Phages cocktail challenge test for biocontrol and determination of bacteriophages insensitive mutants on non-O157 and O157:H7 VETC

BALAGUÉ, CLAUDIA; TOMAT, DAVID DAMIÁN; AQUILI, VIRGINIA; CASABONNE, CECILIA; QUIBERONI, ANDREA DEL LUJÁN

Amsterdam

Simposio; 8th International Simposium on Shiga Toxin (Verocytotoxin) Producing Escherichia coli Infections (VTEC 2012); 2012

Verotoxigenic E. coli strains (VTEC) biocontrol, mediated by phages, may be an interesting tool to control enteric diseases. However, in bacterial populations are found naturally bacteriophage insensitive mutants (BIMs). Challenge test was performed to evaluate cocktails and individual phages biocontrol on non-O157 VTEC and O157:H7 VTEC strains (an O18 food isolate stx1+, stx2+ and a clinical isolate eaeA+, stx2+). BIMs were isolated by extended challenge test (48 h) -secondary culture, SC- of bateriophage/sensitive E. coli strain systems and confirmed by the liquid culture sensitivity test. In addition, emergence frequency, reversion and stability were tested for each confirmed-BIM, through successive subcultures and infections. Ten stock phages were isolated by the technique of double-layer agar (soft-Hershey supplemented with MgSO4) from feces of healthy patients of the Centenary Hospital (LM1-4 and DT1-6) in Rosario city, Argentina. When testing bacteria versus phages, greater reductions were observed after 2 h at 37 ºC in non-O157 VTEC/DT5, DT6 and cocktail challenge tests, being 6.38 log -cocktail- the maximun reduction achieved. On the other hand, for O157:H7 VTEC the greatest reduction was 5.23 log -cocktail- after 6 h of incubation. After 6 and 24 h, all reductions were significant for both strains tested, except for O157:H7 VTEC/DT1 system after 24 h. For both bateriophage/E. coli sensitive strain systems a significant bacterial grown was observed at 37 ºC after 48 h (SC) suggesting BIMs production under these tests conditions. Presumptive BIMs, isolated by SC, showed a 75% of confirmed BIMs and their frequencies, for both evaluated strains, ranged between 3.7x10-7 to 1.8x10-6. Only one BIM showed reversion to phage (DT6) sensitivity, while BIMs evaluated for phage DT1 were resistant. In stability trials, mutants isolated from non-O157 VTEC maintained the resistant phenotype until the second and third -with DT5- and sixth -with DT6- subculture, while BIMs isolated from O157:H7 VTEC up to the fourth -DT6- and only one until seventh -DT1- subculture. We conclude that phages are very effective to eliminate or significantly reduce the population of non-O157 VTEC and O157:H7 VTEC in vitro and both cocktails (DT1+DT6 and DT5+DT6) showed an additive effect. E. coli confirmed-BIMs showed variable reversion and stability and a very low emergence frequency, being no obstacle in using bacteriophages as biocontrol tools against VTEC.