RESUMEN
Abstract The bacteria residing in the gut of honey bees (HB) has demonstrated a significant role in protecting bees against various pathogens, production of honey and wax. However, no information exists about the antibacterial potential of bacterial isolates from gut of Asian HB, Apis cerana Indica F. (Hymenoptera: Apidae), against human pathogens. This study aims to investigate the antibacterial and multienzyme potential of aerobic bacteria from A. cerana gut using culture dependent approach. A total of 12 HB gut bacteria were characterized morphologically and biochemically. These strains were further screened for their antimicrobial activity against pathogenic human microorganisms Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumonia, Bacillus licheniformis and Bacillus subtilis using cross streak (primary screening) and agar well diffusion methods (secondary screening). Preliminary characterization of cell-free supernatant (CFS) of two promising isolates was performed by measuring lactic acid concentrations, enzymatic digestion of antimicrobial compounds, stability over a range of temperature, pH and amplification of spaS (subtilin) and spoA (subtilosin) genes. In primary screening, among 12 HB isolates, eight strains showed statistically significant highest zones of inhibition (p≤0.05) against E. coli, K. pneumoniae and P. aeruginosa. 16S rRNA sequencing revealed that these isolates belong to Bacillus genus, identified as B. tequilensis, B. pumilus, B. xiamenensis, B. subtilis, B. amyloliquefaciens, B. safensis, B. licheniformis, B. altitudinis (Accession numbers: MT186230-MT186237). Secondary screening revealed that among eight isolates, B. subtilis and B. amyloliquefaciens showed statistically significantly strong inhibition (p≤0.05) against all tested pathogens. Antibiotic susceptibility testing revealed that both isolates were resistant to antibiotics and possesses proteolytic, lipolytic and cellulolytic activities. The nature of the compound causing inhibitory activity was found to be proteinaceous and showed stability over a wide range of temperature as well as pH. PCR study confirmed the presence of bacteriocins by successful amplification of important antimicrobial peptide biosynthesis genes spaS and spoA. These results suggest that the HB gut is a home to bacteria that possess antimicrobial activity and important enzymes with antimicrobial potential. To our knowledge, this is the first report demonstrating the antimicrobial potential of bacteria isolated from gut of HB (A. cerana) against human pathogens.
RESUMEN
Enterotoxigenic Escherichia coli (ETEC) strains are leading causes of childhood diarrhea in developing countries. Adhesion is the first step in pathogenesis of ETEC infections and ETEC pili designated colonization factor antigens (CFAs) are believed to be important in the biofim formation, colonization and host cell adhesions. As a first step, we have determined the biofilm capability of ETEC expressing various types of pili (CFA/I, CfaE-R181A mutant/ CfaE tip mutant, CFA/II and CS2). Further, enzyme-linked immunosorbent assay (ELISA) assay were developed to compare the binding specificity of CFA/I, CFA/II (CS1 - CS3) and CS2 of ETEC, using extracted pili and piliated bacteria. CFA/II strain (E24377a) as well as extracted pili exhibited significantly higher binding both in biofilm and ELISA assays compared to non piliated wild type E24377a, CFA/I and CS2 strains. This indicates that co-expression of two or more CS2 in same strain is more efficient in increasing adherence. Significant decrease in binding specificity of DH5αF'lacIq/∆cotD (CS2) strain and MC4100/pEU2124 (CfaE-R181A) mutant strain indicated the important contribution of tip proteins in adherence assays. However, CS2 tip mutant strain (DH5αF'lacIq/pEU5881) showed that this specific residue may not be important as adhesions in these strains. In summary, our data suggest that pili, their minor subunits are important for biofilm formation and adherence mechanisms. Overall, the functional reactivity of strains co expressing various antigens, particularly minor subunit antigen observed in this study suggest that fewer antibodies may be required to elicit immunity to ETEC expressing a wider array of related pili.