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Elimination of symbiotic Aeromonas spp. from the intestinal tract of the medicinal leech, Hirudo medicinalis, using ciprofloxacin feeding
ORIGINAL ARTICLE
BACTERIOLOGY
Elimination of symbiotic Aeromonas spp. from the intestinal tract of the medicinal leech, Hirudo medicinalis, using ciprofloxacin feeding K. Y. Mumcuoglu1, L. Huberman1, R. Cohen1, V. Temper2, A. Adler2, R. Galun1 and C. Block2 1) Department of Parasitology and 2) Department of Clinical Microbiology and Infectious Diseases, Hebrew University-Hadassah Medical School, Jerusalem, Israel
Abstract 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 leechassociated 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. Keywords: Aeromonas, artificial feeding, Hirudo medicinalis, medicinal leech, symbiont Original Submission: 28 November 2008; Revised Submission: 5 January 2009; Accepted: 7 January 2009 Editor: D. Raoult Article published online: 11 June 2009 Clin Microbiol Infect 2010; 16: 563–567 10.1111/j.1469-0691.2009.02868.x Corresponding author and reprint requests: K. Y. Mumcuoglu, PhD, Department of Parasitology, Hebrew University-Hadassah Medical School, PO Box 12272, Jerusalem 91120, Israel E-mail: [email protected]
Introduction The use of the medicinal leech (Hirudo medicinalis) for the treatment of human diseases has been known since ancient Egyptian times. Galen (130–201 AD) used leeches for bloodletting, which was based on the belief that removal of the patient’s blood would correct the humoral imbalance and restore good health [1]. In the 18th and 19th centuries, the popularity of leeching reached its height in Europe. In 1884, Haycraft discovered hirudin, an anti-coagulant substance in leech saliva. Markwardt [2] isolated and characterized this molecule and in 1986 it was produced by genetic engineering [3].
In recent years, recognition of a number of antithrombotic substances and a vasodilator in the saliva of the medicinal leech [4,5] has generated renewed interest in the use of the leech itself to promote venous drainage in tissues whose vitality is threatened by venous congestion and obstruction, especially after plastic and reconstructive surgery. In 2004, this treatment modality received the approval of the Food and Drug Administration in the USA. Hirudotherapy, as it is termed, has been complicated by infections caused by Aeromonas spp. which are considered to be obligatory endosymbionts of the leech [4]. Infections with aeromonads have been observed in 7–20% of patients treated with leeches after reconstructive surgery; in these cases the chances of successful re-implantation or flap survival dropped by 30%. Therefore, antibiotics such as cephalosporins or fluoroquinolones are given prophylactically for the duration of the treatment [1,6].
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Clinical Microbiology and Infection, Volume 16 Number 6, June 2010
In order to reduce the risk of infection, attempts have been made to remove or decrease the number of aeromonads inside and outside leeches, by frequent changes of the water in leech containers, and by submerging leeches in hypochloric acid and antibiotics, however, these treatment modalities were not sufficient to destroy the internal bacterial flora of the leech [7,8]. Antimicrobial prophylaxis for bacteria remaining in the digestive tract is thus necessary to make the leeches suitable for the treatment of patients, preventing their internal bacteria from constituting a source of infection. The purpose of this study was to attempt to suppress the endosymbiotic aeromonads by feeding leeches artificially with a blood-free antibiotic solution, with the ultimate aim of eliminating the need for chemoprophylaxis in the patients. Ciprofloxacin, which is an agent of choice in the therapy and prophylaxis of infection as a result of these organisms, was chosen after preliminary testing showed leech-associated Aeromonas spp. to be fully susceptible.
Materials and Methods
CMI
Feeding chamber Warming water
Blood or arginine
Membrane Water
Feeding leech
FIG. 1. Schematic representation of the artificial feeding chamber for leeches.
blood meal after treatment. After feeding, leeches were maintained individually in sterilized chlorine-free tap water containing ciprofloxacin at a concentration of 20 mg/L and examined periodically for bacterial contamination.
Leeches
Bacteriological examination
Leeches (Hirudo medicinalis) collected in the wild in Turkey (Ergene Ltd, Tekirdag) were kept in holding vessels (40 · 60 · 40-cm plastic containers) in chlorine-free tap water that was changed weekly. (The authors are familiar with recent changes in the taxonomy of Hirudo species in which many medicinally applied leeches have been shown to be H. verbana rather than H. medicinalis. As the leeches used in this study were not formally identified, the latter term was kept for convenience [9]).
Samples were taken from the water in the holding vessels, from the exterior surface of the animals as well as from their alimentary tract and from the feeding solution. Leeches were disinfected externally by immersion in 70% ethyl alcohol for 1 min, followed by transfer to sterile distilled water. The purpose of this was to ensure that external contaminants did not interfere with cultures from the intestinal tract. In order to prevent the entry of alcohol to the digestive tract and to avoid the discharge of gut contents through the mouth or anus, a ligature was applied to the proximal and distal ends (c. 2–3 mm) of the leech. Swabs were taken from the outer surface of the animals, which were then transected c. 10 mm distal to the anterior sucker, and crop fluid was collected using a 10-lL disposable bacteriological loop (Quadloop; Miniplast, Ein Shemer, Israel) inserted into the crop. A second incision about 2 mm proximal to the posterior sucker was made and the intestinal contents were sampled with a loop. Samples were inoculated on 5% sheep blood agar, incubated at 37C and examined after 24 and 48 h. Oxidase positive organisms that grew on MacConkey agar and fermented glucose were identified using API 20NE panels (bioMerieux, Marcy-l’Etoile, France).
Artificial feeding
The artificial feeding device comprised a glass feeding chamber resembling an inverted thistle funnel with a jacket added for temperature control (Fig. 1). The chamber was prewarmed to 38–40C by running warm water through its outer jacket. This apparatus was inserted into a 250-mL plastic container, to which 100 mL of dechlorinated water had been added, through a close-fitting hole cut into its lid. A Parafilm membrane (American National Can, Chicago, IL, USA) was stretched tightly across the lower part of the inverted funnel. The feeding solution was added through the tube of the funnel. This was a 2 g/L arginine solution in 6.4 g/L NaCl, which is a known phagostimulant for leeches [10,11]. For the decontamination experiments the same solution was supplemented with 100 mg/L ciprofloxacin. Outdated transfusion units of human blood replaced the artificial phagostimulant to assess the readiness of leeches to take a
Molecular identification of Aeromonas species
DNA was extracted using a commercial kit (Wizard Genomic DNA Purification Kit; Promega, Inc., Madison, WI, USA). Amplification of the gyrB gene was performed using primers
ª2009 The Authors Journal Compilation ª2009 European Society of Clinical Microbiology and Infectious Diseases, CMI, 16, 563–567
Mumcuoglu et al.
CMI
gyrB3F and gyrB14R as described [12]. The 1100-bp amplicons were purified and sequenced by a commercial laboratory (HyLabs, Rehovot, Israel). The resulting gyrB sequences were then aligned using the ClustalW algorithm to a series of 21 known sequences from 17 different previously characterized Aeromonas species using MEGA software version 4 (http:// www.megasoftware.net/). A radial phylogenetic tree was constructed with the neighbour-joining method, using sequence data for reference strains of Aeromonas spp. derived from two studies [12,13] in which the same primers were used. Statistical methods
Fisher’s exact test was used to compare treated with control leeches.
Results Approximately 80% of the leeches engorged fully when fed artificially either on human blood or on the arginine/NaCl solution with and without antibiotics. In 94.4% of the 106 unfed leeches treated with 70% ethyl alcohol no bacteria were grown from exterior surface samples. However, this treatment caused the death of the leeches as expected. Aeromonas spp. were detected in 57 out of 80 control leeches fed only with arginine/NaCl solution (71.3%), but in none of the 56 leeches treated with ciprofloxacin (p <0.001 using Fisher’s exact test). In the untreated control leeches, bacteria were found in the intestine, but not in the crop, in 35 animals (61.4%); they were confined to the crop in nine animals (15.8%), whereas in 13 (22.8%) they were present both in the crop and intestine. A blood-meal, through artificial feeding, was offered to 56 leeches treated with antibiotics. Thirty-four of them took a blood meal 1–4 weeks after treatment, whereas 22 leeches refused to feed, all of them in the first 2 weeks. All 56 leeches were dissected and examined for Aeromonas spp. All were negative (Table 1). Aeromonads were also not detected in the water in which the leeches were kept during the experimental period. TABLE 1. Presence of aeromonads in leeches treated with ciprofloxacin (100 mg/L) Blood meal offered At 1 week At 2 weeks At 3 weeks At 4 weeks Total
n
Blood meal taken
Aeromonas spp.
Other environmental organisms
6 31 10 9 56
1 14 10 9 34 (61%)
0 0 0 0 0
0 2 6 1 9
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Ten antibiotic-treated leeches were maintained individually in sterilized chlorine-free tap water containing ciprofloxacin at a concentration of 20 mg/L. Three died after 2 months, three after 3 months and four of them survived up to 4 months. Aeromonads were not detected in any of the 15 arginine/ NaCl samples from feeding chambers that were used to feed antibiotic-treated leeches, whereas other environmental microorganisms were found in 16% (9 out of 56) of these leeches. Environmental bacteria such as Ochrobactrum spp. and some filamentous fungi were also isolated from 26.5% of treated leeches that had taken a blood meal 1–4 weeks after treatment (data not shown). All 106 water samples in which leeches were kept before treatment were contaminated with Aeromonas spp. The dominant species was identified by the API 20NE kit as A. sobria. Two different phenotypes within this taxon were studied further: isolates designated 1 and 3. The API 20 NE profile for isolate 1 was 7176755 (99.2%. T 1.0). Additional conventional tests showed the isolate to be Voges-Proskauer positive, DNAse positive and ornithine decarboxylase negative, all properties consistent with an identification of A. veronii biovar sobria [14]. The profile for isolate 3 was 7176744 (84.8%, T 0.72) being much less compatible with the identification of A. sobria. This organism was Voges-Proskauer negative, DNAse negative, ornithine decarboxylase negative and confirmed negative for citrate, a combination much less consistent with an identification of A. veronii biovar sobria. Clinical diagnostic laboratory methods for identification of Aeromonas spp. are known to be inconclusive [14–16], so molecular characterization was undertaken to clarify the taxonomic status of these isolates. Indeed, the gyrB sequences revealed that isolate 1 was very closely matched with A. veronii and that isolate 3 bore most similarity to A. media (Fig. 2). The API reactions and the additional conventional testing of this isolate were consistent with this taxon [14].
Discussion It is generally accepted that the medicinal leech has an obligatory symbiotic relationship with the bacteria of the genus Aeromonas, which live in its intestinal tract. The symbiotic bacteria: (i) contribute enzymes such as proteases, lipases and amylases, which play a major role in digestion; (ii) provide vitamins of the B-complex, which are sparse in blood; and (iii) secrete antimicrobial substances, which prevent the growth of other bacteria and accordingly retard putrefaction so that the leech can store blood for long periods [4,17].
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A. veronii bv. Sobria CECT 4246 A. veronii bv. Veronii CECT 4257 A. veronii bv. Sobria CECT 4906 A. veronii bv. Veronii CECT 4260 Aeromonas 1* A. culicicola MTCC 3249 A. sobria CECT 4245 A. allosaccharophila CECT 4199 A. hydrophila CECT 839 A. jandaei CECT 4228 A. trota CECT 4255 A. caviae CECT 838 A. caviae CECT 4221 Aeromonas 3* A. media CECT 4232 A. media CECT 4234 A. eucrenophila CECT 4224 A. encheleia CECT 4342 A. bestiarum CECT 4227 A. popoffii CECT 5176 A. salmonicida sbsp masoucida CECT 896 A. salmonicida sbsp achromogene CECT 895 A. schubertii CECT 4240
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FIG. 2. Phylogenetic position of Aeromonas isolates 1* and 3* according to gyrB sequences. Phylogenetic tree constructed with the neighbour-joining method using sequence data for reference strains of Aeromonas spp. derived from the studies of Yanez et al., 2003 [12] and Pidiyar et al., 2003 [13], in which the same primers were used. The bar shows 0.01 sub-
0.01
In earlier studies, phenotyping of colonizing bacteria classified all aeromonad isolates from H. medicinalis as A. hydrophila. As identification techniques and classification of these organisms evolved, Graf [18] found A. veronii biovar sobria as a single species in the crop and intestine of this leech. In contrast, Mackay et al. [7] found A. caviae to be the most common species encountered. Using molecular taxonomic techniques, we identified two species among aeromonads colonizing the intestinal tract of a single population of leeches; A. veronii, phenotypically consistent with A. veronii var sobria, and A. media. Recent studies utilizing DNA sequence-based analyses have thrown new light on the complex biology of the bacterial populations of the leech intestine; apart from A. veronii, additional taxa were identified, including genera in the a-, c-, and d-Proteobacteria, Fusobacteria, Firmicutes, and Bacteroidetes. The clearly dominant organisms were A. veronii and a novel clone of a Rikenella-like bacterium of the Bacteroidetes group, designated PW3, whereas the other clones, considered by the investigators to represent transient organisms, were typically present only in low numbers [19–21]. In addition, A. veronii and A. jandaei have been cultured from Hirudo orientalis [22], whereas A. jandaei has been also isolated from Macrobdella decora [23]. In the present study, aeromonads were reduced to levels undetectable by our methods for up to 4 weeks in all
stitutions per nucleotide position.
leeches treated with ciprofloxacin and subsequently fed with human blood. A blood meal is normally followed by proliferation of aeromonads in the gut. Furthermore these bacteria were neither found in the water in which the leeches were kept nor in the blood or arginine solution within the feeding chamber; the latter observation suggests that aeromonads were not present in detectable numbers in the mouthparts or saliva of the leeches. Several groups have attempted to eliminate the bacterial load inside and outside leeches by treating them exteriorly with antibiotics and antiseptics, succeeding only in eliminating bacteria from the surface of the leech [7,8,18,19]. In this study, the artificial feeding of leeches was conducted under non-sterile conditions. It is possible that the bacteria and fungi found in the intestinal tract of the leeches after antibiotic treatment might have had their origin in the environment. However, it is equally conceivable that they were microorganisms that normally exist in very low quantities in the leech intestine as a result of their sensitivity to blood components or antimicrobial substances excreted by the dominant aeromonad, and that flourished after suppression of the aeromonad by ciprofloxacin. Whitlock et al. [24] predicted that aeromonads would be an infection risk in hirudotherapy, and Dickson et al. [25]
ª2009 The Authors Journal Compilation ª2009 European Society of Clinical Microbiology and Infectious Diseases, CMI, 16, 563–567
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Mumcuoglu et al.
described a case after breast reconstruction. Since then, Aeromonas infections after leech application have become well known, including both soft tissue infections and other severe infections such as septicaemia and even meningitis [26–29]. Infections have been observed in 7–20% of patients treated with leeches, reducing the chances of successful re-implantations or flaps by 30% [30]. Therefore, during treatment antibiotics such as cephalosporins or fluoroquinolones are commonly given prophylactically [31]. In the present work, our efforts were directed at detecting an antibiotic effect in treated leeches. It was shown that either Aeromonas spp. were reduced to undetectable levels in our system and possibly eliminated, or that surviving bacteria lost their ability to multiply. As a result of resource limitations at this stage, it was not possible to maximize the sensitivity of detection or to assess whether survivors had entered a ‘viable but not culturable’ state. The dramatic reduction in aeromonad densities was a useful endpoint for the study. It is highly likely that viable but not culturable organisms might be of no clinical consequence in the clinical settings in which hirudotherapy is conducted. In conclusion, the results of the present study suggest that the strategy of feeding leeches artificially with antibiotics reduces the number of aeromonads to undetectable levels for extended periods, and that treated leeches are likely to take a blood meal from patients undergoing hirudotherapy.
Transparency Declaration None of the authors has any relationship (commercial or otherwise) that may constitute a dual or conflicting interest.
References 1. Whitaker IS, Izadi D, Oliver DW, Monteath G, Butler PE. Hirudo medicinalis and the plastic surgeon. Br J Plast Surg 2004; 57: 348– 353. 2. Markwardt F. Untersuchungen ueber Hirudin. Naturwissenschaften 1955; 42: 537–538. 3. Harvey RP, Degryse E, Stefani L et al. Cloning and expression of a cDNA coding for the anticoagulant hirudin from the bloodsucking leech Hirudo medicinalis. Proc Natl Acad Sci USA 1986; 83: 1084. 4. Sawyer RT Leech biology and behavior. Vol. II: feeding biology, ecology and systematics. Oxford, UK: Oxford University Press, 1986. 5. Rigbi M, Levy H, Iraqi F et al. The saliva of the medicinal leech Hirudo medicinalis. I. Biochemical characterization of the high molecular weight fraction. Comp Biochem Physiol B 1987; 87: 567–573. 6. De Chalain TM. Exploring the use of the medicinal leech: a clinical risk-benefit analysis. J Reconstr Microsurg 1996; 12: 165–172. 7. Mackay DR, Manders EK, Saggers GCD, Banducci R, Prinsloo J, Klugman K. Aeromonas species isolated from medicinal leeches . Ann Plast Surg 1999; 42: 275–279.
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8. Aydin A, Nazik H, Kuvat SV et al. External decontamination of wild leeches with hypochloric acid. Infect Dis 2004; 4: 28. 9. Siddall ME, Trontelj P, Utevsky SY, Nkamany M, Macdonald KS. Diverse molecular data demonstrate that commercially available medicinal leeches are not Hirudo medicinalis. Proc Biol Sci 2007; 274: 1481–1487. 10. Galun R, Kindler SH. Chemical specificity of the feeding response in Hirudo medicinalis (L.). Comp Biochem Physiol 1966; 17: 69–73. 11. Galun R. The role of host blood in the feeding behavior of ectoparasites. In: Zuckerman A, ed. Dynamic aspects of host–parasite relationships, Vol. II. New York: John Wiley, 1975; 132–160 12. Yanez MA, Catalan V, Apraiz D, Figueras MJ, Martinez-Murcia AJ. Phylogenetic analysis of members of the genus Aeromonas based on gyrB gene sequences. Int J Syst Evol Microbiol 2003; 53: 875–883. 13. Pidiyar VJ, Jangid K, Dayananda KM et al. Phylogenetic affiliation of Aeromonas culicicola MTCC 3249(T) based on gyrB gene sequence and PCR-amplicon sequence analysis of cytolytic enterotoxin gene. Syst Appl Microbiol 2003; 26: 197–202. 14. Abbott SL, Cheung WK, Janda JM. The genus Aeromonas: biochemical characteristics, atypical reactions, and phenotypic identification schemes. J Clin Microbiol 2003; 41: 2348–2357. 15. Ormen O, Granum PE, Lassen J, Figueras MJ. Lack of agreement between biochemical and genetic identification of Aeromonas spp. APMIS 2005; 113: 203–207. 16. Park TS, Oh SH, Lee EY et al. Misidentification of Aeromonas veronii biovar sobria as Vibrio alginolyticus by the Vitek system. Lett Appl Microbiol 2003; 37: 349–353. 17. Jennings JB, Van der Lande VM. Histochemical and bacteriological studies on digestion in nine species of leeches (Annelida: Hirudinea). Biol Bull 1967; 133: 166–183. 18. Graf J. Symbiosis of Aeromonas veronii biovar sobria and Hirudo medicinalis, the medicinal leech: a novel model for digestive tract associations. Infect Immun 1999; 67: 1–7. 19. Worthen PL, Gode CJ, Graf J. Culture-independent characterization of the digestive-tract microbiota of the medicinal leech reveals a tripartite symbiosis. Appl Environ Microbiol 2006; 72: 4775–4781. 20. Kikuchi Y, Graf J. Spatial and temporal population dynamics of a naturally occurring two-species microbial community inside the digestive tract of the medicinal leech. Appl Environ Microbiol 2007; 73: 1984–1991. 21. Graf J, Kikiuchi Y, Rio RVM. Leeches and their microbiota: naturally simple symbiosis models. Trends Microbiol 2006; 14: 365–371. 22. Laufer AS, Siddall ME, Graf J. Characterization of the digestive-tract microbiota of Hirudo orientalis, a European medicinal leech. Appl Environ Microbiol 2008; 74: 6151–6154. 23. Siddall ME, Worthen PL, Johnson M, Graf J. Novel role for Aeromonas jandaei as a digestive tract symbiont of the North American medicinal leech. Appl Environ Microbiol 2007; 73: 655–658. 24. Whitlock MR, O’Hare PM, Sanders R, Morrow NC. The medicinal leech and its use in plastic surgery: a possible cause for infection. Br J Plast Surg 1983; 36: 240–244. 25. Dickson WA, Boothman P, Hare K. An unusual source of hospital wound infection. Br Med J (Clin Res Ed). 1984; 289: 1727–1728. 26. Bauters TG, Buyle FM, Verschraegen G et al. Infection risk related to the use of medicinal leeches. Pharm World Sci 2007; 29: 122–125. 27. Evans JP, Lunnis J, Gaunt PN, Hanley DJ. A case of septicaemia due to Aeromonas hydrophila. Br J Plast Surg 1990; 43: 371–372. 28. Ouderkirk JP, Bekhor D, Turett GS, Murali R. Aeromonas meningitis complicating medicinal leech therapy. Clin Infect Dis 2004; 38: 36–37. 29. Sartor C, Limouzin-Perotti F, Legre´ R et al. Nosocomial infections with Aeromonas hydrophila from leeches. Clin Infect Dis 2002; 35: E1–E5. 30. Mercer NSG, Beere DM, Bornemisza AI, Thomas P. Medical leeches as sources of wound infections. Br Med J 1984; 294: 937. 31. Eldor A, Orevi M, Rigbi M. The role of the leech in medical therapeutics. Blood Rev 1996; 10: 201–209.
ª2009 The Authors Journal Compilation ª2009 European Society of Clinical Microbiology and Infectious Diseases, CMI, 16, 563–567
BACTERIOLOGY
Elimination of symbiotic Aeromonas spp. from the intestinal tract of the medicinal leech, Hirudo medicinalis, using ciprofloxacin feeding K. Y. Mumcuoglu1, L. Huberman1, R. Cohen1, V. Temper2, A. Adler2, R. Galun1 and C. Block2 1) Department of Parasitology and 2) Department of Clinical Microbiology and Infectious Diseases, Hebrew University-Hadassah Medical School, Jerusalem, Israel
Abstract 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 leechassociated 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. Keywords: Aeromonas, artificial feeding, Hirudo medicinalis, medicinal leech, symbiont Original Submission: 28 November 2008; Revised Submission: 5 January 2009; Accepted: 7 January 2009 Editor: D. Raoult Article published online: 11 June 2009 Clin Microbiol Infect 2010; 16: 563–567 10.1111/j.1469-0691.2009.02868.x Corresponding author and reprint requests: K. Y. Mumcuoglu, PhD, Department of Parasitology, Hebrew University-Hadassah Medical School, PO Box 12272, Jerusalem 91120, Israel E-mail: [email protected]
Introduction The use of the medicinal leech (Hirudo medicinalis) for the treatment of human diseases has been known since ancient Egyptian times. Galen (130–201 AD) used leeches for bloodletting, which was based on the belief that removal of the patient’s blood would correct the humoral imbalance and restore good health [1]. In the 18th and 19th centuries, the popularity of leeching reached its height in Europe. In 1884, Haycraft discovered hirudin, an anti-coagulant substance in leech saliva. Markwardt [2] isolated and characterized this molecule and in 1986 it was produced by genetic engineering [3].
In recent years, recognition of a number of antithrombotic substances and a vasodilator in the saliva of the medicinal leech [4,5] has generated renewed interest in the use of the leech itself to promote venous drainage in tissues whose vitality is threatened by venous congestion and obstruction, especially after plastic and reconstructive surgery. In 2004, this treatment modality received the approval of the Food and Drug Administration in the USA. Hirudotherapy, as it is termed, has been complicated by infections caused by Aeromonas spp. which are considered to be obligatory endosymbionts of the leech [4]. Infections with aeromonads have been observed in 7–20% of patients treated with leeches after reconstructive surgery; in these cases the chances of successful re-implantation or flap survival dropped by 30%. Therefore, antibiotics such as cephalosporins or fluoroquinolones are given prophylactically for the duration of the treatment [1,6].
ª2009 The Authors Journal Compilation ª2009 European Society of Clinical Microbiology and Infectious Diseases
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Clinical Microbiology and Infection, Volume 16 Number 6, June 2010
In order to reduce the risk of infection, attempts have been made to remove or decrease the number of aeromonads inside and outside leeches, by frequent changes of the water in leech containers, and by submerging leeches in hypochloric acid and antibiotics, however, these treatment modalities were not sufficient to destroy the internal bacterial flora of the leech [7,8]. Antimicrobial prophylaxis for bacteria remaining in the digestive tract is thus necessary to make the leeches suitable for the treatment of patients, preventing their internal bacteria from constituting a source of infection. The purpose of this study was to attempt to suppress the endosymbiotic aeromonads by feeding leeches artificially with a blood-free antibiotic solution, with the ultimate aim of eliminating the need for chemoprophylaxis in the patients. Ciprofloxacin, which is an agent of choice in the therapy and prophylaxis of infection as a result of these organisms, was chosen after preliminary testing showed leech-associated Aeromonas spp. to be fully susceptible.
Materials and Methods
CMI
Feeding chamber Warming water
Blood or arginine
Membrane Water
Feeding leech
FIG. 1. Schematic representation of the artificial feeding chamber for leeches.
blood meal after treatment. After feeding, leeches were maintained individually in sterilized chlorine-free tap water containing ciprofloxacin at a concentration of 20 mg/L and examined periodically for bacterial contamination.
Leeches
Bacteriological examination
Leeches (Hirudo medicinalis) collected in the wild in Turkey (Ergene Ltd, Tekirdag) were kept in holding vessels (40 · 60 · 40-cm plastic containers) in chlorine-free tap water that was changed weekly. (The authors are familiar with recent changes in the taxonomy of Hirudo species in which many medicinally applied leeches have been shown to be H. verbana rather than H. medicinalis. As the leeches used in this study were not formally identified, the latter term was kept for convenience [9]).
Samples were taken from the water in the holding vessels, from the exterior surface of the animals as well as from their alimentary tract and from the feeding solution. Leeches were disinfected externally by immersion in 70% ethyl alcohol for 1 min, followed by transfer to sterile distilled water. The purpose of this was to ensure that external contaminants did not interfere with cultures from the intestinal tract. In order to prevent the entry of alcohol to the digestive tract and to avoid the discharge of gut contents through the mouth or anus, a ligature was applied to the proximal and distal ends (c. 2–3 mm) of the leech. Swabs were taken from the outer surface of the animals, which were then transected c. 10 mm distal to the anterior sucker, and crop fluid was collected using a 10-lL disposable bacteriological loop (Quadloop; Miniplast, Ein Shemer, Israel) inserted into the crop. A second incision about 2 mm proximal to the posterior sucker was made and the intestinal contents were sampled with a loop. Samples were inoculated on 5% sheep blood agar, incubated at 37C and examined after 24 and 48 h. Oxidase positive organisms that grew on MacConkey agar and fermented glucose were identified using API 20NE panels (bioMerieux, Marcy-l’Etoile, France).
Artificial feeding
The artificial feeding device comprised a glass feeding chamber resembling an inverted thistle funnel with a jacket added for temperature control (Fig. 1). The chamber was prewarmed to 38–40C by running warm water through its outer jacket. This apparatus was inserted into a 250-mL plastic container, to which 100 mL of dechlorinated water had been added, through a close-fitting hole cut into its lid. A Parafilm membrane (American National Can, Chicago, IL, USA) was stretched tightly across the lower part of the inverted funnel. The feeding solution was added through the tube of the funnel. This was a 2 g/L arginine solution in 6.4 g/L NaCl, which is a known phagostimulant for leeches [10,11]. For the decontamination experiments the same solution was supplemented with 100 mg/L ciprofloxacin. Outdated transfusion units of human blood replaced the artificial phagostimulant to assess the readiness of leeches to take a
Molecular identification of Aeromonas species
DNA was extracted using a commercial kit (Wizard Genomic DNA Purification Kit; Promega, Inc., Madison, WI, USA). Amplification of the gyrB gene was performed using primers
ª2009 The Authors Journal Compilation ª2009 European Society of Clinical Microbiology and Infectious Diseases, CMI, 16, 563–567
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gyrB3F and gyrB14R as described [12]. The 1100-bp amplicons were purified and sequenced by a commercial laboratory (HyLabs, Rehovot, Israel). The resulting gyrB sequences were then aligned using the ClustalW algorithm to a series of 21 known sequences from 17 different previously characterized Aeromonas species using MEGA software version 4 (http:// www.megasoftware.net/). A radial phylogenetic tree was constructed with the neighbour-joining method, using sequence data for reference strains of Aeromonas spp. derived from two studies [12,13] in which the same primers were used. Statistical methods
Fisher’s exact test was used to compare treated with control leeches.
Results Approximately 80% of the leeches engorged fully when fed artificially either on human blood or on the arginine/NaCl solution with and without antibiotics. In 94.4% of the 106 unfed leeches treated with 70% ethyl alcohol no bacteria were grown from exterior surface samples. However, this treatment caused the death of the leeches as expected. Aeromonas spp. were detected in 57 out of 80 control leeches fed only with arginine/NaCl solution (71.3%), but in none of the 56 leeches treated with ciprofloxacin (p <0.001 using Fisher’s exact test). In the untreated control leeches, bacteria were found in the intestine, but not in the crop, in 35 animals (61.4%); they were confined to the crop in nine animals (15.8%), whereas in 13 (22.8%) they were present both in the crop and intestine. A blood-meal, through artificial feeding, was offered to 56 leeches treated with antibiotics. Thirty-four of them took a blood meal 1–4 weeks after treatment, whereas 22 leeches refused to feed, all of them in the first 2 weeks. All 56 leeches were dissected and examined for Aeromonas spp. All were negative (Table 1). Aeromonads were also not detected in the water in which the leeches were kept during the experimental period. TABLE 1. Presence of aeromonads in leeches treated with ciprofloxacin (100 mg/L) Blood meal offered At 1 week At 2 weeks At 3 weeks At 4 weeks Total
n
Blood meal taken
Aeromonas spp.
Other environmental organisms
6 31 10 9 56
1 14 10 9 34 (61%)
0 0 0 0 0
0 2 6 1 9
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Ten antibiotic-treated leeches were maintained individually in sterilized chlorine-free tap water containing ciprofloxacin at a concentration of 20 mg/L. Three died after 2 months, three after 3 months and four of them survived up to 4 months. Aeromonads were not detected in any of the 15 arginine/ NaCl samples from feeding chambers that were used to feed antibiotic-treated leeches, whereas other environmental microorganisms were found in 16% (9 out of 56) of these leeches. Environmental bacteria such as Ochrobactrum spp. and some filamentous fungi were also isolated from 26.5% of treated leeches that had taken a blood meal 1–4 weeks after treatment (data not shown). All 106 water samples in which leeches were kept before treatment were contaminated with Aeromonas spp. The dominant species was identified by the API 20NE kit as A. sobria. Two different phenotypes within this taxon were studied further: isolates designated 1 and 3. The API 20 NE profile for isolate 1 was 7176755 (99.2%. T 1.0). Additional conventional tests showed the isolate to be Voges-Proskauer positive, DNAse positive and ornithine decarboxylase negative, all properties consistent with an identification of A. veronii biovar sobria [14]. The profile for isolate 3 was 7176744 (84.8%, T 0.72) being much less compatible with the identification of A. sobria. This organism was Voges-Proskauer negative, DNAse negative, ornithine decarboxylase negative and confirmed negative for citrate, a combination much less consistent with an identification of A. veronii biovar sobria. Clinical diagnostic laboratory methods for identification of Aeromonas spp. are known to be inconclusive [14–16], so molecular characterization was undertaken to clarify the taxonomic status of these isolates. Indeed, the gyrB sequences revealed that isolate 1 was very closely matched with A. veronii and that isolate 3 bore most similarity to A. media (Fig. 2). The API reactions and the additional conventional testing of this isolate were consistent with this taxon [14].
Discussion It is generally accepted that the medicinal leech has an obligatory symbiotic relationship with the bacteria of the genus Aeromonas, which live in its intestinal tract. The symbiotic bacteria: (i) contribute enzymes such as proteases, lipases and amylases, which play a major role in digestion; (ii) provide vitamins of the B-complex, which are sparse in blood; and (iii) secrete antimicrobial substances, which prevent the growth of other bacteria and accordingly retard putrefaction so that the leech can store blood for long periods [4,17].
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A. veronii bv. Sobria CECT 4246 A. veronii bv. Veronii CECT 4257 A. veronii bv. Sobria CECT 4906 A. veronii bv. Veronii CECT 4260 Aeromonas 1* A. culicicola MTCC 3249 A. sobria CECT 4245 A. allosaccharophila CECT 4199 A. hydrophila CECT 839 A. jandaei CECT 4228 A. trota CECT 4255 A. caviae CECT 838 A. caviae CECT 4221 Aeromonas 3* A. media CECT 4232 A. media CECT 4234 A. eucrenophila CECT 4224 A. encheleia CECT 4342 A. bestiarum CECT 4227 A. popoffii CECT 5176 A. salmonicida sbsp masoucida CECT 896 A. salmonicida sbsp achromogene CECT 895 A. schubertii CECT 4240
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FIG. 2. Phylogenetic position of Aeromonas isolates 1* and 3* according to gyrB sequences. Phylogenetic tree constructed with the neighbour-joining method using sequence data for reference strains of Aeromonas spp. derived from the studies of Yanez et al., 2003 [12] and Pidiyar et al., 2003 [13], in which the same primers were used. The bar shows 0.01 sub-
0.01
In earlier studies, phenotyping of colonizing bacteria classified all aeromonad isolates from H. medicinalis as A. hydrophila. As identification techniques and classification of these organisms evolved, Graf [18] found A. veronii biovar sobria as a single species in the crop and intestine of this leech. In contrast, Mackay et al. [7] found A. caviae to be the most common species encountered. Using molecular taxonomic techniques, we identified two species among aeromonads colonizing the intestinal tract of a single population of leeches; A. veronii, phenotypically consistent with A. veronii var sobria, and A. media. Recent studies utilizing DNA sequence-based analyses have thrown new light on the complex biology of the bacterial populations of the leech intestine; apart from A. veronii, additional taxa were identified, including genera in the a-, c-, and d-Proteobacteria, Fusobacteria, Firmicutes, and Bacteroidetes. The clearly dominant organisms were A. veronii and a novel clone of a Rikenella-like bacterium of the Bacteroidetes group, designated PW3, whereas the other clones, considered by the investigators to represent transient organisms, were typically present only in low numbers [19–21]. In addition, A. veronii and A. jandaei have been cultured from Hirudo orientalis [22], whereas A. jandaei has been also isolated from Macrobdella decora [23]. In the present study, aeromonads were reduced to levels undetectable by our methods for up to 4 weeks in all
stitutions per nucleotide position.
leeches treated with ciprofloxacin and subsequently fed with human blood. A blood meal is normally followed by proliferation of aeromonads in the gut. Furthermore these bacteria were neither found in the water in which the leeches were kept nor in the blood or arginine solution within the feeding chamber; the latter observation suggests that aeromonads were not present in detectable numbers in the mouthparts or saliva of the leeches. Several groups have attempted to eliminate the bacterial load inside and outside leeches by treating them exteriorly with antibiotics and antiseptics, succeeding only in eliminating bacteria from the surface of the leech [7,8,18,19]. In this study, the artificial feeding of leeches was conducted under non-sterile conditions. It is possible that the bacteria and fungi found in the intestinal tract of the leeches after antibiotic treatment might have had their origin in the environment. However, it is equally conceivable that they were microorganisms that normally exist in very low quantities in the leech intestine as a result of their sensitivity to blood components or antimicrobial substances excreted by the dominant aeromonad, and that flourished after suppression of the aeromonad by ciprofloxacin. Whitlock et al. [24] predicted that aeromonads would be an infection risk in hirudotherapy, and Dickson et al. [25]
ª2009 The Authors Journal Compilation ª2009 European Society of Clinical Microbiology and Infectious Diseases, CMI, 16, 563–567
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Mumcuoglu et al.
described a case after breast reconstruction. Since then, Aeromonas infections after leech application have become well known, including both soft tissue infections and other severe infections such as septicaemia and even meningitis [26–29]. Infections have been observed in 7–20% of patients treated with leeches, reducing the chances of successful re-implantations or flaps by 30% [30]. Therefore, during treatment antibiotics such as cephalosporins or fluoroquinolones are commonly given prophylactically [31]. In the present work, our efforts were directed at detecting an antibiotic effect in treated leeches. It was shown that either Aeromonas spp. were reduced to undetectable levels in our system and possibly eliminated, or that surviving bacteria lost their ability to multiply. As a result of resource limitations at this stage, it was not possible to maximize the sensitivity of detection or to assess whether survivors had entered a ‘viable but not culturable’ state. The dramatic reduction in aeromonad densities was a useful endpoint for the study. It is highly likely that viable but not culturable organisms might be of no clinical consequence in the clinical settings in which hirudotherapy is conducted. In conclusion, the results of the present study suggest that the strategy of feeding leeches artificially with antibiotics reduces the number of aeromonads to undetectable levels for extended periods, and that treated leeches are likely to take a blood meal from patients undergoing hirudotherapy.
Transparency Declaration None of the authors has any relationship (commercial or otherwise) that may constitute a dual or conflicting interest.
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