin-biotin is not recommended, because streptavidin can not only bind
to biotin coupled to antibodies, but also to every biotin molecule present in the sample,
which could dramatically increase the background fluorescence (28). To reduce
unspecific binding of the antibody to soil or roots, Bohlool and Schmidt (10)
preincubated the soil samples with gelatin. IF-staining has equal sensitivity as PCR for
the detection of target bacteria at a level of 103 104 cells (83). IF is mainly used for in
situ studies of bacteria e.g. on roots or substrate particles. IF can be used for quantitative
in situ studies and culturable as well as non-culturable cells are stained. A direct
confirmation of positive cells is not possible. Immunofluorescence-colony staining
(IFC) has been developed for the detection of culturable bacteria at a level of 10 - 100
bacteria/ml extract (39, 82). IFC allows the direct confirmation of positive cultures.
However, its application for in situ studies is limited because of its poor spatial
resolution. Examples of the use of immunofluorescence techniques to localize bacteria
in situ are given in Table 1.
-
Image analysis and the use of CCD cameras. The introduction of image processing and
analysis has improved the field of microscopic examinations in microbial ecology. The
benefit provided by these computer-assisted methods ranges from simple contrast
enhancement to automated cell counting and determination of biomass (13). Any
computing of photomicrographs needs a digitized image which can be obtained by
converting images recorded with a video camera. Sieracki et al. (72) used this method to
enumerate planktonic bacteria and also compared different algorithms for accurate cell
sizing (73). A similar approach has been used to estimate the cell size of bacteria in
different soils (9).
Comparison of different analog-to-digital converters revealed charge-coupled
device (CCD) cameras to be superior to video-based systems in detecting low signals of
small fluorescence-stained bacterial ceils (85). CCD cameras have been used to detect
single cells of genetically modified bacteria in soil (74) and in plant material (70).
Scanning confocal laser microscopy. When viewing thick samples in conventional
microscopy, results frequently are severely affected by the limited focus area. An exact
localization of investigated microorganisms is impossible in many cases. The detection
of the fluorescence signals is often hampered by high levels of background fluorescence
conferred by e.g. plant material and organic or clay particles in soil samples. As an
alternative to the thin sectioning of the specimen which may introduce various
artifacts, optical sectioning methods are available. Eliminating out-of-focus information
can be accomplished by processing conventionally obtained images with deconvoluting
algorithms (1).
This strategy however, although proposed to be equally powerful at a lower cost
level (24), has been outcompeted by the use of scanning confocal laser microscopy
80
M. SCHLOTERet al.
(SCLM). The physical properties (86) as well as biological applications of this technique
(71) have been already described. Briefly, single spot illumination of a focused laser
beam as well as a pinhole allowing only in-focus-signals to be detected lead to an
accurate optical section of specimen and virtually eliminate blur originating from
unfocused parts. The instruments available at the moment provide software covering
image processing as well as three-dimensional reconstruction of an object analyzed by a
series of sections. The combination of SCLM with fluorescence labeled antibodies
allowed the three-dimensional reconstruction of the colonization patterns of
Azospirillum brasilense strains on root samples of inoculated wheat plants (66). Using
different laser wavelengths for excitation, it is even possible to perform double or triple
staining of bacteria with different antibodies, or antibodies and oligonucleotide probes
(7).
Immunogold techniques. Immunogold detection is mainly used for in situ detection of
bacteria in ultrathin cuts (70 nm) of imbedded environmental samples with a
transmission electron microscopy (42). The detection limit is about 105 bacteria. If
immunogold detection is combined with silver enhancement techniques thin cuts (5
~m) and light microscopic techniques can be used (32). Some examples for the
application of immunogold techniques to localize bacteria in situ are given in Table 1.
Table 1: Examples of the application of antibodies in the in situ localization of bacteria
in different environments using Immunofluorescence (IF)- or Immunogold
(IG)-techniques
Antibody specificity
Acetobacter diazotrophicus
Azoarcus spp.
Azospirillum brasilense
Bacillus anthracis
Bradyrhizobium japonicum
Enterobacter aroidae
Escherichia coli
Erwinia carotovora
Erwinia chrysanthemi
Flavobacterium sp.
Pseudomonas putida
Rhizobium sp.
Vibrio cholerae
Xanthomonas campestris
Technique
IG
IG
IF, IG
IF
IG
IF
IF
IF
IF, IG
IF
IG
IF, IG
IF
IF
System
plant
plant
rhizosphere
soil
rhizosphere
rhizosphere
lake water
slurry
slurry
soil
water
rhizosphere
water
plant
Reference
31
29
42,66
59
32
36
11
83
82
45,48
60
32
12
3
IMMUNOLOGICAL METHODS AND BACTERIA
81
Identification, Ouantification and Enrichment Techniques in Extraction Susvensions
Extraction methods. In the following section, techniques are described which are based
on the extraction of bacteria from the environment. When microbes are extracted from
a soil matrix, considerable attention should be given to the method used. The methods
described so far for the extraction of microorganisms from environmental samples
employ an initial treatment to disperse inorganic material, especially to disaggregate
clay. A step follows, by which organisms are separated from the bulk of clay before the
final purification by density gradient centrifugation. This step is critical for the controls
and the possibility for scaling up.
Agglutination-techniques. Agglutination-techniques, which make the formation of an
antibody-antigen complex visible, are well known for the identification of bacteria. The
reaction is based on an agglutination of an antigen with an antibody. In most cases,
antibodies are further coupled with latex particles, which results in a better signal (16).
The detection limit of these techniques is about 108 bacteria/ml (63). Due to the weak
detection limit, these techniques cannot be used without first selectively enriching for
the microorganism to be detected.
With the introduction of the agar double diffusion precipitin test by Ochterlony
(53), agar precipitation tests (APT) became widely spread in microbiology. For example
APT has been used to identify strains of Pseudomonas syringae (79) or Pseudomonas
pisi (78). As the detection limit did not improve, compared to the classic agglutination
techniques, selective enrichment of the microorganisms must also be used with APTtechniques.
ELISA techniques. The most commonly used method for estimation of bacteria is the
non-sandwich, indirect Enzyme-Linked Immunosorbent Assay (ELISA). Bacteria are
coupled covalently to microtiter plates either by using an alkaline buffer system (65) or
polylysine (77). The detection of the antigen-antibody complex using enzymatic
reactions results in an increase of sensitivity as compared to fluorometric detection
methods (65). The enzymes that are most often used are peroxidase (65) and alkaline
phosphatase (62). The detection limit depends on the antibody used (see above) and the
substrate for the enzyme. For polyclonal antisera and colorimetric detection of the
b o u n d enzyme a detection limit of 106 bacteria/ml is reported (6). By using a
preenrichment cultivation step for the bacteria, in semiselective media, an increase in
sensitivity can be achieved (9). When the avidin-biotin system was applied an increase
in sensitivity was reported (40). Using monoclonal antibodies with a high affinity
against a cell surface antigen, and highly sensitive chemoluminescence detection based
on b o u n d peroxidase and luminol, a detection limit as low as of 102 bacteria was
achieved (65). As the number of light counts is proportional to the number of bacteria,
82
M. SCHLOTERet al.
this system can be used for a quantitative determination of bacterial numbers in
environmental samples e.g. soil extracts. Other sensitive immunoassays have also been
described, e.g. an enzyme-amplified lanthanide-luminescence immunoassay (22). Thus
the sensitive detection of antigen-antibody complexes using radioisotope techniques
can be avoided.
The dot-immunobinding assay is an enzyme-based method for the qualitative
detection of microorganisms. Nitrocellulose membranes are spotted with samples (60).
The principle design of the dot-immunobinding assay is equal to ELISA. The sensitivity
can be compared with the classic ELISA depending on the enzyme and substrate system
in use.
Some examples of the use of ELISA techniques to detect and quantify bacteria in
situ are given in Table 2.
Table 2: Examples of the application of antibodies for the estimation of bacteria in
different environments using ELISA techniques
Antibody spedficity
Alcaligenes eutrophus
Azospirillum brasilense
Bacillus licheniformis
Bradyrhizobium japonicum
Escherichia coli
Enterobacter cloacae
Erwinia carotovora
Erwinia chrysanthemi
Pseudomonas cepacia
Pseudomonas fluorescens
Pseudomonas putida
Rhizobium japonicum
Rhizobium spp.
Salmonella spp
Thiobacillus [erroxidans
Technique
ELISA
ELISA
ELISA
ELISA
ELISA
ELISA
ELISA
ELISA
ELISA
ELISA
ELISA
ELBA
Dot blot
Dot blot
Dot blot
System
ake water
rhizosphere
seawater
rhizosphere
lake water
soil
slurry
slurry
plant
sediment
soil
rhizosphere
rhizosphere
water
sediment
Reference
33,58
40,65
50
75
11
58
83
82
81
51
60
6
55
17
5
Flow cytometric techniques. Flow cytometry can be u s e d alternatively to study
fluorescence-labeled bacteria. In a flow cytometer, a stream of cells moving in single file
crosses a focused laser beam. A number of arranged photomultiplier tubes can record a
couple of different parameters simultaneously, like scattered light or the signals of the
bacteria labeled with fluorescence-coupled antibodies, fluorescence-coupled
oligonucleotide probes or DAPI. Although recommended several times (69), flow
cytometry is rarely applied in microbiology possibly due to the high cost of the
equipment and the fact that the bacterial cell size is close to the detection limit of most
of the instruments routinely used in cell biology. Flow cytometry enables one to
automatically analyze thousands of cells within a few minutes, providing much better
IMMUNOLOGICAL METHODS AND BACTERIA
83
statistical data than tedious microscopic counting. Most applications reported are in
aquatic microbial ecology, where flow cytometry enables both the monitoring of an
introduced organism (18) and the investigation of natural microflora (47). The use of
flow cytometry in aquatic microbiology has been reviewed recently (80). Not much is
reported to date about flow cytometric analysis of bacterial populations in soil. Page and
Burns (56) have described the recovery of an inoculated Flavobacterium from soil by
fluorescence-coupled antibodies and flow cytometry. However, the cell numbers
detected by flow cytometry were lower than in direct counts. As the desorption and
disaggregation steps from soil matrix are not yet optimized, it appears that the method
needs further improvement.
Immuno-isolation techniques. A serological method for immuno-isolation of Bacillus
polymyxa from root free soil and the rhizosphere of wheat was developed by Mavingui
et al. (46). Microtiter plates were coated with anti-Bacillus polymyxa CF43 antibody.
After the addition of soil samples and several incubation- and washing steps, the bound
bacteria were desorbed with 0.1 M KC1. Using classical methods of enumeration, the
population size of Bacillus polymyxa was less than 0.1% of the total microflora. When
the immunotrapping method was used for enrichment, this percentage was increased
to 20%.
Alun et al. (2) used a immunocapture technique which is based on antibodies
coupled with paramagnetic particles. This technique was originally developed for the
isolation of lymphocyte subsets directly from blood samples (38, 84). Alun et al.
described the use of this method for the isolation of Pseudomonas putida mt 2 directly
from lake water by using bacterial flagella as a target antigen for capture. The
paramagnetic particles had a size of 4.5 ~m. The bacteria marked with the coupled
antibody were isolated from the lake sample with a magnetic particle concentrator.
Important considerations for this technique were not only the density of the antigen on
the cell surface but also the firmness of binding of the antigen to the bacterial surface
which is needed to tolerate the shear forces during the washing steps. The recovery of
target cells released into lake water at concentrations of between 102 and 105 CFU m1-1
was about 20%. The unspecific binding of cells to uncoupled paramagnetic particles
varied between 1% and 0.1%. Christensen et al. (15) used this technique to recover
thermophilic sulfate-reducing bacteria from oil field waters. Antibodies directed against
lipopolysaccharides of Thermodesulfobacterium mobile were coupled to paramagnetic
particles with a size of 2.8 ~m . The prevailing cells immunocaptured with this
antibody were morphologically and serologically similar to Thermodesulfobacterium
mobile type strain cells. Thermodesulfobacterium mobile was not detected in these oil
field waters by classical isolation procedures. Extraction with antibody-coated
paramagnetic particles allowed pure strains to be isolated directly from primary
enrichment cultures without prior time-consuming subculturing and consecutive
84
M. SCHLOTERet al.
transfers to selective media. Dye (19) reported the use of immunomagnetic separation
for the enrichment of Rhizobium strains from soil samples. By using antibodies
coupled to 2.8 ~m paramagnetic particles and some optimization steps of Christensen's
procedure for soil, a 200-fold enrichment of Rhizobium was achieved. In food
technology this method has become a widespread alternative to enrichment broths
mainly for the detection of pathogens in food (44, 76).
Using flow cytometry and fluorescence coupled antibodies, a specific cell sorting
and enrichment should be also possible, but there is not much experience yet available
concerning this application.
CONCLUSION
The diversity of methods available for the tracking of microbes in the
environment has increased greatly since the introduction of recombinant DNA
technology, and improved immunological methods. The greatest advantage of
antibody-based detection is that it allows the visualization of cells in situ, as well as the
option for a sensitive quantification. Depending on the technique used, all cells which
carry a recognizable antigen can be detected, whether the bacterium is culturable or not.
The combination of modern biophysical techniques (flow cytometry, confocal laser
scanning microscopy, image analysis, luminometry) and monoclonal antibodies, which
have a well known antigenic epitope and a defined affinity constant, make it possible to
follow the fate of bacteria introduced in the environment. The sensitivity of ELISA
techniques has increased dramatically and cell densities of 102 per gram of soil can be
detected. Magnetic particles coated to a second antibody offer the possibility of
enrichment and retrieval of living cells.
In many cases, two or more detection methods have to be combined to get precise
information about the fate of a specific bacteria in the environment. It will be very
useful to combine antibody techniques and the oligonucleotide approaches. Combining
methods provides greater certainty for the unambiguous detection of particular target
strains, as well as the dynamics of different parts of populations.
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