Isolation and Molecular Identification of Gram-negative Bacteria from The Leech Limnatis paluda in Iraq

Abstract
Limnatis paluda is a sanguivorous, endoparasitic leech that has the ability to parasitize the mucus membranes of human and domestic animals. Leech has been used for many years to treat a wide range of diseases. However, Leeches' gut is known to harbor variable bacterial species which can cause moderate to severe infections. Thus, we aimed in this study to isolate and study Gram- negative bacteria from L. paluda. From September 2024 to July 2025, 53 leech samples were collected from Karokh mountain's water springs in Erbil province/ Iraq. Morphological identification was done and followed by a molecular study using COI gene. Subsequently, bacteria were isolated from three parts of the leech's body and Gram staining, biochemical tests and molecular methods using 16S rRNA gene were used to identify the isolated bacteria. The results revealed the isolation of 21 Gram- negative bacterial isolate that comprised of 6 species: Aeromonas hydrophila, Aeromonas veronii, Morganella morganii, Serratia sarumanii, Serratia ureilytica and Yokenella regensburgei. It is worth mentioning that depending on NCBI, Serratia sarumanii and Serratia ureilytica were recorded for the first time in Iraq during this study. Moreover, Yokenella regensburgei was molecularly studied and identified using 16S rRNA for the first time in Iraq. Moreover, the results revealed that Aeromonas veronii was the predominant bacteria in the L. paluda. Furthermore, the highest percentage of Gram- negative bacteria was found in crushed buccal cavity swab 9(42.86%). Biofilm assay using microtiter plate method showed that 14(66.67%) were biofilm producers. Antibiotic sensitivity test was performed using 12 antimicrobial agents. The results showed that amikacin, ciprofloxacin, imepenem and netilmicin were the most effective antibiotics against all the isolates. However, all the isolates were 100% resistant to ampicillin, cefoxitin, ceftazidime and ertapenem.