The Hirudo Medicinalis Microbiome Is a Source of New Antimicrobial Peptides.

Antimicrobial peptides (AMPs) are considered a promising new class of anti-infectious agents. This study reports new antimicrobial peptides derived from the Hirudo medicinalis microbiome identified by a computational analysis method applied to the H. medicinalis metagenome. The identified AMPs possess a strong antimicrobial activity against Gram-positive and Gram-negative bacteria (MIC range: 5.3 to 22.4 µM), including Staphylococcus haemolyticus, an opportunistic coagulase-negative pathogen. The secondary structure analysis of peptides via CD spectroscopy showed that all the AMPs except pept_352 have mostly disordered structures that do not change under different conditions. For peptide pept_352, the α-helical content increases in the membrane environment. The examination of the mechanism of action of peptides suggests that peptide pept_352 exhibits a direct membranolytic activity. Furthermore, the cytotoxicity assay demonstrated that the nontoxic peptide pept_1545 is a promising candidate for drug development. Overall, the analysis method implemented in the study may serve as an effective tool for the identification of new AMPs.

Low-Level Antimicrobials in the Medicinal Leech Select for Resistant Pathogens That Spread to Patients.

Fluoroquinolones (FQs) and ciprofloxacin (Cp) are important antimicrobials that pollute the environment in trace amounts. Although Cp has been recommended as prophylaxis for patients undergoing leech therapy to prevent infections by the leech gut symbiont Aeromonas, a puzzling rise in Cp-resistant (Cpr) Aeromonas infections has been reported. We report on the effects of subtherapeutic FQ concentrations on bacteria in an environmental reservoir, the medicinal leech, and describe the presence of multiple antibiotic resistance mutations and a gain-of-function resistance gene. We link the rise of CprAeromonas isolates to exposure of the leech microbiota to very low levels of Cp (0.01 to 0.04 µg/ml), <1/100 of the clinical resistance breakpoint for Aeromonas Using competition experiments and comparative genomics of 37 strains, we determined the mechanisms of resistance in clinical and leech-derived Aeromonas isolates, traced their origin, and determined that the presence of merely 0.01 µg/ml Cp provides a strong competitive advantage for Cpr strains. Deep-sequencing the Cpr-conferring region of gyrA enabled tracing of the mutation-harboring Aeromonas population in archived gut samples, and an increase in the frequency of the Cpr-conferring mutation in 2011 coincides with the initial reports of CprAeromonas infections in patients receiving leech therapy.IMPORTANCE The role of subtherapeutic antimicrobial contamination in selecting for resistant strains has received increasing attention and is an important clinical matter. This study describes the relationship of resistant bacteria from the medicinal leech, Hirudo verbana, with patient infections following leech therapy. While our results highlight the need for alternative antibiotic therapies, the rise of Cpr bacteria demonstrates the importance of restricting the exposure of animals to antibiotics approved for veterinary use. The shift to a more resistant community and the dispersion of Cpr-conferring mechanisms via mobile elements occurred in a natural setting due to the presence of very low levels of fluoroquinolones, revealing the challenges of controlling the spread of antibiotic-resistant bacteria and highlighting the importance of a holistic approach in the management of antibiotic use.

[Infections Associated with Treatment with Hirudo medicinalis - Report of Two Cases and Review of the Literature]. / Infektionen im Zusammenhang mit der Anwendung von Hirudo medicinalis - Zwei Fallberichte und Literaturübersicht.

Medicinal leeches are well-established for promoting venous drainage in transplants/flaps and analgesia in osteoarthritis. Although medicinal leeches are bred and kept under controlled conditions, they are colonised by a genuine species-specific bacterial flora. Therapeutic application of leeches implies skin penetration carrying an a priori risk of infection. We report 2 cases with different indications for medicinal leech therapy. In both cases wound infection occurred in close temporal and spatial correlation or with evidence of a leech-associated germ that could be treated successfully. An unclarified complication rate warrants strict indications for the application of medicinal leeches. Preventive measures are currently tested.

Mucinivorans hirudinis gen. nov., sp. nov., an anaerobic, mucin-degrading bacterium isolated from the digestive tract of the medicinal leech Hirudo verbana.

Three anaerobic bacterial strains were isolated from the digestive tract of the medicinal leech Hirudo verbana, using mucin as the primary carbon and energy source. These strains, designated M3(T), M4 and M6, were Gram-stain-negative, non-spore-forming and non-motile. Cells were elongated bacilli approximately 2.4 µm long and 0.6 µm wide. Growth only occurred anaerobically under mesophilic and neutral pH conditions. All three strains could utilize multiple simple and complex sugars as carbon sources, with glucose fermented to acid by-products. The DNA G+C contents of strains M3(T), M4 and M6 were 44.9, 44.8 and 44.8 mol%, respectively. The major cellular fatty acid of strain M3(T) was iso-C15 : 0. Phylogenetic analysis of full-length 16S rRNA gene sequences revealed that the three strains shared >99 % similarity with each other and represent a new lineage within the family Rikenellaceae of the order Bacteroidales, phylum Bacteroidetes. The most closely related bacteria to strain M3(T) based on 16S rRNA gene sequences were Rikenella microfusus DSM 15922(T) (87.3 % similarity) and Alistipes finegoldii AHN 2437(T) (87.4 %). On the basis of phenotypic, genotypic and physiological evidence, strains M3(T), M4 and M6 are proposed as representing a novel species of a new genus within the family Rikenellaceae, for which the name Mucinivorans hirudinis gen. nov., sp. nov. is proposed. The type strain of Mucinivorans hirudinis is M3(T) ( = ATCC BAA-2553(T) = DSM 27344(T)).

Homolog of allograft inflammatory factor-1 induces macrophage migration during innate immune response in leech.

Allograft inflammatory factor-1 (AIF-1) is a 17-kDa cytokine-inducible calcium-binding protein that, in vertebrates, plays an important role in the allograft immune response. Its expression is mostly limited to the monocyte/macrophage lineage. Until recently, AIF-1 was assumed to be a novel molecule involved in inflammatory responses. To clarify this aspect, we have investigated the expression of AIF-1 after bacterial challenge and its potential role in regulating the innate immune response in an invertebrate model, the medicinal leech (Hirudo medicinalis). Analysis of an expressed sequence tag library from the central nervous system of Hirudo revealed the presence of the gene Hmaif-1/alias Hmiba1, showing high homology with vertebrate aif-1. Immunohistochemistry with an anti-HmAIF-1 polyclonal antibody revealed the constitutive presence of this protein in spread CD68(+) macrophage-like cells. A few hours after pathogen (bacterial) injection into the body wall, the amount of these immunopositive cells co-expressing HmAIF-1 and the common leucocyte marker CD45 increased at the injected site. Moreover, the recombinant protein HmAIF-1 induced massive angiogenesis and was a potent chemoattractant for macrophages. Following rHmAIF-1 stimulation, macrophage-like cells co-expressed the macrophage marker CD68 and the surface glycoprotein CD45, which, in vertebrates, seems to have a role in the integrin-mediated adhesion of macrophages and in the regulation of the functional responsiveness of cells to chemoattractants. CD45 is therefore probably involved in leech macrophage-like cell activation and migration towards an inflammation site. We have also examined its potential effect on HmAIF-1-induced signalling.

Characterization of the digestive tract microbiota of Hirudo orientalis (medicinal leech) and antibiotic resistance profile.

BACKGROUND: There are at least three distinct European leech species used medicinally: Hirudo medicinalis, H. orientalis, and H. verbana. Infection caused by leech microbiota is the most widely reported complication. Few studies have reported the culturable and unculturable bacteria and examined the antibiotic resistances in H. orientalis. METHODS: Following stratified random sampling from a major worldwide leech supplier, Hirudo orientalis leeches were identified by visual comparison and amplification and sequencing the cox1 locus. Combined culture and culture-independent approaches were used to characterize the microbiota of the midgut, and bacterial gyrB sequences from distinct colonies were used to identify the Aeromonas isolates. Nonculturable studies involved clone libraries of 16S rRNA genes, and Etests were used to investigate antibiotic sensitivities. RESULTS: Analysis of 16S rRNA gene clone libraries revealed the presence of several species in the intraluminal fluid of the crop, including a new finding of Morganella morganii, with Rikenella-like (35 percent) and Aeromonas veronii (38 percent) dominant members. The intestinum contained bacteria not previously isolated from the leech: Magnetospirillium species and Roseospira marina. Etests showed all A. veronii isolates were sensitive to ciprofloxacin, with either a complete or intermediate resistance to Augmentin. CONCLUSIONS: The authors show diverse microbiota in the leech digestive tract. The pathogenic potential of the additional gut symbionts isolated in this study is yet to be elucidated; however, M. morganii, which is a known human pathogen, is a new finding. In addition to adding to the knowledge base regarding antibiotic sensitivities, this article serves as an update to the reconstructive surgeon regarding leech therapy.

Flap infection associated with medicinal leeches in reconstructive surgery: two new drug-resistant organisms.

The use of medicinal leeches in reconstructive surgery has proven value for the salvage of flaps with venous congestion but is associated with a risk of leech-acquired infection. The most common leech-associated organism is Aeromonas hydrophila, which antibiotic prophylaxis is typically directed against. The authors describe two new multidrug-resistant organisms acquired from medicinal leech therapy that resulted in flap infection. The evaluation of suspected leech-borne infection and management protocol for this leech-acquired resistant multi-organism infection is presented.

Niabella hirudinis and Niabella drilacis sp. nov., isolated from the medicinal leech Hirudo verbana.

Two Gram-negative, rod-shaped bacteria, strains E96(T) and E90(T), were isolated from medicinal leeches (Hirudo verbana) and characterized by a polyphasic taxonomic approach. Phylogenetic analysis based on the nearly full-length 16S rRNA gene sequence showed that the two strains shared 98.1% sequence similarity and were affiliated with the genus Niabella within the phylum Bacteroidetes, with 94.4-97.6% sequence similarity to type strains of species of the genus Niabella and highest sequence similarity to the type strain of Niabella aurantiaca (97.3 and 97.6%, respectively). Niabella-related 16S rRNA gene sequences were recently detected in the bladders of Hirudo verbana; however, no cultured representatives were so far available. Genomic fingerprint analysis using repetitive element primed (rep)- and randomly amplified polymorphic DNA (RAPD)-PCRs and DNA-DNA hybridization experiments clearly showed that the strains were different from each other (DNA-DNA relatedness values of 39.1%, reciprocal 28.0%) and from the type strains of N. aurantiaca (<19.7%) and Niabella tibetensis (<41.1%). Chemotaxonomic analyses confirmed the affiliation to the genus Niabella. Both strains contained MK-7 as the predominant menaquinone. The major fatty acids of both strains were iso-C15:0, iso-C15:1 G, iso-C17:0 3-OH and summed feature 3 (C16:1ω7c and/or iso-C15:0 2-OH), which is characteristic for the genus Niabella. Based on genotypic, chemotaxonomic and physiological characterization, we propose two novel species of the genus Niabella, Niabella hirudinis sp. nov., with strain E96(T) ( =DSM 25812(T) =CCM 8411(T) =LMG 26956(T)) as the type strain, and Niabella drilacis sp. nov., with strain E90(T) ( =DSM 25811(T) =CCM 8410(T) =LMG 26954(T)) as the type strain.

Ciprofloxacin-resistant Aeromonas hydrophila cellulitis following leech therapy.

We report a case of surgical site infection with ciprofloxacin-resistant Aeromonas hydrophila following leech therapy. Antimicrobial and genetic analyses of leech and patient isolates demonstrated that the resistant isolates originated from the leech gut microbiota. These data suggest that ciprofloxacin monotherapy as a prophylaxis regimen prior to leech therapy may not be effective in preventing infection.

The role of leech water sampling in choice of prophylactic antibiotics in medical leech therapy.

Medical leech therapy (MLT) with Hirudo medicinalis is well established as a treatment for venous congestion of tissue flaps, grafts, and replants. Unfortunately, this treatment is associated with surgical site infections with bacterial species, most commonly Aeromonas hydrophila, which is an obligate symbiot of H. medicinalis. For this reason, prophylactic antibiotics are recommended in the setting of MLT. After culturing Aeromonashydrophila resistant to ciprofloxacin from a tissue specimen from a patient with a failed replant of three digits post-MLT, we performed environmental surveillance cultures and antibiotic susceptibility testing on water collected from leech tanks. This surveillance was performed twice weekly for 2.5 months. Fourteen surveillance cultures demonstrated 21 isolates of Aeromonas species, 71.4% of which were ciprofloxacin susceptible. All isolates were sulfamethoxazole-trimethoprim (SXT) susceptible. The prophylactic antibiotic regimen of choice for leech therapy at our institution is SXT, with culture of tank water to refine antimicrobial choice if necessary. This study demonstrates the importance of regular surveillance to detect resistant Aeromonas species in medical leeches; however optimal practice has not been established.

Luteolibacter cuticulihirudinis sp. nov., isolated from Hirudo medicinalis.

A Gram-negative, coccoid shaped bacterium isolated from the outer surface of the medicinal leech Hirudo medicinalis was characterized. The 16S rRNA gene sequence comparison revealed that the bacterium was closely related to species of the genus Luteolibacter. Luteolibacter pohnpeiensis was the most closely related species (94.6 % sequence similarity), followed by Luteolibacter luojiensis (93.4 %) and Luteolibacter algae (93.3 %). Chemotaxonomic data (major ubiquinone: MK-9; major polar lipids: phosphatidylethanolamine and phosphatidylglycerol; and major fatty acids: iso-C(14:0), C(16:0), iso-C(16:1), and anteiso-C(15:0)) supported the affiliation of the isolate to the genus Luteolibacter. DNA-DNA hybridizations with the type strain of L. pohnpeiensis was 31 % (reciprocal value 30 %). A phenotypic differentiation of strain E100(T) from L. pohnpeiensis and the other Luteolibacter species was possible by several physiological tests. We conclude Strain E100(T) represents a novel species, for which we propose the name Luteolibacter cuticulihirudinis sp. nov. with the type strain E100(T) (=CCM 8400(T) = LMG 26924(T)).

Pseudarcicella hirudinis gen. nov., sp. nov., isolated from the skin of the medical leech Hirudo medicinalis.

A pinkish-pigmented, Gram-stain-negative, rod-shaped, non-spore-forming bacterium, strain E92(T), was isolated from the skin of the medical leech Hirudo medicinalis, on R2A agar. 16S rRNA gene sequence analysis of strain E92(T) showed a relatively low 16S rRNA gene sequence similarity (93.0-93.5 %) to representatives of the genus Arcicella and 91.5-92.0 % to members of the genus Flectobacillus. The polar lipid profile was composed of the major compounds phosphatidylethanolamine, an unidentified aminophospholipid, an unidentified aminolipid and an unidentified polar lipid; glycolipids were not detected. The major quinone was menquinone MK-7, and the major compound in the polyamine pattern was spermidine. The predominant fatty acids were C(16 : 1)ω5c and C(16 : 1)ω7c and/or iso-C(15 : 0) 2-OH (detected as summed feature 3). The isolate did not contain C(14 : 0) or the hydroxyl fatty acid iso-C(17 : 0) 3-OH found in all representatives of the genera Arcicella and Flectobacillus, but did produce C(18 : 1)ω7c and 11-methyl C(18 : 1)ω7c which are not found in these two genera. The DNA G+C content of strain E92(T) was 64.4 mol%. The unique 16S rRNA gene sequence, and specific chemotaxonomic and physiological data revealed that strain E92(T) represents a new genus in the family Cytophagaceae for which we propose the name Pseudarcicella hirudinis gen. nov., sp. nov., with the type strain of the type species as E92(T) (= LMG 26720(T) = CCM 7988(T)).

Cutaneous complications of improper leech application.

INTRODUCTION: The medical leech (Hirudo medicinalis) has been used throughout the centuries and continues to be used today, mainly in reconstructive surgery and microsurgery. Easy access to these animals may entail an improper use of this therapeutic method by patients as a form of self-treatment. CASE REPORT: A man who presented with skin erythema and oedema due to the application of a medical leech. DISCUSSION: Infection is considered the most common complication of hirudotherapy, even though bacteriological examination of leech applied by the patient showed neither bacterial nor parasitic infection. The skin lesions were probably of allergic origin; whether this reaction was due to substances released from the leech or as a consequence of reusing the same leech, remains to be determined.

Acute postoperative infection with Aeromonas hydrophila after using medical leeches for treatment of venous congestion.

INTRODUCTION: Venous convulsion after reconstructive microsurgery procedures is one major complication a surgeon has to deal with. Today, especially in the field of reconstructive microsurgery, medicinal leech therapy enjoys a renaissance. The potential risks such as infections associated with leech therapy are generally underestimated and not sufficiently discussed in literature. METHOD/PATIENTS: We present five male patients with an average age of 47 years. All patients suffered from a trauma incident, which had to be treated as an emergency. Three patients showed, postoperatively, a venous congestion after the reconstructive procedures. Another two patients with flap reconstruction and flap training developed venous problems after 12 and 14 days. In all five cases, the indication was given to use medical leeches (Hirudo medicinalis). In all the patients, a local infection of the injured extremity could be regarded after beginning with the leech treatment. The treatment duration with medical leeches for postoperative venous congestion was an average of 6 days. RESULTS: The reconstructive procedures in all five cases were unfortunately unsuccessful as major local infections were observed. Microbiological analyses showed, in all cases, an infection with Aeromonas hydrophila. CONCLUSION: We recommend making a considered indication for leech therapy, to diagnose wound infections early and to think about prophylactic antibiotics in patients with leech application.

Aeromonas septicemia after medicinal leech use following replantation of severed digits.

Medicinal leeches are used to control venous congestion. Aeromonas in the leech gut are essential for digestion of blood. This case report describes a patient who had Aeromonas bacteremia develop after leeching. He had an injury to his hand that required replantation of his thumb. Following the surgery, leech therapy was started with ampicillin-sulbactam prophylaxis. Sepsis developed. Blood cultures were positive for Aeromonas that were resistant to ampicillin-sulbactam. The antibiotic was changed to ciprofloxacin on the basis of the sensitivity profile of the organisms. Cultures from the leech bathwater confirmed it as the source of the Aeromonas. Clinicians who use leech therapy must be aware that leeches can harbor Aeromonas species resistant to accepted prophylactic antibiotics and that sepsis may occur.

Elimination of symbiotic Aeromonas spp. from the intestinal tract of the medicinal leech, Hirudo medicinalis, using ciprofloxacin feeding.

The use of the medicinal leech (Hirudo medicinalis) in promoting venous drainage in tissues whose vitality is threatened by venous congestion and obstruction, especially in plastic and reconstructive surgery, has been complicated by infections caused by Aeromonas spp. These are leech endosymbionts for which patients undergoing hirudotherapy frequently receive systemic chemoprophylaxis. In order to evaluate the possibility of rendering leeches safe for use on patients, H. medicinalis were fed artificially with a 2 g/L arginine solution (used as a phagostimulant) supplemented with ciprofloxacin (100 mg/L). Aeromonads were detected in 57 out of 80 control leeches (71.3%), but in none of the 56 leeches treated with ciprofloxacin (p <0.001). Treated leeches survived for up to 4 months. Tested weekly, 61% of these leeches took human blood for at least 4 weeks after treatment and all remained negative for aeromonads. All water samples in which leeches were kept before treatment were contaminated with Aeromonas spp.; none were detected in any of the NaCl/arginine solutions with which treated animals were fed. Molecular characterization of two phenotypically distinct isolates using gyrB sequencing showed that one clustered tightly with A. veronii and the other was closely related to A. media. Other environmental bacteria and fungi were isolated from 26.5% of treated leeches that had taken a blood meal 1-4 weeks after treatment. Ciprofloxacin reduced the number of leech-associated aeromonads to undetectable levels for extended periods. Most treated leeches were ready to take a blood meal after treatment, suggesting the possibility of using ciprofloxacin-treated leeches instead of chemoprophylaxis in patients undergoing hirudotherapy.

Microbial challenge promotes the regenerative process of the injured central nervous system of the medicinal leech by inducing the synthesis of antimicrobial peptides in neurons and microglia.

Following trauma, the CNS of the medicinal leech, unlike the mammalian CNS, has a strong capacity to regenerate neurites and synaptic connections that restore normal function. In this study, we show that this regenerative process is enhanced by a controlled bacterial infection, suggesting that induction of regeneration of normal CNS function may depend critically upon the coinitiation of an immune response. We explore the interaction between the activation of a neuroimmune response and the process of regeneration by assaying the potential roles of two newly characterized antimicrobial peptides. Our data provide evidence that microbial components differentially induce the transcription, by microglial cells, of both antimicrobial peptide genes, the products of which accumulate rapidly at sites in the CNS undergoing regeneration following axotomy. Using a preparation of leech CNS depleted of microglial cells, we also demonstrate the production of antimicrobial peptides by neurons. Interestingly, in addition to exerting antibacterial properties, both peptides act as promoters of the regenerative process of axotomized leech CNS. These data are the first to report the neuronal synthesis of antimicrobial peptides and their participation in the immune response and the regeneration of the CNS. Thus, the leech CNS appears as an excellent model for studying the implication of immune molecules in neural repair.

Identification of Aeromonas veronii genes required for colonization of the medicinal leech, Hirudo verbana.

Most digestive tracts contain a complex consortium of beneficial microorganisms, making it challenging to tease apart the molecular interactions between symbiont and host. The digestive tract of Hirudo verbana, the medicinal leech, is an ideal model system because it harbors a simple microbial community in the crop, comprising the genetically amenable Aeromonas veronii and a Rikenella-like bacterium. Signature-tagged mutagenesis (STM) was used to identify genes required for digestive tract colonization. Of 3,850 transposon (Tn) mutants screened, 46 were identified as colonization mutants. Previously we determined that the complement system of the ingested blood remained active inside the crop and prevented serum-sensitive mutants from colonizing. The identification of 26 serum-sensitive mutants indicated a successful screen. The remaining 20 serum-resistant mutants are described in this study and revealed new insights into symbiont-host interactions. An in vivo competition assay compared the colonization levels of the mutants to that of a wild-type competitor. Attenuated colonization mutants were grouped into five classes: surface modification, regulatory, nutritional, host interaction, and unknown function. One STM mutant, JG736, with a Tn insertion in lpp, encoding Braun's lipoprotein, was characterized in detail. This mutant had a >25,000-fold colonization defect relative to colonization by the wild-type strain at 72 h and, in vitro, an increased sensitivity to sodium dodecyl sulfate, suggesting the presence of an additional antimicrobial property in the crop. The classes of genes identified in this study are consistent with findings from previous STM studies involving pathogenic bacteria, suggesting parallel molecular requirements for beneficial and pathogenic host colonization.

Infection risk related to the use of medicinal leeches.

OBJECTIVE: To assess the incidence of postoperative wound infections related to treatment with medicinal leeches at Ghent University Hospital. METHOD: A 2-year retrospective analysis of bacteriologic culture results of soft tissue infections in patients treated with medicinal leeches. RESULTS: Cultures of suspected wound infections were taken and susceptibility testing of isolates was performed on 17 of 47 patients (36.2%). Aeromonas was frequently isolated (18.5%). CONCLUSIONS: A high incidence of infection during and after application of medicinal leeches, despite their external decontamination, necessitates an antibiotic prophylaxis. In particular Aeromonas must be covered, as soft tissue infections with these bacteria can give serious complications. The prophylactic antibiotic should cover the most frequent isolated species taking into account the importance of Aeromonas and the susceptibility pattern. Based on the results, fluoroquinolones seem to be a good choice. The authors believe that practical recommendations to hospital pharmacists on prophylaxis during Hirudo medicinalis treatment, might enhance the safety of it's use by reducing the number of infections.