Silage review: Using molecular approaches to define the microbial ecology of silage.

Ensiling of forages was recognized as a microbial-driven process as early as the late 1800s, when it was associated with the production of "sweet" or "sour" silage. Classical microbiological plating techniques defined the epiphytic microbial populations associated with fresh forage, the pivotal role of lactic acid-producing bacteria in the ensiling process, and the contribution of clostridia, bacilli, yeast, and molds to the spoilage of silage. Many of these classical studies focused on the enumeration and characterization of a limited number of microbial species that could be readily isolated on selective media. Evidence suggested that many of the members of these microbial populations were viable but unculturable, resulting in classical studies underestimating the true microbial diversity associated with ensiling. Polymerase chain reaction-based techniques, including length heterogeneity PCR, terminal RFLP, denaturing gradient gel electrophoresis, and automated ribosomal intergenic spacer analysis, were the first molecular methods used to study silage microbial communities. Further advancements in whole comparative genomic, metagenomic, and metatranscriptomic sequencing have or are in the process of superseding these methods, enabling microbial communities during ensiling to be defined with a degree of detail that is impossible using classical microbiology. These methods have identified new microbial species in silage, as well as characterized shifts in microbial communities with forage type and composition, ensiling method, and in response to aerobic exposure. Strain- and species-specific primers have been used to track the persistence and contribution of silage inoculants to the ensiling process and the role of specific species of yeast and fungi in silage spoilage. Sampling and the methods used to isolate genetic materials for further molecular analysis can have a profound effect on results. Primer selection for PCR amplification and the presence of inhibitors can also lead to biases in the interpretation of sequence data. Bioinformatic analyses are reliant on the integrity and presence of sequence data within established databases and can be subject to low taxonomic resolution. Despite these limitations, advancements in molecular biology are poised to revolutionize our current understanding of the microbial ecology of silage.

Impact of adding Saccharomyces strains on fermentation, aerobic stability, nutritive value, and select lactobacilli populations in corn silage.

Bacterial inoculants can improve the conservation and nutritional quality of silages. Inclusion of the yeast Saccharomyces in the diet of dairy cattle has also been reported to be beneficial. The present study assessed the ability of silage to be used as a means of delivering Saccharomyces strains to ruminants. Two strains of Saccharomyces cerevisiae (strain 1 and 3)and 1 strain of Saccharomyces paradoxus (strain 2) were inoculated (10(3) cfu/g) individually onto corn forage that was ensiled in mini silos for 90 d. Fermentation characteristics, aerobic stability, and nutritive value of silages were determined and real-time quantitative PCR (RT-qPCR) was used to quantify S. cerevisiae, S.paradoxus, total Saccharomyces, fungal, and bacterial populations. Fermentation characteristics of silage inoculated with S1 were similar to control silage. Although strain 3 inoculation increased ash and decreased OM contents of silage (P = 0.017), no differences were observed in nutrient composition or fermentation profiles after 90 d of ensiling. Inoculation with Saccharomyces had no detrimental effect on the aerobic stability of silage. In vitro DM disappearance, gas production, and microbial protein synthesis were not affected by yeast inoculation.Saccharomyces strain 1 was quantified throughout ensiling, whereas strain 2 was detected only immediately after inoculation. Saccharomyces cerevisiae strain 3 was quantified until d 7 and detectable 90 d after ensiling. All inoculants were detected and quantified during aerobic exposure. Inoculation with Saccharomyces did not alter lactobacilli populations. Saccharomycetales were detected by RT-qPCR throughout ensiling in all silages. Both S. cerevisiae and S. paradoxus populations increased during aerobic exposure, demonstrating that the density of these yeast strains would increase between the time that silage was removed from storage and the time it was fed.

Growth and Survival of Acid-Resistant and Non-Acid-Resistant Shiga-Toxin-Producing Escherichia coli Strains during the Manufacture and Ripening of Camembert Cheese.

Growth and survival of acid-resistant (AR) and non-acid-resistant (NAR) Shiga-toxin-producing Escherichia coli (STEC) strains were investigated during the manufacture and ripening of microfiltered milk Camembert cheeses. The induction of acid resistance of the STEC strains in cheeses was also studied. Six different mixtures of AR and/or NAR STEC strains were inoculated separately into microfiltered milk at a level of 10(3) CFU mL(-1). The STEC counts (AR and NAR) initially increased by 1 to 2 log(10) CFU g(-1) during cheese-making. Thereafter, the populations stabilized during salting/drying and then decreased during the early stages of ripening. Exposing the STEC strains in artificially inoculated cheeses to simulated gastric fluid (SGF - pH: 2.0) reduced the number of NAR strains to undetectable levels within 40 minutes, versus 120 minutes for the AR STEC strains. AR and NAR STEC were able to survive during the manufacture and ripening of Camembert cheese prepared from microfiltered milk with no evidence of induced acid tolerance in NAR STEC strains.

Fate of acid-resistant and non-acid resistant Shiga toxin-producing Escherichia coli strains in experimentally contaminated French fermented raw meat sausages.

Both pathogenic and nonpathogenic E. coli exhibit a stress response to sublethal environmental stresses. Several studies have reported acid tolerance and survival characteristics of E. coli O157:H7 in foodstuffs, but there are few reports about the tolerance of non-O157 serogroups (STEC) to organic acids in foods. The purpose of this study was to examine the effect of the manufacturing process of French fermented raw meat sausages on the growth and survival of acid-resistant (AR) and non-acid resistant (NAR) STEC strains. The six strains, 3 AR and 3 NAR, were inoculated separately into raw sausage mixture at a level of 10(4)-10(5) CFU/g. A total of 19 batches of sausages were manufactured. A rapid and similar decrease in the number of both AR and NAR STEC strains, from less than 1 to 1.5 log(10) CFU/g, was observed during the first 5 days of fermentation at 20-24 degrees C. This rapid decrease was followed by a more gradual but continuous decrease in STEC counts after drying at 13-14 degrees C, up to day 35. The STEC counts were <10 CFU/g after 35 days for the NAR strains and the same concentration for the AR strains on the best before date (day 60). It was not possible to detect any NAR STEC after 60 days. The present study shows that the process used in the manufacture of French sausages results in a complete destruction of NAR STEC strains after 60 days, but it does not have the same effect on the AR STEC strains.