Closed Genome Sequence of Aeromonas veronii Strain Hm21, an Isolate from the Medicinal Leech Hirudo verbana.

Aeromonas veronii strain Hm21 was isolated from the medicinal leech Hirudo verbana and is used for genetic studies. We present here the 4.71-Mbp genome with a 56-kb plasmid and identify the mutations present in strains commonly used for genetic engineering.

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.

Host Matters: Medicinal Leech Digestive-Tract Symbionts and Their Pathogenic Potential.

Digestive-tract microbiota exert tremendous influence over host health. Host-symbiont model systems are studied to investigate how symbioses are initiated and maintained, as well as to identify host processes affected by resident microbiota. The medicinal leech, Hirudo verbana, is an excellent model to address such questions owing to a microbiome that is consistently dominated by two species, Aeromonas veronii and Mucinivorans hirudinis, both of which are cultivable and have sequenced genomes. This review outlines current knowledge about the dynamics of the H. verbana microbiome. We discuss in depth the factors required for A. veronii colonization and proliferation in the leech crop and summarize the current understanding of interactions between A. veronii and its annelid host. Lastly, we discuss leech usage in modern medicine and highlight how leech-therapy associated infections, often attributable to Aeromonas spp., are of growing clinical concern due in part to an increased prevalence of fluoroquinolone resistant strains.

Draft Genome Sequence of Pedobacter sp. Strain Hv1, an Isolate from Medicinal Leech Mucosal Castings.

The Pedobacter sp. Hv1 strain was isolated from the medicinal leech, Hirudo verbana, mucosal castings. These mucosal sheds have been demonstrated to play a role in horizontal symbiont transmission. Here, we report the draft 4.9 Mbp genome sequence of Pedobacter sp. strain Hv1.

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)).

Metagenomic analysis of the medicinal leech gut microbiota.

There are trillions of microbes found throughout the human body and they exceed the number of eukaryotic cells by 10-fold. Metagenomic studies have revealed that the majority of these microbes are found within the gut, playing an important role in the host's digestion and nutrition. The complexity of the animal digestive tract, unculturable microbes, and the lack of genetic tools for most culturable microbes make it challenging to explore the nature of these microbial interactions within this niche. The medicinal leech, Hirudo verbana, has been shown to be a useful tool in overcoming these challenges, due to the simplicity of the microbiome and the availability of genetic tools for one of the two dominant gut symbionts, Aeromonas veronii. In this study, we utilize 16S rRNA gene pyrosequencing to further explore the microbial composition of the leech digestive tract, confirming the dominance of two taxa, the Rikenella-like bacterium and A. veronii. The deep sequencing approach revealed the presence of additional members of the microbial community that suggests the presence of a moderately complex microbial community with a richness of 36 taxa. The presence of a Proteus strain as a newly identified resident in the leech crop was confirmed using fluorescence in situ hybridization (FISH). The metagenome of this community was also pyrosequenced and the contigs were binned into the following taxonomic groups: Rikenella-like (3.1 MB), Aeromonas (4.5 MB), Proteus (2.9 MB), Clostridium (1.8 MB), Eryspelothrix (0.96 MB), Desulfovibrio (0.14 MB), and Fusobacterium (0.27 MB). Functional analyses on the leech gut symbionts were explored using the metagenomic data and MG-RAST. A comparison of the COG and KEGG categories of the leech gut metagenome to that of other animal digestive-tract microbiomes revealed that the leech digestive tract had a similar metabolic potential to the human digestive tract, supporting the usefulness of this system as a model for studying digestive-tract microbiomes. This study lays the foundation for more detailed metatranscriptomic studies and the investigation of symbiont population dynamics.

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.

Draft Genome Sequence of Aeromonas veronii Hm21, a Symbiotic Isolate from the Medicinal Leech Digestive Tract.

Aeromonas veronii strain Hm21 was isolated from the digestive tract of the medicinal leech Hirudo verbana and has been used to identify genes that are important for host colonization. This species is also a symbiont in the gut of zebrafish and is a pathogen of mammals and fish. We present here a 4.68-Mbp draft genome sequence for Hm21.

Investigation into the physiologies of Aeromonas veronii in vitro and inside the digestive tract of the medicinal leech using RNA-seq.

Host-associated microbial communities are widespread in nature and vital to the health and fitness of the host. Deciphering the physiology of the microbiome in vivo is critical to understanding the molecular basis of the symbiosis. Recently, the development and application of high-throughput sequencing techniques, particularly RNA-seq, for studying microbial communities has enabled researchers to address not only which microbes are present in a given community but also how the community functions. For microbes that can also be cultivated in the laboratory, RNA-seq provides the opportunity to identify genes that are differentially expressed during symbiosis by comparing in vitro to in vivo transcriptomes. In the current study, we used RNA-seq to identify genes expressed by the digestive-tract microbiome of the medicinal leech, Hirudo verbana, and by one of the two dominant symbionts, Aeromonas veronii, in a rich medium. We used a comparative approach to identify genes differentially expressed during symbiosis and gain insight into the symbiont's physiology in vivo. Notable findings include evidence for the symbionts experiencing environmental stress, performing arginine catabolism, and expressing noncoding RNAs that are implicated in stationary phase survival, a state in which A. veronii persists for months within the host.

Bacterial symbioses of the medicinal leech Hirudo verbana.

Gastrointestinal microbiomes play important roles in the health and nutrition of animals and humans. The medicinal leech, Hirudo verbana, serves as a powerful model for the study of microbial symbioses of the gut, due to its naturally limited microbiome compared with other popular models, the ability to cultivate the most abundant microbes, and genetically manipulate one of them, Aeromonas veronii. This review covers the relevance and application of leeches in modern medicine as well as recent discoveries detailing the nature of the gut microbiome. Additionally, the dual life-style of A. veronii allows one to do direct comparisons between colonization factors for beneficial and pathogenic associations, and relevant findings are detailed with respect to their role within the host and pathogenicity to other animals.

Medicinal leeches and the microsurgeon: a four-year study, clinical series and risk benefit review.

BACKGROUND: There are case reports and small series in the literature relating to the use of medicinal leeches by plastic surgeons; however, larger series from individual units are rare. The aim of this article is to present a comprehensive 4-year case series of the use of medicinal leeches, discuss the current evidence regarding indications, risks, and benefits and highlight the recent updates regarding leech speciation. METHODS: Patients prescribed leeches in a 4-year period (July 2004-2008) were collated from hospital pharmacy records (N = 35). The number of leeches used, demographic, clinical, and microbiological details were retrospectively analyzed. RESULTS: Thirty-five patients were treated with leeches. The age range was 2 to 98 years (mean = 49.3). Leeches were most commonly used for venous congestion in pedicled flaps and replantations. Blood transfusions were necessary in 12 cases (34%) [mean = 2.8 units, range 2-5 units]. Our infection rate was 20% (7/35) including five infections with Aeromonas spp. (14.2%). The proportion of patients becoming infected after leech therapy was significantly greater in the group of patients that did not receive prophylactic antibiotic treatment (Fisher's Exact test P = 0.0005). In total, 14 cases (40%) were salvaged in entirety, in 7 cases 80% or more, in 2 cases 50 to 79%, and in 1 case less than 50% of the tissues were salvaged. In 11 cases (31%), the tissues were totally lost. CONCLUSION: Our study highlights both the benefits and the risks to patients in selected clinical situations and also the potential risks. The routine use of antibiotic prophylaxis is supported. In view of the emerging evidence that Hirudo verbana are now used as standard leech therapy, and the primary pathogen is Aeromonas veronii, until a large prospective multicenter study is published, large series of patients treated with leeches should be reported.

Symbiont succession during embryonic development of the European medicinal leech, Hirudo verbana.

The European medicinal leech, Hirudo verbana, harbors simple microbial communities in the digestive tract and bladder. The colonization history, infection frequency, and growth dynamics of symbionts through host embryogenesis are described using diagnostic PCR and quantitative PCR. Symbiont species displayed diversity in temporal establishment and proliferation through leech development.

Stratified bacterial community in the bladder of the medicinal leech, Hirudo verbana.

Most animals harbour symbiotic microorganisms inside their body, where intimate interactions occur between the partners. The medicinal leech, Hirudo verbana, possesses 17 pairs of excretory bladders that harbour a large number of intracellular and extracellular symbiotic bacteria. In this study, we characterized the bladder symbionts using molecular phylogenetic analyses, transmission electron microscopy (TEM) and fluorescence in situ hybridization (FISH). Restriction fragment length polymorphism (RFLP) and sequence analyses of 16S rRNA gene clone libraries suggested that six bacterial species co-colonize the leech bladders. Phylogenetic analyses revealed that these species belong to the alpha-Proteobacteria (Ochrobactrum symbiont), beta-Proteobacteria (Beta-1 and Beta-2 symbionts), delta-Proteobacteria (Bdellovibrio symbiont) and Bacteroidetes (Niabella and Sphingobacterium symbionts). Species-specific PCR detection and FISH confirmed the localization of the symbiotic bacteria in the bladders. The Ochrobactrum, Beta-1, Bdellovibrio and Sphingobacterium symbionts were consistently detected in 13 leeches from two populations, while infection rate of the other symbionts ranged between 20% and 100% in the two leech populations. Transmission electron microscopy observations of the bladders revealed epithelial cells harbouring a number of intracellular bacilli and an additional type of extracellular, rod-shaped bacteria in the luminal region. Fluorescence in situ hybridization with group-specific oligonucleotide probes revealed the spatial organization of the bacterial species in the bladder: the Ochrobactrum symbiont was located intracellularly inside epithelial cells; the Bacteroidetes were localized close to the epithelium in the lumen of the bladder; and the Bacteroidetes layer was covered with dense beta-proteobacterial cells. These results clearly demonstrate that a simple but organized microbial community exists in the bladder of the medicinal leech.

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.

Characterization of the digestive-tract microbiota of Hirudo orientalis, a european medicinal leech.

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

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.

Characterization of a catalase gene from Aeromonas veronii, the digestive-tract symbiont of the medicinal leech.

The catalase gene katA of the medicinal leech symbiont Aeromonas veronii bv. sobria was cloned, sequenced, and functionally characterized. Southern hybridization, using an A. veronii katA-specific hybridization probe, suggested the presence of a single gene copy in many Aeromonas species. A. veronii katA consisted of 1446 nt encoding a protein with a high degree of similarity to the small-subunit group III bacterial catalases. A catalase-null mutant (JG186) was constructed through gene-replacement mutagenesis. In the parent strain (HM21R), catalase activity was only detected in extracts of cells grown to early exponential phase following H(2)O(2) induction, in which the ability to induce activity was inversely related to optical density. In contrast, induced JG186 cells were very sensitive to oxidative stress, with survival being affected even at low H(2)O(2) concentrations. In contrast to the findings of previous reports of other symbiotic systems, the catalase mutant was not defective in its ability to competitively colonize or persist within its host, in both co-inoculation and sole-colonization assays. This body of evidence suggests either that oxidative stress, in the form of H(2)O(2) exposure, is not encountered by the microbial partner under the examined symbiotic conditions or that compensatory mechanisms exist. The data suggest that although many colonization factors reoccur, each symbiotic system has also evolved specific mechanisms that affect symbiont-host dynamics.

Spatial and temporal population dynamics of a naturally occurring two-species microbial community inside the digestive tract of the medicinal leech.

The medicinal leech, Hirudo verbana, is one of the simplest naturally occurring models for digestive-tract symbioses, where only two bacterial species, Aeromonas veronii bv. sobria (gamma-Proteobacteria) and a Rikenella-like bacterium (Bacteroidetes), colonize the crop, the largest compartment of the leech digestive tract. In this study, we investigated spatial and temporal changes of the localization and microcolony structure of the native symbionts in the crop, after ingestion of a sterile blood meal, by fluorescence in situ hybridization. The population dynamics differed between the two symbiotic bacteria. A. veronii was detected mainly as individual cells inside the intraluminal fluid (ILF) during 14 days after feeding (daf) unless it was found in association with Rikenella microcolonies. The Rikenella-like bacteria were observed not only inside the ILF but also in association with the luminal surface of the crop epithelium. The sizes of Rikenella microcolonies changed dynamically through the 14-day period. From 3 daf onward, mixed microcolonies containing both species were frequently observed, with cells of both species tightly associating with each other. The sizes of the mixed microcolonies were consistently larger than the size of either single-species microcolony, suggesting a synergistic interaction of the symbionts. Lectin staining with succinylated wheat germ agglutinin revealed that the planktonic microcolonies present in the ILF were embedded in a polysaccharide matrix containing N-acetylglucosamine. The simplicity, symbiont-symbiont interaction, and mixed microcolonies of this naturally occurring, digestive-tract symbiosis lay the foundation for understanding the more complex communities residing in most animals.

Novel role for Aeromonas jandaei as a digestive tract symbiont of the North American medicinal leech.

The gut bacteria of the North American medicinal leech, Macrobdella decora, were characterized. Biochemical tests and DNA sequences indicated that Aeromonas jandaei is the dominant culturable symbiont in leeches from a broad geographic area. In this work we identified a new habitat for A. jandaei, and here we suggest that there is unexpected specificity between leeches and Aeromonas species.

Culture-independent characterization of the digestive-tract microbiota of the medicinal leech reveals a tripartite symbiosis.

Culture-based studies of the microbial community within the gut of the medicinal leech have typically been focused on various Aeromonas species, which were believed to be the sole symbiont of the leech digestive tract. In this study, analysis of 16S rRNA gene clone libraries confirmed the presence of Aeromonas veronii and revealed a second symbiont, clone PW3, a novel member of the Rikenellaceae, within the crop, a large compartment where ingested blood is stored prior to digestion. The diversity of the bacterial community in the leech intestinum was determined, and additional symbionts were detected, including members of the alpha-, gamma-, and delta-Proteobacteria, Fusobacteria, Firmicutes, and Bacteroidetes. The relative abundances of the clones suggested that A. veronii and the novel clone, PW3, also dominate the intestinum community, while other clones, representing transient organisms, were typically present in low numbers. The identities of these transients varied greatly between individual leeches. Neither time after feeding nor feeding on defibrinated blood caused a change in identity of the dominant members of the microbial communities. Terminal restriction fragment length polymorphism analysis was used to verify that the results from the clone libraries were representative of a larger data set. The presence of a two-member bacterial community in the crop provides a unique opportunity to investigate both symbiont-symbiont and symbiont-host interactions in a natural model of digestive-tract associations.