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

Background: Hirudo 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. Results: Fourteen 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 Gramnegative 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. Conclusion: Chloramphenicol, 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.

the blood from coagulation during feeding (Sig et al., 2017).
Digestion of blood is completed through the action of many digestive enzymes and complex microbial community present in the gut of animal which influences the metabolism of an animal. These organisms absorb nutrients from the lumen and survive in the digestive tract condition. Moreover, they create a barrier for other microbes and prevent their proliferation on the surface (Graf, 2016). Leech biting transfers saliva along with some amount of gut microbes (Giltner, Bobenchik, Uslan, et al., 2013). Complications like bacterial infections in leech therapy were reported in 2% to 36% cases (Marden, McClure, Beka, & Graf, 2016). Such infectious bacteria considered to have come from the gut of the leech. Gut microbes reside in the mucosal membrane of the digestive tract of the leech and they transmit through saliva (Whitaker et al., 2014). Gut microbial community explains immunity of animal (here leech) and also provide information against the pathogen colonization. Moreover, gut microbes play an important role in symbiosis with host animal and also help in the digestion of nutrients (Worthen, Gode, & Graf, 2006).
Antimicrobial resistance is a serious problem nowadays for the humans, which occured due to the extensive use of antimicrobial agents for human welfare (Beka, Fullmer, Colston, et al., 2018). Medicinal leech is treated with a prophylactic antibiotic before application for medicinal purposes. These antibiotics reduced the chances of several bacterial infections (Marden et al., 2016). It triggers the chance of multidrug resistance (MDR) and also the transmission of such MDR bacteria among the hosts (Giltner et al., 2013). Therefore, antibiotics assay and genetic characterization of gut microbes become important to conserve such multitasking therapeutic invertebrates. In the present study, an attempt was made to isolate cultivable bacteria from leech saliva and their antibiotic assay in order to propose the broad-spectrum antibiotic for promising prophylaxis. Molecular characterization by RAPD-PCR was also carried out to evaluate random sequence-based evolutionary relationship analysis.

Materials and methods
Organism of the study Indian medicinal leech Hirudinaria granulosa were collected from Ahmadabad (Surekh Education, M85/ 1009, Panchvati apartment, Sola Road, Naranpura, opp. Rameshwar app., Near Indian oil petrol pump, between Bhuyandev to Parasnagar, Ahmadabad). Leech was divided into two categories. One, before any skin treatment named as 'unused' (Coded as UTL or U) while the second was after skin disease treatment named as 'used' (coded as TL or T) for the present study.

Culture maintenance
Leeches were stored and maintained in a transparent glass container. The container should be sufficiently big enough and filled up pure dechlorinated tap water up to 3/4th of its capacity, allowing enough space for the movement of leech. The water was changed after two successive days. The lid of the container must contain fine holes for air exchange.

Preparation of leech for experiment
The purification of leeches was carried out by putting the leeches in dechlorinated water containing a pinch of turmeric for 60 min to increase the appetite and bloodsucking power.

Saliva extraction from used and unused leach
Both used and unused leeches were starved for 3 weeks before the extraction of saliva. Starved leeches were fed with phagostimulatory solution [0.01 M Arginine in 0.15 M sodium chloride kept at 37°C] through sterile glass funnel wrapped with ultra-violet (UV) sterilized parafilm sheets (Bemis, Neenah WI 54956). Leeches were introduced near the parafilm sheet and allowed attaching itself. After the leech becomes fully satiated, it detached itself from the parafilm. Leeches were kept in sterile ice container for 15-20 min, during the incubation time, leech became completely paralyzed and regurgitated all the solution it had already sucked. Then leeches were squeezed from the posterior end toward the anterior end in order to collect the remaining sucked solution under sterile condition. Collected saliva was centrifuged at 10,000 rpm for 15 min. Sediment was used for bacterial isolation. Leeches regained their activity on immersing them in warm dechlorinated water (37°C) for 15-30 min.

Isolation of gut/saliva microflora
The bacterial suspension was serially diluted in sterile distilled water up to 10 (−10) dilution and 0.1 ml of inoculum was spread on the nutrient agar plate. Inoculated media plates were incubated at 37°C for 24 h. Colonies with different morphological appearances were picked up after incubation.

Gram staining and biochemical test for bacterial isolates
All bacterial isolates collected from the saliva of unused and used leech were categorized by Gram staining (Hucker's modification). All the Gram-positive bacteria were inoculated with commercially available biochemical identification API kit (HiMedia product code: KB013).
Similarly, Gram-negative bacteria were inoculated on API kit (HiMedia product code: KB002) for rapid identification of the organism. Both kits were incubated at 37°C for 48 h.

Antibiotic assays
Antibiotic assays of bacterial isolates were carried out using universal antibiotic combo disc (IC001* procured from HiMedia, Mumbai). For assays, each bacterial culture was grown in nutrient broth until OD 600 reaches up to 1.00. Next day, bacterial culture was spread on nutrient agar medium prepared in 20-cm Petri plate and the antibiotic disc was placed on the surface of the medium after inoculation. Plates were incubated at 37°C overnight and next day, plates were checked for zone of inhibition.

DNA isolation and quantification
Bacterial genomic DNA was extracted by the modified Green and Sambrook method for bacterial genomic DNA extraction (Green & Sambrook, 2012). Briefly, bacterial (both Gram-negative and Gram-positive) cells were harvested by centrifugation at 8000 rpm for 10 min. Pellet obtained was re-suspended in 300 μl of SET buffer  (made up of 2 ml 1 M Tris, 1 ml 5 M NaCl, and 0.2 ml 0.5 M ethylenediaminetetraacetic acid (EDTA) in the final volume of 100 ml in double-distilled water) followed by incubation at 37°C for an hour. In order to break open the cells and remove RNA from mixture, 10 μl of lysozyme and RNase each were added to it respectively. Mixture was incubated at 37°C for 90 min and later, 23 μl sodium dodecyl sulfate (SDS) and 10 μl Proteinase-K was added and kept at 55°C for 2-h incubation. Later, 85 μl of 5 M NaCl was added and after 5 min 85 μl Chloroform was added followed by mixing by inverting the tunes. Mixture was kept at room temperature for 45 min and then centrifuged at 10,000 rpm for 10 min to separate phases. Aqueous phaseupper phase was transferred to a fresh 1.5-ml Eppendorf tube and added equal volume of 80% ethanol to precipitate DNA. Allow tubes to air dry under laminar airflow. DNA was resuspended in 40 μl tris-EDTA (TE) Buffer. DNA concentrations obtained from each bacterial sample were quantified (in ng/μl) spectrophotometrically using UV-visible spectrophotometer (Shimadzu UV VIS 1800-A114548) by checking absorption at 260 nm and 280 nm followed by calculating ratio (OD 260 /OD 280 ).

RAPD-PCR
The purity of extracted DNA was checked by separating on 1% agarose gel. One μl of (15 ng purified) genomic DNA and 2.7 μl Master Mix (EmeraldAmp GT PCR Master mix-2X Premix, Takara, Tokyo) were taken in  fresh PCR tube along with 0.6 μl of OPL-14 (random) primer [5'-TCG CTC CGT T-3'] and 5.7 μl sterile distilled water was added to get the final volume of reaction mixture to 10 μl. PCR amplification was done by keeping 94°C for 1 min as initial denaturation step followed by 37°C annealing for 1 min, 40 cycles of extension at 72°C with 2 min duration and final extension for 10 min again at 72°C (Padmalatha & Prasad, 2006).

Agarose gel electrophoresis
Agarose gel (1%) prepared in TAE buffer (1X) was used to check the purity and RAPD pattern analysis. Two μl of PCR amplicon was used for electrophoresis with 1 kb ladder agarose gel containing EtBr (5 μl/ml of gel) at 60 V. DNA banding pattern was visualized in UV trans-illuminator and was documented using BioRed Gel documentation system, Molecular Imager®, ChemiDoc™ XRS + equipped with Image Lab™ Software version 6.0.0 Bio-Red Laboratories Inc.

Phylogenetic tree construction by neighbor end-joining method
Phylogenetic relationships between isolates were evaluated by constructing the phylogenetic tree using PyElph GUI software version 1.4, according to the neighbor end-joining method.

Isolation and morphological characterization of isolates
Fourteen bacterial isolates with different morphological characters were obtained from the saliva of unused leech which was coded as UTL-1 to UTL-14. Colonial characters observed visually are described in Table 1. From used leech, thirteen bacterial isolates obtained were   coded as TL-1 to TL-13 and colonial characteristic of each isolated were presented in Table 2. Each of the isolates was categorized according to their Gram's reaction. Out of fourteen isolated from unused leech, five were Gram-negative while the other nine were Gram-positive. Gram-negative used leech salivary isolates were five while rests of eight were Gram-positive (Tables 3 and 4).

Biochemical characterization of isolates
Bacterial cultures isolated from leech were categorized as Gram-positive and Gram-negative. Biochemical characterization of each isolates were described in Tables 5, 6, 7 and 8. Positive biochemical tests of each isolates were compared with the manual provided with identification kit for preliminary identification. The list of probably identified bacteria was also included in Tables 5, 6, 7 and 8.
Bacteria isolated from unused leech were quite different from those found in used leech. Interestingly, it was observed that certain species of bacteria which were not present initially become part of the digestive tract of leech after treatment. In the present experiment, leech was starved for 15 days to remove possible bacteria which were carrying temporarily. Bacillus species was found in both types of leech saliva more commonly. Pseudomonas species were present higher in number before the treatment. Micrococcus species which was found in unused leech which completely disappears in used leech. Vibrio species were also found in both the leech.

Antibiotic assay of isolates
The antibiotic assay was carried out for the bacteria isolated from the leech saliva ( Figs. 1 and 2). Clear zone around the antibiotic disc indicated that bacteria were sensitive at a given concentration. It was observed that all isolates were sensitive to most of the antibiotics. In general, chloramphenicol (30 μg) and norfloxacin (10 μg) showed highest sensitivity for bacteria from unused leech. While bacteria isolated from used leech showed the highest sensitivity towards sparfloxacin (5 μg) followed by cefaclor (30 μg).

RAPD-PCR and phylogenetic tree
In the present study, RAPD-PCR was performed using random primer OPL-14 to screen intra-species variation among the isolates from both used and unused leech. Variation was screened with respect to random primer OPL-14 (Chacon, Martinez, Wachsman, et al., 2013;Fernanda, Da, Biscola, et al., 2016). There were 20 amplified band patterns were observed using OPAL14 primer (Fig  3). The variation in band patterns for each successfully characterized isolate as indicated by RAPD-PCR possibly suggests the existence of sequence within the species (Adeleke, Olaitan, Abiona, et al., 2014). Figure 3 showed the RAPD band pattern with a ladder (1 kb) at the first lane. Phylogenetic tree construction (Fig. 4) by neighbor end-joining method reveals that isolate U7, U4, and U3 were phylogenetically different than U11, U8, U10, U9, U12, and U6. Similarly, T9B, T8, T12, and T10 contain different genetic makeup than T4 and T1. Branches of these isolates indicated that they were evolved to survive under similar conditions. U2, T2, T9A, and T10 showed no amplification, which indicated absence of primers sequence.

Discussion
Herudotherapy or leech therapy for skin diseases is an ancient process (Das, 2014). These blood-sucking animals serve the human kind for since long. The saliva of medicinal leech contains many bioactive compounds and it was reviewed in detail in the past years (Siddall, Trontelj, Utevsky, Nkamany, & Macdonald-III, 2007;Das, 2014;Sig et al., 2017). These bioactive compounds are of many medicinal properties for which leeches were exploited (Siddall et al., 2007). But saliva does not only contain bioactive compounds but it is also the habitat of some gut microbes. Such gut microbes are normal flora of them or might becomes from the environment in which the leeches live. These microfloras might be pathogenic to humans. For example, Aeromonas which is commensal in the leech digestive system is pathogenic for human (Marden et al., 2016). It reported to cause pneumonia, gastroenteritis, and septicemia (Das, 2014). Therefore, medicinal leech, before the treatment of skin diseases, treated with antibiotic solutions to eliminate possibilities of post-operational bacterial infections (Das, 2014). Microevolution process changes the genetics of these bacteria as they divide in a very short time and the problem of drug resistance arises. Nowadays, due to the excessive use of many antibiotics, these drug resistance phenomena changed in multidrug resistance (MDR). The bacterium which was sensitive to a certain drug becomes resistant to those antibiotics. MDR become the global threat (Giltner et al., 2013). MDR is a genetic phenomenon. Therefore, it becomes important to evaluate the salivary microbiota for their genetic profile and antibiotic sensitivity. Conventional microbiological techniques were used to isolate the bacteria from the saliva of the leech. For the present study, two types of leech were used. One leech was before the skin treatment and another was after the skin treatment. Bacterial isolates were also tested biochemically to find out the genus name of them. It was noted that salivary bacterial makeup was partially changed. Aeromonas and Burkholderia were isolated from used leech which was thought to be present in the digestive tract of leech as symbiotic bacteria (Graf, 2016;Ott, Dacks, Ryan, & Rio, 2016;Fig. 3 RAPD fingerprint of all isolates using OPL-14 as a random primer