The libraries from cycloheximide-treated samples were more diverse, and consisted of a variety of species that included A. cultriforme, Acanthamoeba, Sterkiella histriomuscorum, Spathidium stammeri, O. flexilis, V. costatus, S. stammeri and the fungal species Galactomyces geotrichum. In our experimental model system, the reduction of E. coli O157:H7 in nonsterile cow manure compost was significantly faster than that observed in a sterile sample (Fig. 1), strongly suggesting that the naturally present microbial communities played a major role in the decline of E. coli O157:H7 cell numbers. Our most significant finding in this study was
Selleck SP600125 that the addition of cycloheximide, which is a protein synthesis inhibitor in eukaryotes, significantly improved the survival of E. coli O157:H7 in compost at 25 °C. Previous research has
also observed an improvement in the survival of microorganisms such as Xanthomonas campestris and E. coli K-12 in soil amended with cycloheximide (Habte & Alexander, 1975; Johannes Sørensen et al., 1999); however, we are unaware of any previous studies suggesting that cycloheximide-sensitive microorganisms were capable of inhibiting E. coli O157:H7 in compost. While cycloheximide is a general inhibitor of eukaryotic populations, we feel that two pieces of data suggest that the protist populations, and not the fungal populations, have the most dramatic effect on E. coli O157:H7 reduction in our model system. First, the DGGE patterns DNA Damage inhibitor do not show very remarkable differences in the complexity of the fungal populations at 25 °C (Fig. 3) between cycloheximide-treated and -untreated samples. Second, survival of E. coli O157:H7 improves in compost models that have a lower moisture content than the one used here (data not shown), and lower moisture is expected to promote the growth of fungal species over protists (Kouyeas, 1964; Bardgett & Griffiths, 1997). The survival in low
moisture was not improved by the addition of cycloheximide, suggesting that in dry environments, the protists play a less significant role in pathogen reduction (data not shown). As our system the likely has a much higher moisture content than that routinely present during commercial or on-farm composting, future work is needed to identify what moisture levels promote the protist-mediated decline of E. coli O157:H7 counts. Clone library sequence analysis revealed significant diversity within the cycloheximide-treated samples that initially seemed to contradict DGGE data (Fig. 3). We speculate that in the absence of cycloheximide, a limited number of E. coli O157:H7 antagonistic protist species dominate and that this correlates with the lower diversity observed. Cycloheximide treatment may have eliminated the dominant inhibitory species, but not the low-abundance species that cannot be visualized by DGGE. The coverage values (Table 1) suggest that all the species were not identified by this method and, therefore, other species inhibitory to E.
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