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Appl Environ Microbiol. Oct 2008; 74(19): 6151–6154.
Published online Aug 8, 2008. doi:  10.1128/AEM.00795-08
PMCID: PMC2565982

Characterization of the Digestive-Tract Microbiota of Hirudo orientalis, a European Medicinal Leech[down-pointing small open triangle]

Abstract

FDA-approved, postoperative use of leeches can lead to bacterial infections. In this study, we used culture-dependent and culture-independent approaches to characterize the digestive-tract microbiota of Hirudo orientalis. Surprisingly, two Aeromonas species, A. veronii and A. jandaei, were cultured. Uncultured Rikenella-like bacteria were most similar to isolates from Hirudo verbana.

Medicinal leeches have made a surprising comeback in modern medicine (2, 13, 24, 25). A dangerous complication from reconstructive microvascular surgery is venous congestion, reducing blood flow, consequently leading to tissue necrosis and a localized immunocompromised condition. When medicinal leeches are applied to relieve the symptoms, they secrete powerful anticoagulants and vasodilators while actively removing blood (18). The application greatly increases the chance for a successful outcome, and these benefits have led to the approval of the medicinal leech Hirudo medicinalis as a medical device by the FDA (16). The postoperative application of leeches also carries the risk of an infection with Aeromonas veronii, a symbiont of the most widely used medicinal leech, Hirudo verbana (1, 2, 7, 11, 12). Preemptive antibiotic treatment often prevents these infections; however, the increase of antibiotic-resistant aeromonads is of concern and necessitates a better understanding of the bacterial symbionts.

While the FDA has approved H. medicinalis and most suppliers of medicinal leeches market the animals as H. medicinalis, a recent study has revealed that European medicinal leeches are comprised of at least three distinct species: H. medicinalis, Hirudo orientalis, and H. verbana (19). Of these three species, H. verbana is the most commonly available one from commercial suppliers. Previously we characterized the microbiota of H. verbana and discovered an unusually simple microbial community (10, 27). Two species, A. veronii and a Rikenella-like bacterium, dominate the microbial community in the crop, which is the largest compartment of the digestive tract and where the ingested blood meal is stored. A. veronii is a gammaproteobacterium that is found in mutualistic associations with the medicinal leech, in pathogenic associations with humans and cold-blooded vertebrates, and free living in fresh water (5, 9). The anaerobic Rikenella sp. bacteria are members of the Bacteroidetes, and while difficult to culture, rRNA surveys indicate they are commonly found in digestive-tract environments (5, 15, 27). This unusual simplicity is due to the complement system of the ingested blood meal, leech macrophage-like cells that phagocytose sensitive bacteria, and other yet-to-be-identified factors of host or symbiont origin (5, 8, 20, 21). Because the geographic ranges of the European medicinal leeches are not fully characterized, suppliers inadvertently ship other Hirudo species. In light of various antibiotic resistance profiles for Aeromonas species, it is important to know whether the different species of Hirudo contain the same microorganisms.

Leeches were identified as H. orientalis first by visual comparison to published descriptions of external color patterns (22) and then through DNA barcoding. The latter involved the amplification and sequencing of 630 bp from the eukaryotic mitochondrial cox1 locus and comparison to previously sequenced representatives of European medicinal leeches (19). As in our previous studies of H. verbana, we used a combined culture-based and culture-independent approach to characterize the microbiota in the crop and intestinum (4, 27). The crop is a large organ in which the ingested blood meal is stored and where water and salts are absorbed (18). The digestion of the blood meal is thought to occur in the intestinum. Leeches were fed a sterile blood meal and kept at room temperature, 21°C (4). At various time points after feeding, animals were sacrificed, intraluminal fluid of the crop was obtained aseptically, and the intestinum was dissected intact (27). DNA was extracted using the Qiagen DNA tissue kit (Qiagen, Germantown, MD) with modifications for gram-positive bacteria, and an aliquot was serially diluted and plated on blood agar plates under aerobic, anaerobic, and microaerophilic conditions. The plates were incubated at 21°C until growth was observed.

Bacteria cultured from five H. orientalis leeches were initially screened for Aeromonas strains by restriction fragment length polymorphism (RFLP) analysis of the 16S rRNA gene (27). Because of the limited usefulness of the 16S rRNA gene to identify Aeromonas strains to the species level (14), four isolates from the crop, Ho599 and Ho636, or intestinum, Ho603 and Ho635, from two leeches were further characterized by sequencing gyrB, which was shown to be a good marker to identify Aeromonas strains to the species level (28). The gyrB gene was amplified by PCR as described by Yañez et al. (28) and sequenced as described by Silver et al. (20). Sequences were aligned using ClustalW in the Vector NTI software package (Invitrogen, Carlsbad, CA). Aligned sequences were analyzed phylogenetically with PhyML (6), employing 100 bootstrap replicates and using a general time reversible model of DNA sequence evolution previously determined to be appropriate for this genus of bacteria (19a). Reisolation of DNA and resequencing of gyrB verified that the samples were not switched.

Surprisingly, the analysis of the gyrB sequences revealed the presence of both A. veronii, strains Ho635 and Ho636, and Aeromonas jandaei, strains Ho599 and Ho603, in H. orientalis (Fig. (Fig.1).1). The sequences differed by 1.7 to 2.3% and 1.9 to 2.1% from those of the type strains within each clade, respectively. This divergence falls within the observed level of nucleotide substitutions observed within Aeromonas species (0 to 2.6%) (28). The high bootstrap support values (100% and 90%, respectively) of the clades containing the definitive strains of A. veronii and A. jandaei further support this conclusion. Thus, our data suggest that two Aeromonas species, belonging to distinct clades, can colonize H. orientalis. Our limited sampling depth did not allow us to draw conclusions about the relative abundances of the two species. Finding A. veronii in H. orientalis is consistent with this microbe being the digestive-tract symbiont of H. verbana. The discovery of A. jandaei, also recently isolated (19a) from the distantly related North American medicinal leech, Macrobdella decora, was not expected.

FIG. 1.
Unrooted phylogram of gyrB sequences from Aeromonas strains, including those cultured in this study. The tree was constructed using a maximum-likelihood analysis, and bootstrap support (100 replicates) is shown for nodes with >50%. Strains ...

This investigation revealed that two closely related leech species, H. orientalis and H. verbana, harbor different symbiotic microorganisms. We evaluated the abilities of A. jandaei and A. veronii isolates to colonize an alternate host, H. verbana, using a competition assay (20). Spontaneous streptomycin resistance (100 μg/ml) (Smr) mutants from Ho603 and Ho635 were isolated (17). The generation times of these strains and the rifampin-resistant HM21R (4) and Smr HM21S isolates from H. verbana were determined at 30°C in LB in a gyratory shaker (200 rpm) and ranged from 31 to 33 min. Each of the Smr strains was coinoculated with HM21R into heat-inactivated blood. A portion of the blood meal was incubated in a microcentrifuge tube, while the remainder of the blood was fed to H. verbana. The animals and in vitro controls were incubated at room temperature (21°C) for 41 h. The competitive index was determined as described previously (20). Interestingly, the A. jandaei strain Ho603S was outcompeted approximately 10-fold both in the blood control and inside the leech by Hm21R (Fig. (Fig.2),2), indicating that compared to the A. veronii isolates this strain had a decreased ability to proliferate in blood and that this growth defect was likely responsible for its animal phenotype. Because the blood was heat inactivated, neither the complement system nor sheep macrophages would be expected to be responsible. While the A. veronii isolate from H. orientalis showed a trend of outcompeting the H. verbana strain, Hm21R, in the blood control, it was not statistically significant.

FIG. 2.
Competitive colonization assay with H. verbana. Strains obtained from H. orientalis, Ho603S (A. jandaei) and Ho635S (A. veronii), and from H. verbana, Hm21S (A. veronii), were competed against Hm21R. The competitive index was assessed 41 h after inoculating ...

The composition of the gut microbiota may depend not as much on the microbial species but rather on the guild or the functional niche the bacteria fill (3). While the presumed beneficial function that Aeromonas provides for the host is still under investigation, factors that the bacteria require for successful colonization have been identified (8, 20, 21). Included among these traits are resistance to serum and a type III secretion system (T3SS) (8, 20). Four isolates from H. orientalis, Ho599, Ho603, Ho635, and Ho636, were tested for resistance to serum (21), and the four strains were resistant to undiluted, fresh serum. Previously we have shown that a T3SS is essential for A. veronii in escaping phagocytosis by leech hemocytes that patrol the crop and phagocytose sensitive bacteria. Diagnostic PCR for ascV was done (23). Both A. veronii strains, Ho635 and Ho636, tested positive for ascV, while no PCR product was obtained from DNA isolated from the two A. jandaei strains. The annealing temperature was lowered, but no band of the correct size was detected. These negative data do not conclusively demonstrate that the A. jandaei strains do not possess a T3SS, because we have recently shown that the diagnostic PCR primers do not amplify the ascV gene from all Aeromonas strains (A. C. Silver and J. Graf, submitted). Other factors that might affect A. jandaei's ability to grow in blood could be lower levels of hemolysis, a reduced ability to acquire iron or other key nutrients. Alternatively, A. veronii could secrete a compound that negatively affects the proliferation of A. jandaei.

The discovery of two distinct Aeromonas species colonizing different leech species is of importance for the medical application of leeches, because A. veronii is usually considered to be one of the three most virulent Aeromonas species; however, A. jandaei has also been shown to be a human pathogen (9). We tested four H. orientalis isolates, Ho599, Ho603, Ho635, and Ho636, for their sensitivities to antibiotics using Sensidics (Becton, Dickinson and Company, Sparks, MD) according to the manufacturer's instructions. Each isolate, along with A. veronii strain HM21 (also known as Hv391) from H. verbana, was sensitive to all antibiotics tested (i.e., chloramphenicol, sulfate trimethoprim, gentamicin, cefuroxime, ceflotaxime, naladixic acid, and ciprofloxacin). As such, current preemptive antibiotic treatments would be effective regardless of the leech species used.

In order to more thoroughly characterize the crop flora of H. orientalis, we constructed two clone libraries of the 16S rRNA gene from one animal 6 days after feeding (27) (Table (Table1).1). Clones first were characterized by RFLP-PCR, after which representative clones were sequenced. In the crop, a Rikenella-like sequence, that of clone AL5CE1, accounted for 98.5% of the clones. The sequence was similar to that of clone PW3 (7.26% divergence) from the crop of H. verbana. Two related clones were discovered in the intestinum, AL5ID2 and AL5IB3. These clones formed a highly supported clade in the Rikenella clade (data not shown). Rikenella-like bacteria appear to be widespread in the digestive tracts of numerous animals, including humans (27). We did not detect an Aeromonas sequence in the crop of this particular H. orientalis individual using the culture-independent approach. These animals were starved for many months prior to feeding, which likely decreases the number of Aeromonas bacteria more than those of Rikenella (10). Sampling an animal 6 days after a single feeding might not provide sufficient time for the Aeromonas population to rebound to a level comparable to that of Rikenella. Nonetheless, the culture-based approach did succeed in culturing Aeromonas from the same leech, indicating their presence, albeit below the limit of detection for the RFLP library. Aeromonas and Desulfovibrio 16S rRNA genes were detected in the intestinum. The Desulfovibrio sequence was most similar to a sequence we cloned from the intestinum of H. verbana (12% divergence). While the role of Desulfovibrio in the intestinum is not clear, its presence indicates an anaerobic environment, perhaps containing sulfate, but some Desulfovibrio bacteria can grow in the absence of sulfate in syntrophy with methanogens (26).

TABLE 1.
Bacterial phylotypes recovered from 16S rRNA gene clone libraries constructed from the digestive tract of the medicinal leech H. orientalis 6 days after feeding

Our experiments revealed the presence of two distinct Aeromonas species in H. orientalis and that the two isolates tested can colonize an alternate host, H. verbana, although the A. jandaei strain colonized at significantly lower levels. This defect was probably due to a reduced ability to grow in blood, but its biological basis and importance in the natural association remain to be shown. More detailed studies of the leech microbiota in different leech species are needed to ensure a comprehensive understanding of the potential risks of leech therapy.

Acknowledgments

We thank Adam Silver for independently confirming the gyrB data and testing for the presence of the T3SS.

This research was supported by NSF Career Award MCB 0448052 to J.G. and by DEB-0640463 to M.E.S. from the National Science Foundation. A.S.L. was supported by an ASM undergraduate fellowship, a SURF fellowship from the University of Connecticut, and an REU supplement from the National Science Foundation to J.G.

Footnotes

[down-pointing small open triangle]Published ahead of print on 8 August 2008.

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