Evaluation of Polymicrobial Involvement Using Fluorescence In Situ Hybridization (FISH) in Clinical Practice

Alexander Swidsinski and Vera Loening-Baucke

The involvement of microorganisms in infection is often deduced from their presence during disease and absence in healthy humans or animals, which is an oversimplification. The proof of direct involvement is decisive. Fluorescence in situ hybridization (FISH) combines the specific identification of microorganisms and the morphological aspect of the host tissues and is as a consequence especially helpful for these purposes.
The present manuscript describes FISH methods which we use in ambulatory patients for Polymicrobial Infections and Bacterial Biofilms of the Charite Hospital to visualize pathogens (pathogenic consortia) in clinical samples.

The contemporary understanding of infections is based on identifying microorganisms in diseased persons that are absent in healthy persons. However, the presence of a bacterium (or bacteria) in health does not mean that it is healthy or at least harmless (chapter by Benedetta Bottari et al. "FISHing for Food Microorganisms").

Neisseria meningitides is part of the normal nonpathogenic flora in the nasopharynx of up to 5–15 % of adults. Its causative involvement in meningitis is however beyond doubt, since it is the only bacterium found in the inflamed cerebrospinal fluid.

Detecting bacteria at the site of an infection is more appropriate for identifying the infectious agent than its absence within normal colonization. Difficulties arise when multiple organisms are present at the infection site. In this case, the criminological experiment is decisive. A transfection of the suspected bacteria to healthy animals helps to uncover potential pathogens.

However, when none of the involved microorganism causes infection, does this exclude the harmful potential of a group? No.

Figure 1
Figure 1
Isolated islands of bacteria attached to desquamated epithelial cells 1,000: mouth and surgically removed material; universal bacterial probes (Eub338 FITC, green fluorescence),and Burkholderia (Burkho-Cy3, yellow fluorescence) on the left.
Unspecific DAPI stain of the DNA is overlaid with Burkholderia fluorescence on the right.

A well-known example is the induction of Vincent’s angina by Rosebury, who transferred plaque-infected material holding different components While single microorganisms were innocuous and incapable to initiate infection, it was possible to cause disease with the combination of different species. The required consortium was called the “Pathogenic Quartet” and included the following species that were isolated from a patient diagnosed with Vincent’s angina: a spirochete, a fusiform Bacillus, a Vibrio, and an anaerobic Streptococcus. Rosebury’s conclusion was that each of these species is a member of healthy indigenous flora, but they may cooperate and form an unmanageable complex structure.

In nature, microorganisms build diverse consortia in which single participants complement each other and display specific properties, which cannot be discovered in one of the participants or in other associations. Can some of these consortia be pathogenic? Yes.

We should await the presence of such consortia on surfaces which contact the outer world such as the skin, mouth, intestine, vagina, etc.

Figure 2
Figure 2
Multicolor FISH of superficial tonsils infiltrates 400.
Gamma proteobacteria as a part of superficial infiltrate (Gam42-Cy5, red fluorescence).
The main group involved in infiltration is a Fusobacterium nucleatum (Fnuc Cy3, yellow fluorescence)

Can the role of these consortia be proved in transfection experiments? Presently, no.

Rosebury transfected not really a consortia but a mix of isolated cultured single strains. This should be only in exceptional cases successful. The problem is that until now, we are unable to cultivate polymicrobials. When more than three bacterial strains are incubated in the same culture, their growth is getting unpredictable, and one of the strains suppresses and overgrows the others. Polymicrobial culture is a challenge for future research.

In the absence of polymicrobial cultures, a link between the consortium of distinct species and their involvement in disease can be established directly by visualizing pathogenic consortia within biofilms and microbial infiltrates in host tissues via fluorescence in situ hybridization (FISH).

Figure 3

Figure 3
Prolific bacterial biofilm covers the colonic mucosa in a patient with Crohn’s disease 1000 multicolor FISH:
(A) DAPI stain of DNA structures;
(B) Bac303 Cy3; Bacteroides; orange fluorescence;
(C) EREC Cy5; Eubacterium rectale; and red fluorescence

We have successfully used this approach in case of colonic cancer, inflammatory bowel disease, FISH combines the specific identification of microorganisms and the morphological aspect and is especially helpful for identifi- cation of polymicrobial consortia involved in local infection. Each single bacterium possesses 103–105 ribosomes of which each ribosome owns the same copy of ribosomal RNA. Some of the regions of the rRNA are strain-specific; others are universal for species, families, or even kingdoms. Oligonucleotides synthesized complimentary to rRNA sequences and labeled with fluorescent dye are called FISH probes. When added to samples containing bacteria FISH probes hybridize with the rRNA of the bacterial ribosomes. No additional enhancement of fluorescence is necessary and bacteria can be visualized directly with a fluorescence microscope due to the large number of ribosomes in each bacterium.

Multicolor FISH enables the identification of potentially all bacterial groups in spatial relation to each other and in relation to histological structures of the host. Any biological material can be studied for in situ presence of bacteria and bacterial biofilms, including smears from tonsils or vagina, desquamated epithelial cells in the urine, tissue biopsies, surgically removed tissues, saliva, perspiration, exudation, sperm samples, and medical devices removed from the body (Figs.1, 2, 3).

FISH protocols described here are standard protocols, which are used for ambulatory patients in the Laboratory for Molecular Genetics, Polymicrobial Infections, and Bacterial Biofilms at the Charite Hospital in Berlin, Germany.

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