Leech Poecilobdella manillensis protein extract ameliorated hyperuricemia by restoring gut microbiota dysregulation and affecting serum metabolites.

BACKGROUND: Hyperuricemia (HUA) is a public health concern that needs to be solved urgently. The lyophilized powder of Poecilobdella manillensis has been shown to significantly alleviate HUA; however, its underlying metabolic regulation remains unclear. AIM: To explore the underlying mechanisms of Poecilobdella manillensis in HUA based on modulation of the gut microbiota and host metabolism. METHODS: A mouse model of rapid HUA was established using a high-purine diet and potassium oxonate injections. The mice received oral drugs or saline. Additionally, 16S rRNA sequencing and ultra-high performance liquid chromatography with quadrupole time-of-flight mass spectrometry-based untargeted metabolomics were performed to identify changes in the microbiome and host metabolome, respectively. The levels of uric acid transporters and epithelial tight junction proteins in the renal and intestinal tissues were analyzed using an enzyme-linked immunosorbent assay. RESULTS: The protein extract of Poecilobdella manillensis lyophilized powder (49 mg/kg) showed an enhanced anti-trioxypurine ability than that of allopurinol (5 mg/kg) (P < 0.05). A total of nine bacterial genera were identified to be closely related to the anti-trioxypurine activity of Poecilobdella manillensis powder, which included the genera of Prevotella, Delftia, Dialister, Akkermansia, Lactococcus, Escherichia_Shigella, Enterococcus, and Bacteroides. Furthermore, 22 metabolites in the serum were found to be closely related to the anti-trioxypurine activity of Poecilobdella manillensis powder, which correlated to the Kyoto Encyclopedia of Genes and Genomes pathways of cysteine and methionine metabolism, sphingolipid metabolism, galactose metabolism, and phenylalanine, tyrosine, and tryptophan biosynthesis. Correlation analysis found that changes in the gut microbiota were significantly related to these metabolites. CONCLUSION: The proteins in Poecilobdella manillensis powder were effective for HUA. Mechanistically, they are associated with improvements in gut microbiota dysbiosis and the regulation of sphingolipid and galactose metabolism.

Leech management before application on patient: a nationwide survey of practices in French university hospitals.

Background: Leech therapy in plastic/reconstructive microsurgery significantly improves a successful outcome of flap salvage but the drawback is a risk of severe infection that results in a drop of the salvage rates from 70-80% to below 30%. We report the results of a national survey conducted in all the French university hospitals to assess the current extent of use of leech for medical practices in the hospital and to investigate maintenance, delivery practices and prevention of the risk of infection. Methods: Data concerning conditions of storage, leech external decontamination, microbiological controls, mode of delivery and antibiotic prophylaxis were collected from all the French university hospitals in practicing leech therapy, on the basis of a standardized questionnaire. Results: Twenty-eight of the 32 centers contacted filled the questionnaire, among which 23 practiced leech therapy, mostly with a centralized storage in the pharmacy; 39.1% of the centers declared to perform leech external decontamination and only 2 centers recurrent microbiological controls of the water storage. Leech delivery was mostly nominally performed (56.5%), but traceability of the leech batch number was achieved in only 39.1% of the cases. Only 5 centers declared that a protocol of antibiotic prophylaxis was systematically administered during leech therapy: either quinolone (2), sulfamethoxazole/trimethoprim (2) or amoxicillin/clavulanic acid (1). Conclusions: Measures to prevent infectious complications before application to patient have to be better applied and guidelines of good practices are necessary.

Preventing infective complications following leech therapy: elimination of symbiotic Aeromonas spp. from the intestine of Hirudo verbana using antibiotic feeding.

BACKGROUND: Hirudotherapy is often used successfully in modern medicine, especially in plastic and reconstructive surgery. However, Aeromonas infections are the most common complications of post-operative leech application. Hence, prophylactic antibiotic administration is recommended before and during leech therapy. It has been confirmed that patient safety and achieving the desired therapeutic effect depend mainly on the microbiologic purity of the animals used. The aims of this study were to find a safe and practical way to eradicate symbiotic Aeromonas spp. occuring in the intestine of Hirudo verbana. METHODS: Leeches were fed artificially with 1.5 mL of sterile defibrinated sheep blood supplemented with ciprofloxacin (CIP) or cefotaxime (CTX), at bacteriostatic concentrations of 0.2 mcg/mL or 1.5 mcg/mL, and bactericidal concentrations of 20 mcg/mL or 50 mcg/mL, respectively. Bacteria were isolated from the leech intestines before and after feeding at different time intervals: 1, 7, 14, 21, and 28 d. RESULTS: Biochemical identification of bacterial isolates from water samples and intestines of H. verbana using the API-NE20 test showed that A. veronii biovar sobria was predominant. Bacteria belonging to the genus Aeromonas were detected in all control leeches. The results showed that optimum eradication of bacteria from leech intestines was obtained using 20 mcg/mL of CIP and 50 mcg/mL of CTX, which decreased the number of Aeromonas spp. to undetectable levels for two weeks after feeding in all treated leeches. A statistically significant reduction in the number of bacterial colonies (p<0.0001) was observed in leeches treated with bacteriostatic concentrations of CIP or CTX; no bacterial growth was found on the plates after only seven days of feeding with antibiotics. All water samples in which the leeches were kept before treatment were contaminated with Aeromonas spp., whereas these samples were negative after antibiotic feeding of animals. CONCLUSIONS: All leeches were ready to take a blood meal after treatment, suggesting the possibility of using ciprofloxacin-treated or cefotaxime-treated leeches instead of chemoprophylaxis in patients undergoing hirudotherapy.

Endemic scrub typhus-like illness, Chile.

We report a case of scrub typhus in a 54-year-old man who was bitten by several terrestrial leeches during a trip to Chiloé Island in southern Chile in 2006. A molecular sample, identified as related to Orientia tsutsugamushi based on the sequence of the16S rRNA gene, was obtained from a biopsy specimen of the eschar on the patient's leg. Serologic analysis showed immunoglobulin G conversion against O. tsutsugamushi whole cell antigen. This case and its associated molecular analyses suggest that an Orientia-like agent is present in the Western Hemisphere that can produce scrub typhus-like illness. The molecular analysis suggests that the infectious agent is closely related, although not identical, to members of the Orientia sp. from Asia.

Preventing infective complications following leech therapy: is practice keeping pace with current research?

BACKGROUND: Despite several publications strongly advocating prophylactic antibiotics during leech therapy, and recent primary articles shedding new light on the microbiota of leeches, many units either do not use antibiotic prophylaxis, or are continuing to use ineffective agents. METHODS: A 5-year follow-up of plastic surgery units in the United Kingdom and the Republic of Ireland was conducted in 2007 to ascertain current practice regarding the use of prophylactic antibiotics with leech therapy. A comprehensive literature search investigated primary research articles regarding the microbiota of leeches to update the reconstructive surgery community. RESULTS: Despite published evidence to support the use of prophylactic antibiotics during leech therapy, 24% of units do not use antibiotic prophylaxis and 57% of those using antibiotics are using potentially ineffective agents. Advanced molecular genetic techniques, which allow accurate characterization of both culturable and non-culturable microbiota of the leech digestive tract, show a wider diversity than at first thought, with variable antibiotic resistance profiles. CONCLUSIONS: Despite infection due to leech therapy being a well known and relatively common complication, many units are not using appropriate antibiotic prophylaxis.

Delayed leech-borne infection with Aeromonas hydrophilia in escharotic flap wound.

Medicinal leeches (Hirudo medicinalis) are commonly used in plastic surgery for the salvage of congested flaps and replanted parts compromised by venous congestion. Infection associated with leech therapy is a documented complication of leech application, with reported incidences ranging from 2.4 to 20% [De Chalain TM. Exploring the use of the medicinal leech: a clinical risk-benefit analysis. J Reconstr Microsurg 1996;12(3):165-72.1]. We describe a case of delayed leech-borne infection, from the escharotic portion of a latissimus dorsi flap, which developed several days after stopping leech therapy for venous congestion in a reconstructed breast.

Transcriptomic analysis in the leech Theromyzon tessulatum: involvement of cystatin B in innate immunity.

At the present time, there is little information on mechanisms of innate immunity in invertebrate groups other than insects, especially annelids. In the present study, we have performed a transcriptomic study of the immune response in the leech Theromyzon tessulatum after bacterial challenge, by a combination of differential display RT (reverse transcriptase)-PCR and cDNA microarrays. The results show relevant modulations concerning several known and unknown genes. Indeed, threonine deaminase, malate dehydrogenase, cystatin B, polyadenylate-binding protein and alpha-tubulin-like genes are up-regulated after immunostimulation. We focused on cystatin B (stefin B), which is an inhibitor of cysteine proteinases involved in the vertebrate immune response. We have cloned the full-length cDNA and named the T. tessulatum gene as Tt-cysb. Main structural features of cystatins were identified in the derived amino acid sequence of Tt-cysb cDNA; namely, a glycine residue in the N-terminus and a consensus sequence of Gln-Xaa-Val-Xaa-Gly (QXVXG) corresponding to the catalytic site. Moreover, Tt-cysb is the first cystatin B gene characterized in invertebrates. We have determined by in situ hybridization and immunocytochemistry that Tt-cysb is only expressed in large coelomic cells. In addition, this analysis confirmed that Tt-cysb is up-regulated after bacterial challenge, and that increased expression occurs only in coelomic cells. These data demonstrate that the innate immune response in the leech involves a cysteine proteinase inhibitor that is not found in ecdysozoan models, such as Drosophila melanogaster or Caenorhabditis elegans, and so underlines the great need for information about innate immunity mechanisms in different invertebrate groups.

Molecular characterization of two novel antibacterial peptides inducible upon bacterial challenge in an annelid, the leech Theromyzon tessulatum.

Two novel antimicrobial peptides named theromacin and theromyzin were isolated and characterized from the coelomic liquid of the leech Theromyzon tessulatum. Theromacin is a 75-amino acid cationic peptide containing 10 cysteine residues arranged in a disulfide array showing no similarities with other known antimicrobial peptides. Theromyzin is an 86-amino acid linear peptide and constitutes the first anionic antimicrobial peptide observed in invertebrates. Both peptides exhibit activity directed against Gram-positive bacteria. Theromacin and theromyzin cDNAs code precursor molecules containing a putative signal sequence directly followed by the mature peptide. The enhancement of theromacin and theromyzin mRNA levels has been observed after blood meal ingestion and upon bacterial challenge. In situ hybridization revealed that both genes are expressed in large fat cells in contact with coelomic cavities. Gene products were immunodetected in large fat cells, in intestinal epithelia, and at the epidermis level. In addition, a rapid release of the peptides into the coelomic liquid was observed after bacterial challenge. The presence of antimicrobial peptide genes in leeches and their expression in a specific tissue functionally resembling the insect fat body provide evidence for the first time of an antibacterial response in a lophotrochozoan comparable to that of holometabola insects.

Aeromonas meningitis complicating medicinal leech therapy.

Medicinal leeches have an important and expanding role in medicine, but infection can complicate their use. We describe a unique case of Aeromonas meningitis associated with the use of leech therapy to salvage a skin flap after central nervous system surgery.

Nosocomial Infections with Aeromonas hydrophila from Leeches.

The manner in which leeches are maintained before they are used for therapy has not been studied as a factor contributing to nosocomial infections. A 5-year retrospective survey of Aeromonas hydrophila nosocomial infections at a hospital in Marseille, France, revealed infections in 5 (4.1%) of an estimated 122 patients treated with leeches in the Hand Surgery Unit and 2 (2.4%) of an estimated 85 patients treated with leeches in other hospital units. The retrospective survey showed that the Hand Surgery Unit was the only unit that had its own aquarium for maintaining leeches; this aquarium was filled with tap water contaminated with Aeromonas species and was not regularly disinfected or cleaned. Leeches used in other units were maintained in noncarbonated water in a transport device. Use of leeches kept in aquariums that are filled with tap water and not disinfected or cleaned regularly may be linked to A. hydrophila infections.

Bacterial flora of Hirudo medicinalis and their antibiotic sensitivities in the Middle Black Sea Region, Turkey.

The rate of infectious complications of leech therapy is almost 20% because Hirudo medicinalis has endosymbiotic bacteria. The aim of this study was to determine the bacterial flora of H. medicinalis and their antibiotic sensitivities in a region in Turkey. Sixteen adult leeches were collected in Middle Black Sea region, Turkey. They were rubbed onto blood agar plates directly under ether anesthesia to obtain surface cultures. They were then killed to obtain mouth and gut cultures. Culture swabs were applied to blood agar, eosin methylene blue agar, and ampicillin blood agar plates. Gut contents were applied to blood culture medium as well. Bacteria were isolated in 15 of 16 leech surfaces, in 7 of 16 mouths, and in 15 of 16 guts. Isolated bacteria were identified with Analytical Profile Index 32 E and Analytical Profile Index 20 NE (fermentative and nonfermentative respectively). Most common types of cultured bacteria were Aeromonas hydrophila (N = 25), Ochrobacter anthropi (N = 23), nonfermenting Gram-negative rods (N = 12), Acinetobacter lwoffi (N = 3), and A. sobria (N = 2) in 73 isolates. A standard disk diffusion test was performed on isolated bacteria. All isolates were 100% susceptible to ciprofloxacin, cefotaxime, ceftazidime, gentamicin, and trimethoprim/sulfamethoxazole. Because leeches are carriers of Aeromonas and other bacteria, appropriate antibiotic prophylaxis should be administrated to the patient who needs leech therapy. Antibacterial agents can be determined by the resistance pattern of the bacterial flora of regional H. medicinalis.

Ingested blood contributes to the specificity of the symbiosis of Aeromonas veronii biovar sobria and Hirudo medicinalis, the medicinal leech.

Hirudo medicinalis, the medicinal leech, usually carries in its digestive tract a pure culture of Aeromonas veronii bv. sobria. Such specificity is unusual for digestive tracts that are normally colonized by a complex microbial consortium. Important questions for the symbiotic interaction and for the medical application after microvascular surgery are whether other bacteria can proliferate or at least persist in the digestive tract of H. medicinalis and what factors contribute to the reported specificity. Using a colonization assay, we were able to compare experimentally the ability of clinical isolates and of a symbiotic strain to colonize H. medicinalis. The symbiotic A. veronii bv. sobria strain proliferated well and persisted for at least 7 days inside the digestive tract. In contrast, the proliferation of Pseudomonas aeruginosa and Staphylococcus aureus was inhibited inside the animal compared to growth in the in vitro control, indicating that the ingested blood was modified within the digestive tract. However, both strains were able to persist in the digestive tract for at least 7 days. For an Escherichia coli strain, the viable counts decreased approximately 1, 000-fold within 42 h. The decrease of viable E. coli could be prevented by interfering with the activation of the membrane-attack complex of the complement system that is present in blood. This suggests that the membrane-attack complex remained active inside H. medicinalis and prevented the proliferation of sensitive bacteria. Thus, antimicrobial properties of the ingested vertebrate blood contribute to the specificity of the A. veronii-H. medicinalis symbiosis, in addition to modifications of the blood inside the digestive tract of H. medicinalis.

Aeromonas species isolated from medicinal leeches.

Aeromonas hydrophila infections are a recognized complication of the use of medicinal leeches. The authors performed an experiment designed to find a safe and practical way to sterilize the leech gut of pathogenic organisms. Leeches were incubated for a 12-hour period in solutions of antibiotic effective against A. hydrophila. The incubations in the antibiotic solutions failed to eradicate pathogenic bacteria from the gut of the leeches. The authors examined cultures of bacteria isolated from the guts of the commonly used Hirudo medicinalis (European leech) and found a wide variety of pathogenic organisms. A. hydrophila is widely believed to be the most common enteric pathogen, but the authors found A. sobria more frequently in their experiment. They also cultured the guts of the leech H. michaelseni recently used clinically in South Africa. A. caviae was the most common pathogen encountered in these leeches. A. caviae and A. sobria cause a spectra of disease similar to A. hydrophila. The authors endorse the current recommendation that all patients who have leech therapy for congested flaps or replants receive broad-spectrum prophylactic antibiotics. This appears to be the safest and simplest way to prevent leech-related infections.

Leech-borne Serratia marcescens infection following complex hand injury.

Leeches are commonly used in the postoperative course of plastic surgical operations where there is venous congestion in a pedicled or free flap. They provide a temporary relief to venous engorgement whilst venous drainage is re-established. It is known that leeches can carry Aeromonas hydrophila infection, and a second or third generation cephalosporin antibiotic has traditionally been given as prophylaxis against infection. We report a new observation that leeches can carry Serratia marcescens and give rise to clinically significant infection. The implication for prophylaxis and treatment of leech-associated cellulitis is discussed.

Intestinal flora of the medicinal leech Hirudinaria manillensis.

Medicinal leeches are widely used to treat venous congestion in microvascular surgery. Aeromonas hydrophila infection, following application of the leech species Hirudo medicinalis, is a recognized complication. Administration of antibiotics directed at Aeromonas has been successful in minimizing complications of infection from this organism. A different leech species, Hirudinaria manillensis, has recently been introduced for microsurgical use. A study of the enteric content of 30 of these leeches showed that Aeromonas hydrophila was isolated in only 20 percent of animals, while the majority of remaining positive cultures were single and mixed gram-negative rods. All organisms isolated were sensitive to current recommended coverage for Aeromonas hydrophila. This study suggests that the enteric flora of different leech species may be variable and should be carefully characterized, to direct appropriate prophylactic therapy prior to release of new species for clinical use.