Detection of virulence attributes of Burkholderia pseudomallei.
OBJECTIVE
Melioidosis caused by Burkholderia pseudomallei is an emerging disease in India. This study examined the toxin activity of bacteria-free culture filtrate in three different cell lines (cytotoxic assay) and its effect on Caenorhabditis elegans (nematode toxicity assay). Endotoxic activity of the viable bacteria was also studied in C. elegans (co-culture killing assay).


METHODS
For toxin studies, serial doubling dilutions of unheated, heated crude and ultra filtrate of bacteria-free culture supernatants of B. pseudomallei were tested in 96-well microtitre plate containing confluent mono layers of McCoy, Hep-2 and HeLa cell lines. For the effects on C. elegans, the worms were exposed to heated and unheated bacteria-free culture supernatants in 24-well microtitre plate for 24h and then transferred to OP50 Escherichia coli lawn culture. The endotoxic activity of the live bacterium was studied by feeding the worms in the lawn culture of B. pseudomallei.


RESULTS
All the clinical isolates (n=38) produced cytotoxic changes in all the cell lines. No difference was observed in the cytotoxicity of unheated, heated and ultra-filtered culture supernatant. The septicaemic isolates were observed to produce cytotoxic changes in high dilutions (1:160) of culture filtrate. None of the unheated and heated crude filtrate had deleterious effect on C. elegans, while all the live bacteria were found to be lethal to the nematode.


CONCLUSIONS
The culture supernatants, though produced cytopathic effect in various tissue cultures, failed to have any deleterious effect on the worms. However, live bacteria were lethal to the worms B. pseudomallei. Use of C. elegans model to detect virulence attributes of B. pseudomallei is recommended as an alternative to tissue culture methods as this can be carried out in laboratories where a tissue culture set up is not available.
Balaji
V
V
Department of Clinical Microbiology, Christian Medical College & Hospital, Vellore, India. micro@cmcvellore.ac.in
Jesudason
Mary V
MV
Sridharan
G
G
Subramanian
K
K
D000818:Animals; D001427:Bacterial Toxins / Q000378:metabolism; D016957:Burkholderia pseudomallei / Q000254:growth & development / Q000302:isolation & purification / Q000378:metabolism / Q000472:pathogenicity*; D017173:Caenorhabditis elegans / Q000382:microbiology / Q000502:physiology; D002460:Cell Line; D018920:Coculture Techniques; D004731:Endotoxins / Q000378:metabolism; D005098:Exotoxins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D018805:Sepsis / Q000382:microbiology
D001427:Bacterial Toxins; D004731:Endotoxins; D005098:Exotoxins