Antibiotic sensitivity and RAPD-PCR studies on cultivable gut bacteria from Indian Medicinal Leech\u2014Hirudinaria granulosa

Abstract

BackgroundHirudo granulosa—an Indian cattle leech, is frequently used for the treatment of psoriasis and eczema. During treatment which followed by leech biting, it transfers some amount of gut microbes along with saliva. This may result in bacterial infection at the treatment site. Antibiotics used as post-surgical hirudotherapy is the reason for drug resistance. Drug resistance is the result of the change in the genetic makeup of bacteria. Therefore, it is necessary to study antibiotic sensitivity of gut bacteria and characterized them genetically.ResultsFourteen bacterial isolates were obtained from unused leech in which five were Gram-negative and the other nine were Gram-positive. Similarly from the used leech, thirteen were isolated in which five were Gram-negative and the other eight were Gram-positive. Biochemical analysis reveals that isolates from unused leech saliva belong to Pseudomonas, Micrococcus, Streptococcus, and Vibrio species, while the used leech salivary bacteria were the member of genus Pseudomonas, Comamonas, Escherichia, Citrobacter, Aeromonas, Providencia, Enterobacter, and Yersinia. Antibiotic sensitivity tests for isolates indicated that chloramphenicol (30 μg) and norfloxacin (10 μg) were effective for unused leech isolates while sparfloxacin (5 μg) and Cefaclor (30 μg) were effective against used leech salivary isolates. Random primer (OPL-14)-based random amplified polymorphic DNA (RAPD) fingerprint showed twenty amplified regions among all tested bacteria. Most of the bacteria contain the tested sequence except U2, T2, T9A, and T10 which showed no amplification indicated the absence of primers sequence.ConclusionChloramphenicol, norfloxacin, sparfloxacin, and Cefaclor antibiotics alone or in combination were possibly used to treat post-therapy infections. Bacteria from treated and untreated leeches were clustered at nearby branch in neighbor end-joining phylogenetic tree, which indicates the similar (but not exact) genetic makeup. Therefore, it can be concluded that these antibiotics were possibly used against most of them.