Leishmaniasis remains as a public health problem worldwide due to its morbidity and geographical distribution  . Transmission of the disease is complex and involves not only the participation of different species of Leishmania parasites but also sandflies vector insects   and mammalian species that serve as reservoirs for the parasite. The infection in humans generates various clinical manifestations, being visceral leishmaniais (VL) one of the clinical forms with greater impact in the Americas specifically in countries like Colombia, Brazil and Venezuela for the possibility of causing the death of patients if not diagnosed and treated early  .
Currently, the VL presents difficulties associated with treatment, diagnostic tests and surveillance and control strategies of insect vectors  . This problem is attributed mainly to the emergence of drug-resistant strains of the L. infantum parasite, as well as the ubiquity and adaptability of vector insects, Lu. longipalpis, and Lu. evansi, and the existence of different eco-epidemiological settings where transmission can occur   . Therefore, it is necessary to explore alternatives aimed at interrupting the transmission of the infection and thereby reduce the impact of leishmaniasis in public health  . An alternative option to the chemical control of vectors or to the synthetic generation of vaccines and treatments is to understand the “intestinal microbiota” of sandflies vectors   .
From a holistic point of view, it is suggested to integrate the isolation of bacterial communities and the study of the action or activity of bacteria by generating secondary metabolites and bacterial peptides that can impact directly (antileishmanial activity) or indirectly (immune system) the development of Leishmania parasites, being decisive in the modulation of the transmission or vector competence of Lutzomyia spp   . There are several studies on intestinal microbiota in sandflies aimed at finding molecules with antileishmanial activity. Among these, the study of lytic effects generated in L. chagasi (syn L. infantum) by its interaction with Serratia marcescens  , the variability of molecules like defensins in Phlebotomus duboscqi induced by changes in the microbiota  , the generation of reactive oxygen species mediated by S. marcescens against L. mexicana in the digestive tract of Lu. Longipalpis  and most recently, the in vitro activity of Pseudozyma sp., Asaia sp., and Ochrobactrum intermedium against the development of promastigotes of L. Mexicana  . In Colombia, there are not known studies that have explored the usefulness of the intestinal microbiota of insects that transmit Leishmania spp.
Lu. evansi, is a vector recognized species for transmitting parasites that generate cutaneous and VL in rural and urban environments of the Caribbean coast of Colombia   . Its abundance and epidemiological importance made it an attractive biological model for the preliminary study of the microbiota using a culture dependent approach under aerobic conditions. This strategy allowed the isolation of bacterial strains P. ananatis, O. anthropi and E. cloacae, arousing interest either by being dominant in the digestive tract of Lu. evansi (E. cloacae), by being symbionts (P. ananatis) or by their reports on antitrypanosomal activity (Ochrobactrum sp.)  . Therefore, this study aimed to evaluate the leishmanicidal and antibacterial activity of extracts and whole bacteria (P. ananatis, O. anthropi and E. cloacae), isolated from the digestive tract of Lu. evansi and their susceptibility to antibiotics.
2.1. Ethics Statement
Sand fly collection was performed in accordance with the parameters of Colombian decree number 1376, which regulates specimen collection of biologically diverse wild species for non-commercial research. No specific permits were required for this study. The sand flies were collected on private property and permission was received from landowners prior to sampling.
2.2. Identification of Bacterial Isolates and Estimate Cell Concentration
P. ananatis, O. anthropi and E. cloacae all Gram negative strains were isolated from the digestive tract of adults and immatures from natural populations of Lu. evansi, associated with a peri-urban biotype from the municipality of Ovejas (Sucre department, Caribbean coast of Colombia), classified as a tropical dry forest ecosystem. The adult specimens were collected using Shannon-type extra-domiciliary white light traps that remained active between 18:00 h and 22:00 h. Prior to gut dissection, adult specimens were washed with 50 μL of 1X PBS and Tween 20, centrifuged at 3000 g for 5 minutes, and submerged in a 70% ethanol wash for one minute to remove excess microvilli, dust and exogenous bacteria.
The guts of adult Lu. evansi specimens were removed aseptically with sterile stilettos under a stereoscope in 1X PBS buffer. Isolates were cultured under aerobic conditions (33˚C for 24 and 48 hours) by surface plating intestinal homogenates on Luria-Bertani (LB) agar (Merck). The selected isolates were purified, characterized by macro and microscopic appareance of the colony, Gram stained (Figures 1(a)-(c)) and molecularly by analyzing the spacer region (ITS) between the 23S and 16S ribosomal gene, the 16S rRNA and (Figure 1(d)) gyrB genes partial nucleotide sequences. Estimated concentrations of 107 CFU/ml and 108 CFU/ml were calculated to challenge the isolates in the in vitro activity test against promastigotes of L. infantum. The cell concentration of bacteria was estimated with commercial McFarland turbidity standard pattern (BBL McFarland Turbidity Standard No. 0.5).
2.3. Reactivation of Leishmania infantum Fluorescent Promastigotes, Fluorescence Emission Estimation and Calculation of Cell Concentration
The BCN-GFP strain of L. infantum transfected with green fluorescent protein was thawed and planted in biphasic modified Novy, Nicolle and McNeal (NNN) medium for growth of promastigotes, verifying their viability by observation with a fluorescence inverted microscope (Nikon eclipse TS100)  . GFP-expressing promastigotes were analyzed flow cytometrically in 10,000 gated events and the numeric data were processed by using WinMDI software. L. infantum promastigotes were incubated at 26˚C, performing successive sub-cultures to obtain parasites with 98% of fluorescence, which allow estimating the action of bacterial isolates in vitro by flow cytometry.
Figure 1. Colony morphology (left panel), Gram stain (right panel) of the strains P. ananatis (a); E. cloacae (b); O. anthropi; (c) isolated from the gut of Lu. evansi and NJ dendrogram (d) of partial nucleotide sequences of 16S gene, illustrating the taxonomic confirmation of the bacterial isolates.
2.4. In Vitro Antileishmanial Activity Assay of Bacterial against Stationary and Exponential Promastigotes of L. infantum
Metacyclic-like (6 days of culture, stationary) and procyclic-like (3 days of culture, exponential) promastigotes of L. infantum were centrifuged at 1500 g for 10 minutes, washed twice with sterile PBS buffer for carbohydrate removal and then re suspended in single phase RPMI liquid culture medium without antibiotic at a final concentration of 3 × 106 parasites/ml for each co-culture and activity assay.
Cultures of P. ananatis, O. anthropi and E. cloacae grown in liquid LB medium (Merk) to 108 CFU/ml and 107 CFU/ml were obtained. These concentrations have been the most used in studies that evaluate the leishmanicidal activity of bacteria obtained from the digestive tract of insects   . The cell pellet (concentrated by centrifugation at 6500 g for 5 minutes) was washed twice with sterile PBS buffer. Bacteria were re-suspended in PBS to a final concentration of 107 CFU/ml and incubated for 24 hours at 27˚C with metacyclic-like or procyclic-like L. infantum promastigotes in RPMI. The trials were independent for each strain and in triplicate for each stage of development of the parasite. Cell viability controls consisted of PBS with promastigotes and the three bacterial strains re-suspended in PBS independently.
2.5. Quantification of the Bacterial Isolates Activity on L. infantum Promastigotes
The action of bacterial isolates on the viability of L. infantum promastigotes were determined by flow cytometry on a Cytomics FC 500MPL using an argon laser at 488nm of excitation and 525nm of emission, counting at least 10,000 events to calculate the number of fluorescent promastigotes. The acquired data was analyzed using the CXP (Beckman Coulter, Fullerton, CA, USA) software.
2.6. Evaluation of Antibacterial Activity from Extracts Secreted by P. ananatis, O. anthropi and E. cloacae
Production and evaluation of secondary metabolites secreted was performed following the method previously described  . An Erlenmeyer containing 50 ml of 2% LB broth (w/v), 2% Amberlite resin XAD-16 (W/V), was inoculated with 0.5 ml of each strain culture and grown overnight   . This Amberlite, allows adsorption of organic substances of small and medium molecular weight in aqueous solutions. This is a macroreticular resin nonionic that absorbs and releases substances through hydrophobic and polar interactions  . These resins have been used successfully in the identification and characterization of antibiotics   and other secondary metabolites.
After seven days of incubation at 30˚C and 180 rpm, the resin was decanted from the culture medium and washed with distilled water and the absorbed products were eluted with 40 ml of 100% methanol for 30 minutes  . Each extract was then concentrated to 1.5 ml in a rotating evaporator at 40˚C (Heidolph Efficient Rotary Evaporator Laborota 4001).
The bacteria used were reference strains: Escherichia coli, Enterococcus faecalis, Bacillus cereus, Pseudomonas aeruginosa, S. marcescens and Staphylococcus aureus subsp. aureus (Table 2). Psychrobacter sp. CP25 isolates were used as controls (positive control from Microbiop reference strain collection, National University of Colombia), Methanol (negative control) and the antibiotic chloramphenicol (10 ug/ml, positive control).
Diffusion test in agar was used  . Sterile Whatman No.1 filter paper discs, 6 mm diameter, were impregnated with 10, 20 and 50 ul of each extract and placed on the surface of Petri dishes containing Mueller-Hinton agar (Becton Dickinson), previously inoculated with a liquid culture of the target strains at a concentration of 1.2 × 108 CFU/ml (absorbance 600 nm = 0.1). The plates were incubated at 37˚C for 18 hours and the diameter of the growth inhibition halo around each disk was measured. Determination of the antibacterial activity was performed following the procedure described by Bauer et al. 1966 and amended by the Clinical and Laboratory Standards Institute   . The antibacterial activity assays were performed in duplicate in two independent experiments.
2.7. Antibiotic Susceptibility Test
The antibiotic susceptibility tests for the bacterial isolates (P. ananatis, O. anthropi and E. cloacae), were developed with Mueller Hilton agar plates (Bckton Dickinson). An inoculum of 108 CFU/ml of each bacterial isolate was used (0.5 units on the McFarland scale, McFarland Turbidity Standard BBL). All isolates were tested against 14 different antibiotics of known concentration classified as follows: Rifampicin (RD 5; Oxoid, 5 ug), Tetracycline (Te 30, Valtek, 30 mcg), Gentamicin (CN120; Oxoid; 120 ug - 10 ug ); Penicillin (P 30; Comprolab; 30 FMU), Chloramphenicol (C 30; Oxoid, 30 ug), Sulbactam Cefopeazone (SFC 105; Oxoid, 105 ug), Cefepime (CEP; Oxoid, 30 ug); Cefoperazone (PIC; Oxoid, 75 ug), Cefuroxime (CXM, Oxoid; 30 ug), Cephazolin (KZ; Oxoid, 30 ug), Ceftriaxone (CRO; Oxoid, 30 ug), Cefoxitin (FOX; Oxoid, 30 ug) Ceftazidime (CAZ; Oxoid, 30 ug). The plates were incubated at 30˚C for 24 hours and the bacterial growth inhibition halos were measured by the diameter in mm. The percentage inhibition was calculated with reference to the measurement of the diameter of the inhibition zone, established for gram positive and gram negative bacteria (M100-S25 protocol- Performance Standards for Antimicrobial Susceptibility Testing).
2.8. Data Analysis
Statistical analysis of the antibacterial activity was estimated by a two-way ANOVA with the GraphPad Prism version 4.0 program using the extracts and targeted pathogenic bacteria as factors. Based on the diameter of the antibiotics inhibition halos (growth inhibition), bacteria were categorized as susceptibility, moderately susceptibility, highly susceptibility and resistant according to the M100-S25 protocol (Performance Standards for Antimicrobial Susceptibility Testing).
3.1. In Vitro Bacterial Test with Procyclic and Metacyclic Promastigotes
The co-culture of the three bacterial isolates with promastigotes of L. infantum caused inhibition of procyclic-like but not metacyclic-like promastigotes. A greater impact on the inhibition percentage of the promastigotes using the bacterial cell concentration of 1 - 2 × 108 CFU/ml (Table 1) was observed. The standard deviation calculated for triplicate assays of co-culturing bacteria and promastigotes was low SD = 0.5 and 5.3, respectively, indicating that the experimental design is robust.
A greater impact of cell concentration of 1 - 2 × 108 CFU/ml of E. cloacae and P. ananatis on the percent inhibition of procyclic-like parasites is further noted, with values of 70.17 ± 1.1 and 77.29 ± 0.6 respectively (Table 1), whereas E. cloacae also significantly altered the development of procyclic-like promastigotes with bacterial concentrations of 1 - 2 × 107 CFU/ml (Table 1). O. anthropi had the lowest inhibitory activity on procyclic-like (62.33 ± 2.0; 38.13 ± 1.4) and metacyclic-like promastigotes (32.95 ± 5.3; 36.81 ± 3.2), with respect to the other two isolates analyzed. P. ananatis was the only bacterial isolate that presented inhibitory activity of 50.01% of metacyclic- like promastigotes.
3.2. Antimicrobial Activity Test of Crude Methanolic Extracts
The three extracts produced by O. anthropi, P. ananatis and E. cloacae exhibited similar antimicrobial activity patterns against all bacteria tested, with inhibition zones between 13 mm and 22 mm (Table 2, Figure 2). Highly significant differences between the inhibition zones associated with gram-positive bacteria used were found (P < 0.003557). The species most susceptibility to the extracts produced by the isolates from the digestive tract of Lu. evansi was B. cereus, with inhibition halos of 22 mm with others less susceptibility to the extracts activity like E. coli and E. faecalis, with inhibition halos between 13 mm and 15 mm. Figure 2(a) shows the antibacterial activity of the E. cloacae extract (more active) with the clinical isolate B. cereus.
Table 1. In vitro activity of three bacterial isolates from the gut of Lu. evansi against procyclic and metacyclic promastigotes of Leishmania infantum.
The data represents the average value (X) ± standard deviation (SD) of two experiments each in triplicate. Symbols: CFU/ml colony forming units per milliliter; % Percentage; ± standard deviation; MP metacyclic promastigotes of Leishmania infantum; PP procyclic promastigotes of Leishmania infantum. Note: Number of parasites in each trial = 3 × 106 parasites/ml.
Table 2. Antibacterial activity of extracts produced by strains O. anthropi, E. cloacae and P. ananatis isolated from the gut of Lu. evansi.
C+: positive control; C−: negative control; mm: diameter of the inhibition zones, average of the replicates per sample.
Figure 2. Agar diffusion assay of the antibacterial activity of the extracts produced by P. ananatis (140), E. cloacae (139), O. anthropi (102): (a) antibacterial activity against B. cereus; (b) antibacterial activity against E. faecalis; (c) antibacterial activity against E. coli; (d) antibacterial activity against P. aeruginosa; and. antibacterial activity against S. aureus subsp. aureus; F. antibacterial activity against S. marcescens; C− = methanol; C+ = Chloramphenicol; CP25 = Psychrobacter.
3.3. Antibiotic Susceptibility Test
The E. cloacae and P. ananatis isolates showed resistance to penicillin and rifampicin, while O. anthropi presented antibiotic resistance to Cephazolin and Cefoxitin (Table 3). Additionally, O. anthropi presented a greater number of resistance patterns to anti- biotics, being resistant to penicillin, sulbactam, cefopeazone, cefuroxime, cephazolin, ceftriaxone, cefoxitin and ceftazidime (Table 3). E. cloacae and P. ananatis had higher susceptibility, mainly with Beta-lactams, cephalosporins, chloramphenicol and whereas O. anthropi only presented high susceptibility with tetracyclines and aminoglycosides (Table 3).
The bacterial isolates P. ananatis, O. anthropi and E. cloacae, obtained from the intestinal microbiota of Lu. evansi assessed in this study exhibited differential activity against L. infantum as well as a differential susceptibility to antibiotics and against clinical isolates. The high inhibition percentage (72.29%) is generated by E. cloacae against procyclic-like promastigotes of L. infantum, when co-cultured under in vitro conditions is emphasized. This is the first study demonstrating the in vitro activity of E. cloacae against promastigotes of Leishmania, from studies that recognize its importance in the vector competence of some insects   . It is suggested that the action of E. cloacae can be derived from the expression of peptides or molecules with lytic activity on the surface of prokaryotes, by the action of enterococcal cytolysins (hemolysin)  . However, this hypothesis needs further studies.
Table 3. Antibiotic sensitivity patterns of the strains O. anthropi, E. cloacae and P. ananatis isolated from the gut of Lu. evansi.
Symbols: R resistant (full growth); +Sensitive (Halo 10 - 17 mm); ++Moderately sensitive (Halo 18 - 27mm); +++Highly sensitive (Halo of 28 - 37 mm).
In this sense, some studies have reported that the protective response of L. infantum procyclic promastigotes associated with the generation of glycoconjugates (proteophos- phoglycans, acid phosphatase, lipophosphoglycans, metalloproteins)  , is not suffi- cient for protection against enzymes or highly pathogenic bacterial peptides expressed by E. cloacae. According to the literature, in this state lifecycle (24 - 48 hrs), procyclical promastigotes of L. infantum present a lower degree of specialization and adaptation with respect to the metacyclic promastigotes (infective stage), which produce stronger enzymes such as chitinases that may even degrade the insects stomodeal valve and have a defence system resistant to mammalian complement factors and greater mobility  . The in vitro activity of E. cloacae on procyclic-like promastigotes of Leishmania is consistent and can justify their use in paratrasgenesis to express antitrypanosomal peptides, because other reports state that the bacteria also block the development of other parasites as Plasmodium falciparum in Anopheles gambiae and the sporogonic development of P. vivax in An. albimanus   .
Similar to E. cloacae, the symbiont P. ananatis showed a significant activity over the survival (70.17%) of the procyclic promastigotes of L. infantum. P. ananatis only has reported entomopathogenic activity for other insects   . These aspects are intere- sting because these bacteria could be used to disrupt the life cycle of sandflies and the transmission of Leishmania spp, by the rapid spread and adaptation of these arthropods   , as previously described in a study in which P. agglomerans (family Entero- bacteriaceae) was genetically modified, to express and secrete two anti-plasmodium effectors proteins (pelB, hly) in infected mosquitoes  .
The dissemination of P. ananatis symbiont to organs or complex structures of insects suggests that it is a specialized bacterium  , which is supported by its pan-genome that incorporates a large number of protein encoding genes that enable P. ananatis to colonize, persist and secrete a wide range of peptides  . This can also be related to the better activity over the survival of metacyclic promastigotes (50.01%) compared to E. cloacae and O. anthropi. O. anthropi had lower activity against metacyclic (32.95%) and procyclic promastigotes (62.33%). Unlike our results, the activity of other Ochro- bactrum species (O. intermedium, Ochrobactrum sp., AK strain) presented greater impact (~90%) on the survival of L. mexicana promastigotes in co-infection trials with Lu. longipalpis and in vitro assays   .
The crude methanolic extracts exhibited similar antimicrobial activity patterns against target bacteria, with a difference appreciated mainly against the growth of B. cereus (22 mm), suggesting that the isolates O. anthropi, E. cloacae and P. ananatis are important sources of promising antimicrobial compounds with a wide biological activity spectrum. In this sense these compounds or secreted peptides, can provide selective advantages to these bacteria in different environmental niches (including the digestive tract of sandflies) and be important for colonization, providing virulence factors and defence systems to keep its niche or prevent invasion from other bacterial strains   .
Gram negative bacteria, such as those used in this study, currently have six types of protein secretion systems reported (T1SS to T6SS) associated with bacterial compete- tion   . Among these systems, T6SS has a role in cytotoxicity, biofilm formation, antimicrobial peptide transport and interaction with host cells. This system has recently been described for P. ananatis, being responsible for their potential virulence and antimicrobial activity  .
Some members of the Enterobacteriaceae family are known to produce bacteriocins (3% to 26%) such as enterocins, colicins and antimicrobial lipopeptides produced by different species of Enterobacter, with great biopharmaceutical potential   suggesting that bacteriocins are produced by these bacteria as part of their defense mechanism to survive complex environments such as the digestive tract of different kinds of insect vectors (Lutzomyia, Phlebotomus, Anopheles, Aedes) where E. cloacae is a dominant taxonomic unit  .
Additionally, O. anthropi, which also exhibits antimicrobial activity against Gram positive and Gram negative bacteria, is of great interest for bioremediation and for their ability to degrade organophosphates  . In this sense, knowledge on antimicrobial peptides secreted by O. anthropi is interesting because this bacterium can transfer pesticide resistance factors to sandflies or simply remove pesticides by degradation   . O. anthropi secretes detoxification enzymes, reactive oxygen species and nucleo- sides of great interest for their anti-tumoral, antiviral, antibiotic and antiparasitic activity   .
Few reports inform about the susceptibility of antibacterial compounds from O. anthropi. In our study, this isolate was resistant to most cephalosporins and penicillins, but susceptible to rifampicin, chloramphenicol, some cephalosporins (cefepime, cefoperazone), tetracycline and gentamicin. The latter two antibiotics were the most active on O. anthropi. Our results are consistent with other studies reporting multi-re- sistance patterns present in O. anthropi   . However some strains of O. anthropi exhibit resistance patterns to cefepime  and only in few cases they are susceptibility to cefoperazone  .
Unlike O. Anthropi, the E. cloacae and P. ananatis isolates exhibited fewer resistance patterns to the antibiotics tested, and agreed in their response to penicillin and rifampicin, while P. ananatis was also resistant to cephazolin and cefoxitin. Both bacteria are reported as multiresistant for its environmental ubiquity and invasion of different hosts including soils, plants, animals and insects  . The greatest susceptibility pattern of these two isolated correspond to cephalosporins, although some reports indicate their resistance to cefuroxime  . Although bacterial resitencia is analyzed in vitro in this study, the result may indicate competitive factors and/or growth of bacteria in the gut, which may favour the development or block Leishmania promastigotes.
The antibiotic susceptibility tests of the intestinal microbiota of insect vectors are important for co-infection based assays with parasites or viruses, in order to evaluate drugs, vaccines or to determine the autonomous vector competence of the insect. In this sense, to remove or modulate the resident intestinal microbiota depends on the resistance state to certain antibiotics, and allows to access the functional relationships between gut microbiota and their hosts.
The ability of E. cloacae and P. ananatis to inhibit the growth of procyclic-like promastigotes of L infantum in co-culture and the similar susceptibility patterns shown by O. anthropic, suggest that these isolates are promising for future control strategies aimed at evaluating the parasite load in Lutzomyia species when exposed to E. cloacae and P. ananatis, in order to provide new ways to reduce the transmission of leishma- niasis.
Acknowledge the support by Luisa Montoya (Microbiodiversity and Bioprospection Research Group, Cellular and Molecular Biology Laboratory, National University of Colombia). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.
CXMH, GECR and SUS: Designed the study, analyzed the data and contributed to write the manuscript. RJV, SR: Designed the study, performed the experiments, analyzed the data and contributed to write the manuscript. VO: Performed the experiments and analyzed the data.
Administrative Department of Science, Technology and Innovation-COLCIENCIAS (Grant CT-695-2014 and Doctoral studies 528-2011); Grupo de Microbiodiversidad y Bioprospección and Grupo de Investigación en Sistemática Molecular, Universidad Nacional de Colombia, Sede Medellín.
Conflict of Interest
There is no conflict of interest from other co-others in the publication of this manuscript in this journal. All the co-others have contributed in the preparation of the manuscript up to the submission stage.