Enzymatic, antimicrobial, and leishmanicidal bioactivity of gram-negative bacteria strains from the midgut of Lutzomyia evansi, an insect vector of leishmaniasis in Colombia

Abstract

Knowledge regarding new compounds, peptides, and/or secondary metabolites secreted by bacteria isolated from the intestine of phebotominae has the potential to control insect vectors and pathogens (viruses, bacteria, and parasites) transmitted by them. In this respect, twelve Gram-negative bacteria isolated from the intestine of Lutzomyia evansi were selected and screened for their enzymatic, antimicrobial, and leishmanicidal activity. E. cancerogenus, E. aerogenes, P. otitidis, E. cloacae, L. soli, and P. ananatis exhibited enzymatic activity. 83.3% of the isolates displayed lipolytic and nitrate reductase activity and 58.3% of the isolates displayed protease activity. Hemolytic activity (17%) was identified only in E. hormaechei, and P. ananatis. E. cancerogenus, A. calcoaceticus, and P. otitidis showed cellulolytic activity. A. gyllenbergii, P. aeruginosa, and E. hormaechei showed amylolytic activity. In general, the totality of methanolic extracts exhibited antimicrobial activity, where E. hormaechei, A. calcoaceticus, and E. cancerogenus presented the highest activity against the evaluated reference bacteria strains. Cell-free supernatants (CFS_S) of the Gram-negative bacteria showed higher growth inhibitory activity against the reference Gram-positive bacteria. The CFS of A. gyllenbergii was the most active antimicrobial in this study, against S. aureus (AA_ODs = 95.12%) and E. faecalis (AA_ODs = 86.90%). The inhibition percentages of CFS against Gram-positive bacteria showed statistically significant differences (repeated measure ANOVA df= 2; F= 6.095; P= 0.007832). The E. hormaechei methanolic extract showed leishmanicidal activity (CE-50 μg/ml = 47.7 + 3.8) against metacyclic promastigotes of Leishmania braziliensis (UA301). Based on this finding, we discuss the possible implications of these bacteria in digestion and physiological processes in the Lu. evansi intestine. P. ananatis, E. cloacae, E. hormaechei, and P. otitidis were considered the most promising bacteria in this study and they could potentially be used for biological control.

Keywords: Antimicrobial screening; Bacteria-intestine interaction; Le. braziliensisenzymatic bioactivity; Leishmanicidal activity.

Fig. 1

Growth curves of B. cereus (clinical isolate) against the exposure of cell-free supernatants (CFSs) of Pseudomonas otitidis.Bc + LB:B. cereus in LB culture medium; Bc+70: B. cereus in presence of CFSs of Pseudomonas otitidis (isolate 70); Bc + Ant:B. cereus in LB medium supplemented with antibiotic (Chloramphenicol 30 μg/ml).

Fig. 2

Predictive model of the interactions and functions of Gram-negative bacteria in the intestine of Lutzomyia evansi, based on in vitro evaluation of enzymatic, antimicrobial, and leishmanicidal activity. a) Intestinal bacteria such as P. ananatis and E. hormaechei display hemolytic activity, suggesting that they might be related to erythrocyte lysis. b) Hemoglobin could be released and used by P. ananatis, E. hormaechei, P. otitidis, E. cloacae, E. cancerogenus, L. soli, and E. aerogenes for nutrient supplementation. Erythrocytes are also an useful source of iron for egg maturation. c) E. aerogenes, A. calcoaceticus, E. cancerogenus, E. cloacae, P. otitidis, O. anthropi, L. soli, E. hormaechei, and A. gyllenbergii are able to reduce nitrates to nitrites and/or molecular nitrogen, from decomposing organic matter, possibly during the development of the immature states of Lu. evansi. This process allows for the synthesis of some necessary amino acids for protein and glycoprotein synthesis, which could also permit formation of the peritrophic matrix in the adult phase. d) P. aeruginosa, E. hormaechei, and P. otitidis are responsible for carbohydrate hydrolysis, processing starch to obtain glucose and produce energy. e) Cellulose is hydrolyzed by P. otitidis, E. cancerogenus, and A. calcoaceticus as an additional source of glucose.) L. soli, P. aeruginosa, E. cancerogenus, P. putida, E. aerogenes, P. otitidis, and A. gyllenbergii showed lipolytic activity that might be associated with fat degradation from the fatty abdominal body, which is filled with nutrients and allows for development and/or reproduction, as fats are the precursors of the synthesis of most proteins and metabolites secreted into the hemolymph. g) E. hormaechei showed activity against metacyclic promastigotes of Leishmania braziliensis. The majority of Gram-negative bacteria show antimicrobial activity, which is a determining aspect in intestinal microbiota dynamics.