LEECH THERAPY INDONESIA

presently unable to be cultivated [36,37]. Technological
developments in the culture-independent profiling of
microbial community complexity and diversity have
revealed a plethora of novel cohabiting microorganisms
that far outnumber the culturable organisms. These developments
have also greatly advanced our understanding of
the residential microbiota of digestive tracts, to which
essential roles in host biology have been attributed, such
as the provision of essential nutrients (reviewed in Ref.
[3]), development [38], energy balance [39] and the priming
of immunity [40,41]. Interesting parallels in the digestivetract
microbial composition among various host species
have been described [39,42], which raises the question of
whether their functional roles are universal or tailored to
the different hosts.
A recent culture-independent study discovered the presence
of a second symbiont in the crop that we have been
unable to cultivate in the laboratory [43]. The 16S rRNA
gene sequence indicates that it is a relative of Rikenella,
members of the Bacteroidetes that have been found in
several different digestive tracts. An exciting aspect about
the discovery of a second symbiont is the natural occurrence
of a restricted – but not monospecific – digestive-tract
microbiota, which will enable us not only to investigate
microbe–host interactions but also to investigate the interaction
between different microbial species. Relevant features
of the Aeromonas and Rikenella species that
comprise the basic two-member microbial community in
the crop are discussed here.
Aeromonadaceae
Aeromonas species are motile, Gram-negative rods that
belong to the family Aeromonadaceae [44]. A widely noted
characteristic of Aeromonas spp. is the production of a
large number of exported hydrolytic enzymes that could
aid in the breakdown of nutrients inside the digestive tract
of animals. This family currently consists of 17 facultatively
anaerobic species that occupy a spectrum of niches
ranging from free-living occupants of freshwater to opportunistic
pathogens of fish, amphibians and humans
(reviewed in Ref. [45]), and to the digestive tract symbionts
of a variety of blood feeders including mosquitoes, the
medicinal leech and the vampire bat [17,31,34,46]. Three
Aeromonas species including A. veronii are associated with
a range of maladies including wound infection, septicemia
and diarrhea in humans [45]. Therefore, A. veronii seems to
have an innate ability to infect the digestive tracts of
multiple host species where manifestations of infection
span from pathogenesis to cooperative.
Rikenellaceae
The recurring identification of 16S rRNA gene sequences
that belong to the Rikenellaceae from a wide range of
digestive tracts is suggestive of both evolutionary adaptation
and physiological contributions towards digestivetract
ecosystems (Figure 2). All of the isolates or sequences
were obtained from a variety of gastrointestinal environments
including goat rumen, termite gut, murine cecum
and the human colon [39,47,48]. Knowledge of the
Rikenella genus is further obscured because of their
fastidious growth and obligate anaerobic requirements.
A novel Rikenella species, related to Rikenella microfusus
isolated from the cecal and fecal samples of Japanese fowl
[49], has been identified as one of two dominant residents
of the medicinal leech crop [43]. An intriguing question is
whether the leech crop is sufficiently anaerobic to support
the growth of the Rikenella symbiont or if A. veronii
has to remove residual oxygen from the ingested blood
meal to prime the microenvironment for the Rikenella
symbiont.
The presence of a basic two-member microbial community
in the leech digestive crop provides an exciting and
unique opportunity to further extend knowledge of
Rikenella species, albeit indirectly. For example, differential
antibiotic regimens might be used selectively to clear
the Aeromonas or Rikenella symbiont. The reintroduction
of various concentrations of A. veronii and/or isogenic
mutants into the host can reveal whether spatial or quantitative
alterations of the Rikenella population occur by
employing techniques such as fluorescence in situ hybridization
and quantitative PCR. Host fitness assays after
differential antibiotic treatments to examine classical life
history traits such as reproductive output, growth rate and
viability could also prove valuable towards the elucidation
of microbial functional roles.
Factors that contribute to a limited microbial
complexity
Factors that contribute to the unusual simplicity of the
leech digestive symbiosis could be derived from three
sources: the ingested blood, the host and/or the symbiotic
bacteria [17,50,51]. The complement system of vertebrate
blood contains powerful antimicrobial properties [52]. Two
lines of evidence suggest that the ingested complement
system remains active for some time inside the leech
and contributes to the specificity of the microbiota.
Heat-inactivation of the blood before feeding enables
colonization by some bacterial species that were unable to
colonize when fed to the leech in fresh blood [50]. Furthermore,
the importance of the Aeromonas lipopolysaccharide
(LPS) layer in protecting against the antimicrobial properties
of the complement system has been demonstrated [53]
by observing that serum-sensitive Aeromonasmutants with
a defect in their LPS had a dramatically reduced ability to
colonize the leech [51].
Other bacteria such as Pseudomonas aeruginosa and
Staphylococcus aureus were tested for their ability to
colonize the leech digestive tract and were able to persist
inside it but had a dramatically reduced ability to grow,
independent of the activity of the complement system,
which suggests the presence of a second layer of defense
[50]. The discovery of the Aeromonas symbiont led to
speculation that this symbiont might release antimicrobial
compounds [26]. As part of a culture-independent characterization
of the leech digestive system, the microbiota of
the intestinum in which the actual digestion of the blood
occurs was also characterized. The intestinum harbored a
more diverse microbial community with an average of eight
species detected [43]. The microbial community of the
intestinum, similar to the crop, was dominated by the
Rikenella and Aeromonas symbionts. The presence of a
more diverse microbiota despite the presence of the crop
Review TRENDS in Microbiology Vol.14 No.8 369
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