Characterization and Antimicrobial Activity of Serratia Marcescens Endosymbionts of Rhabditis Nematodes Against Selected Bacterial and Fungal Pathogen

Abstract

Serratia is a genus of gram-negative non-sporulating gamma proteobacteria of the family Enterobacteriaceae. They are the newest addition to the well-known nematode-bacterium association with the two most common endosymbionts being Xenorhabdus and Photorhabdus species. Serratia marcescens is associated with the entomopathogenic nematodes of the genus Oscheius. They produce many secondary metabolites including antibiotics, enzymes, prodigiosin, serrawettins, siderophores, bacteriocins, anticancer agents, acids and toxins which have shown cytotoxicity against various microbes. This study aimed to characterize the nematode-associated bacteria and determine their efficacy against selected antibiotic-resistant bacteria as well as phytopathogenic fungal species. To obtain the bacteria, five late instars of Galleria mellonella larvae were placed on the surface of a Whatman filter paper lined in a Petri dish followed by the addition of 5 drops of nematode suspension per Petri dish, sealed with parafilm and incubated in the dark at room temperature for 4 days. The hemolymph from dead larvae was streaked on Nutrient bromothymol blue 2, 3, 5-triphenyltetrazolium chloride agar (NBTA) and MacConkey agar, making five replicates and incubated at 28°C for 48hrs. Pure colonies obtained were used in the subsequent bioassay involving antimicrobial susceptibility tests against selected pathogens. All the bacterial isolates were gram-negative with green circular, smooth, colonies on NBTA media. They were all positive for motility test on NBTA and MAC media pathogenicity test, catalase test, Simmons citrate agar (SCA) test, dye adsorption (on both NBTA and MAC) as well as pigment production test. Other biochemical tests including oxidase test, urease test and bioluminescence (visualization in the dark) gave negative results in all four bacterial isolates. The Cetyl Trimethyl Ammonium Bromide (CTAB) method was employed in the isolation of the total genomic DNA followed by amplification of the 16S rRNA gene using universal primers, B27F and 1492R as the forward and reverse primers, respectively. The PCR products were sent for sequencing at the University of Nairobi’s Institute of Tropical and Infectious Disease (UNITID) laboratories. Molecular analysis results established that the four sampled enteric bacteria were as follows; isolate 93A had a 99.26% identity to Klebsiella oxytoca strain 127, while isolates 93B and 93C scored 99.26% and 98.22% identity to Serratia marcescens strain B195 and Serratia marcescens strain RS, respectively and finally isolate 93D had a 99.80% similarity to Citrobacter freundii strain UIS1115. The two species of Serratia in the current study were thus named Serratia marcescens strain 93B and S. marcescens strain 93C. All the bacterial isolates did not significantly differ with respect to

antimicrobial efficacy towards the tested bacterial and fungal pathogens based on the size of the inhibition zones. Isolate 93A showed the lowest inhibition on P. aeruginosa (15.33 mm), S. aureus (18.00 mm), MRSA (21.00 mm), E. coli (23.33 mm), C. albicans (27.00 mm) and highest activity on B. cereus (34.00 mm). The cell-free suspension (100% concentration) of isolate 93B resulted in inhibition zones with the following diameters: MRSA (16.00mm), P. aeruginosa (16.33 mm), S. aureus (18.33 mm), E. coli (26.67 mm), C. albicans (27.67 mm) and B. cereus (33.67 mm). Additionally, the diameters of the zones of inhibition obtained from the effect of the 100% concentration of isolate 93C were, P. aeruginosa (17.33 mm), MRSA (17.67 mm), S. aureus (20.67 mm), E. coli (25.67 mm), B. cereus (31.33 mm) and C. albicans (36.33 mm). Isolate 93D inhibited the growth of bacterial test pathogens as follows: P. aeruginosa (16.00 mm), MRSA (19.67 mm), S. aureus (19.67 mm), E. coli (24.00 mm), B. cereus (33.33 mm) and C. albicans ( 35.33 mm). The susceptibility of phytopathogenic fungi to the 100% concentration of isolate 93A were as follows: F. verticillioides (0.00 mm), F. oxysporum (13.33 mm), F. solani (14.33 mm), P. chrysogenum (17.00 mm), Penicillium spp. (19.00 mm) and A. flavus (21.33 mm). Isolate 93B inhibited fungal growth with diameters as follows: F. verticillioides (0.00 mm), F. solani (15.00 mm), A. flavus (15.33 mm), P. chrysogenum (17.00 mm), Penicillium spp. (18.00 mm) and F. oxysporum (18.33 mm). The inhibitory effects of the full-strength concentration of isolate 93C against the six test fungi were: F. verticillioides (4.67 mm), F. solani (12.67 mm), A. flavus (13.33 mm), Penicillium spp. (16.67 mm), P. chrysogenum (18.33 mm) and F. oxysporum (19.67 mm). The 100% cell-free suspension of isolate 93D showed the following inhibitory diameters against the six pathogenic fungal species: F. verticillioides (0.67 mm), A. flavus (15.33 mm), F. solani (16.00 mm), F. oxysporum (17.67 mm), P. chrysogenum (18.33 mm) and Penicillium spp. (18.67 mm). The current study concluded that the nematode-associated enterobacteria have potential to control both bacterial human pathogens as well as the plant pathogenic fungal species and should therefore be exploited in integrated pest management of plant pathogens and control of antibiotic resistant human pathogens.