Optimizing Methods for Recovering Heat-injured Enterococcus faecium and Indigenous Enterococci in Turkey Litter Compost

Introduction: In order to validate the physical heat treatment applied to eliminate Salmonella spp. in poultry litter as biological soil amendments, there is a need to use a surrogate strain for the pathogen. Enterococcus faecium NRRL B-2354 has been used as a Salmonella surrogate in thermal process validation studies. Since bacterial cells may be injured during heat treatment, an effective recovery step should be incorporated to allow the resuscitation of heat-injured cells for an accurate enumeration.

Purpose: The objective of this study was to optimize the recovery method for isolating heat-injured indigenous enterococci and desiccation-adapted Salmonella surrogate E. faecium from turkey litter compost.

Methods: The turkey litter compost samples with and without inoculation of Salmonella surrogate E. faecium NRRL B-2354 (desiccation-adapted) in the Tyvek pouches were exposed to 75°C for up to 1 h. Recovery media for heat-injured indigenous enterococci were bile esculin agar (BEA) or enterococcosel agar (EA); modified two-step overlay (OV) method (OV/BEA and OV/EA); and modified thin agar layer (TAL) method (TAL/BEA and TAL/EA). To recover heat-injured E. faecium, BEA and EA, in the above recovery media, were supplemented with rifampin (R; 100 µg/ml).

Results: The initial populations of E. faecium and indigenous enterococci in turkey litter compost were 8.21 and 5.45 log CFU/g, respectively. Among those six recovery methods, OV/EA-R plates yielded the highest populations (P< 0.05) of E. faecium (5.09±0.33 log CFU/g). For heat-treated turkey litter compost without inoculation, background microorganisms growing on OV/EA, OV/BEA, TAL/EA, and TAL/BEA caused interference with the enumeration of indigenous enterococci. Therefore, BEA and EA were selected to recover the heat-injured indigenous enterococci.

Significance: The selected recovery methods for heat-injured E. faecium and indigenous enterococci can be used in the thermal process validation studies on poultry litter.