12 Bacterial Flora of the Medicinal Leech (Hirudo medicinalis)

Intestinal Symbiosis of the Medicinal Leech

The modern physician likes to practice medicine under strict conditions of precision and sterility. However, when administering treatments with live animals, it is impossible to achieve the customary level of sterility because all animals are colonized by natural bacteria. These symbionts help the animal by synthesizing essential nutrients, digesting food, and killing harmful organisms [22, 44]. The human intestine exhibits impressive bacterial diversity. Hundreds of different bacterial strains have been isolated from the gut of humans by culture techniques, and culture-independent methods suggest that the actual number of intestinal strains may be twice as high [17, 18, 22]. The intestine of the medicinal leech is likewise colonized by bacteria but exhibits very little bacterial diversity compared to many other animals [15]. In this chapter, we will describe the current state of knowledge on the bacterial flora and antimicrobial mechanisms of the intestine of Hirudo medicinalis.

The so-called stomach (crop) of the leech, which occupies the majority of the space in the digestive tract, serves as the storage site for ingested blood [39]. The leech’s stomach is unlike that of vertebrate animals in that it extracts water and salt from the blood. In this capacity, it works similar to the human intestine, where the reabsorption of fluids and salts occurs. The leech stomach is also able to store ingested red blood cells. The actual process of digestion and nutrient absorption takes place in the intestine [37], which is much smaller than the stomach. Therefore, most investigators who studied the bacterial flora in the leech’s digestive tract probably isolated the bacteria from the leech’s stomach. However, this cannot always be determined with certainty because of the sometimes minimalist descriptions of study methods.

Surprisingly, only a single bacterial species was isolated from the leech stomach in the first microbiological studies reported. This bacterial sym biont was named Bacterium hirudinicolum by Lehmensick and Hornborstel in 1941 [29]. Ten years later, Büsing and co-workers renamed it Pseudo monas hirudinis [6, 7]. The presence of only a single bacterial species in the leech contrasts sharply with the great diversity of bacterial flora found in the digestive tract of most other animals. The bacteria isolated from leeches tested positive for beta-hemolysis and were found to produce extracellular proteases and lipases. Because of the pure culture of these enzymes, which could be important for blood digestion, these bacteria were classified as symbionts.

The exact role of the symbiotic bacteria in the symbiosis is still not fully understood, but three potential functions have been proposed [7, 15, 16]:

1. The bacteria may help to digest ingested blood.

2. The bacteria may produce essential nutrients for the leech.

3. The bacteria may prevent the growth of other bacteria.

The classic proof that a bacterium is involved in blood digestion or nutrient synthesis is antibiotic inactivation of the bacterium. Antibiotic susceptibility tests can be used to determine whether the bacterium is involved in the synthesis of a nutrient such as vitamin B₁₂, which is present in low concentrations in the blood. Two groups of researchers have studied the effects of different antibiotics on the physiology of leech digestion. Büsing and coworkers [7], who treated medicinal leeches with chloramphenicol (1 mg per mL of blood), observed a reduction in water loss and nitrogen excretion after feeding. Two factors to consider are:

1. Chloramphenicol passes relatively quickly from the intestine into the blood (at least in humans).

2. The animals were given relatively high doses of the drug.

Consequently, it is impossible to ascertain with certainty whether the antibiotic actually had a direct effect on the leeches. Zebe et al. observed a reduction in oxygen uptake and NH₃ excretion in leeches treated with kanamycin [47]. Again, very high doses of the drug were used (1 mg per mL of kanamycin). However, unlike chloramphenicol, kanamycin is poorly absorbed by the intestine, so it probably had a smaller effect on the animals [16]. All in all, these findings at least suggest that the symbionts influence the metabolism and physiology of the medicinal leech.

Aeromonas, Intestinal Symbiont of the Medicinal Leech

In the 1960s, the symbiont discovered by Lehmensick was renamed Aeromonas hydrophila [27]. Aeromonas taxonomy has since undergone complex changes and many new species have been described [2, 8, 23, 25, 26]. Classical biochemical identification is not always straightforward, and very discrete differences in 16S rRNA gene sequences of the different strains must be detected [1, 13, 34]. Another well-known problem is incorrect identification of the different Aeromonas species by commercial test kits, especially the older systems. Most investigators who studied the bacterial flora of the medicinal leech in the past only used commercial test kits to identify the symbionts, most of which were identified as Aeromonas hydrophila [5, 11, 30, 31, 33, 35, 38, 41]. Some of the isolates that were identified as Aeromonas sobria [12, 33] probably should have been classified as Aeromonas veronii biovar sobria because the 16S rRNA gene sequence is different. Our group identified the symbionts as Aeromonas veronii biovar sobria using commercial test kits supported by additional tests; identification was subsequently confirmed by 16S rRNA gene sequencing [14]. Our analysis revealed that many species isolated from the leech stomach had atypical biochemical test reactions consistent with the presence of Aeromonas hydrophila. For example, one group of symbionts tested positive for esculin hydrolysis, which is characteristic of Aeromonas hydrophila, but its 16S rRNA sequence was identical with that of Aeromonas veronii biovar sobria. Other investigators recently reported similar difficulties in biochemical identification of Aeromonas strains [1]. Furthermore, leeches from other breeders or from other regions may be—colonized by different bacterial species.

Exact taxonomic identification is probably of lesser importance in clinical medicine because Aeromonas hydrophila and Aeromonas veronii biovar sobria cause similar infections in humans and the same safety precautions apply for both leech species [25]. To my knowledge, no reports of notable infections occurring after application of leeches to well-perfused parts of the body of individuals with an intact immune system have been mentioned in the literature. If blood supply to the application site is impaired, there is an increased risk of wound infection which, however, can be prevented by prophylactic administration of antibiotics. Quinolone antibiotics such as ciprofloxacin and ofloxacin or third-generation cephalosporins such as ceftriaxone should preferably be used for this purpose [19, 21, 28, 38, 42, 45]. First-generation cephalosporins and penicillin are not recommended because resistance to these drugs is a widespread problem. Still, resistance to the recommended antibiotics can also develop [40, 45]. Accurate differentiation between the two Aeromonas species is important for microbiologists because investigators still have not succeeded in distinguishing virulent Aeromonas strains from benign types. Both Aeromonas

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