BackgroundThe broad ecological distribution of L. casei makes it an insightful subject for research on genome evolution and lifestyle adaptation. To explore evolutionary mechanisms that determine genomic diversity of L. casei, we performed comparative analysis of 17 L. casei genomes representing strains collected from dairy, plant, and human sources.ResultsDifferences in L. casei genome inventory revealed an open pan-genome comprised of 1,715 core and 4,220 accessory genes. Extrapolation of pan-genome data indicates L. casei has a supragenome approximately 3.2 times larger than the average genome of individual strains. Evidence suggests horizontal gene transfer from other bacterial species, particularly lactobacilli, has been important in adaptation of L. casei to new habitats and lifestyles, but evolution of dairy niche specialists also appears to involve gene decay.ConclusionsGenome diversity in L. casei has evolved through gene acquisition and decay. Acquisition of foreign genomic islands likely confers a fitness benefit in specific habitats, notably plant-associated niches. Loss of unnecessary ancestral traits in strains collected from bacterial-ripened cheeses supports the hypothesis that gene decay contributes to enhanced fitness in that niche. This study gives the first evidence for a L. casei supragenome and provides valuable insights into mechanisms for genome evolution and lifestyle adaptation of this ecologically flexible and industrially important lactic acid bacterium. Additionally, our data confirm the Distributed Genome Hypothesis extends to non-pathogenic, ecologically flexible species like L. casei.

Currently, the bacterial composition of raw milk in tanker trucks and the outcomes of transfer and storage of that milk at commercial processing facilities are not well understood. We set out to identify the bacteria in raw milk collected for large-scale dairy product manufacturing. Raw bovine milk samples from 899 tanker trucks arriving at two dairy processors in San Joaquin Valley of California during three seasons (spring, summer, and fall) were analyzed by community 16S rRNA gene sequencing. This analysis revealed highly diverse bacterial populations, which exhibited seasonal differences. Raw milk collected in the spring contained the most diverse bacterial communities, with the highest total cell numbers and highest proportions being those of Actinobacteria. Even with this complexity, a core microbiota was present, consisting of 29 taxonomic groups and high proportions of Streptococcus and Staphylococcus and unidentified members of Clostridiales. Milk samples were also collected from five large-volume silos and from 13 to 25 tankers whose contents were unloaded into each of them during 2 days in the summer. Transfer of the milk to storage silos resulted in two community types. One group of silos contained a high proportion of Streptococcus spp. and was similar in that respect to the tankers that filled them. The community found in the other group of silos was distinct and dominated by Acinetobacter. Overall, despite highly diverse tanker milk community structures, distinct milk bacterial communities were selected within the processing facility environment. This knowledge can inform the development of new sanitation procedures and process controls to ensure the consistent production of safe and high-quality dairy products on a global scale.

Validated methods are needed to detect spoilage microbes present in low numbers in foods and ingredients prior to defect onset. We applied propidium monoazide combined with 16S rRNA gene sequencing, qPCR, isolate identification, and pilot-scale cheese making to identify the microorganisms that cause slit defects in industrially produced Cheddar cheese. To investigate milk as the source of spoilage microbes, bacterial composition in milk was measured immediately before and after high-temperature, short-time (HTST) pasteurization over 10-h periods on 10 days and in the resulting cheese blocks. Besides HTST pasteurization-induced changes to milk microbiota composition, a significant increase in numbers of viable bacteria was observed over the 10-h run times of the pasteurizer, including 68-fold-higher numbers of the genus Thermus. However, Thermus was not associated with slit development. Milk used to make cheese which developed slits instead contained a lower number of total bacteria, higher alpha diversity, and higher proportions of Lactobacillus, Bacillus, Brevibacillus, and Clostridium. Only Lactobacillus proportions were significantly increased during cheese aging, and Limosilactobacillus (Lactobacillus) fermentum, in particular, was enriched in slit-containing cheeses and the pre- and post-HTST-pasteurization milk used to make them. Pilot-scale cheeses developed slits when inoculated with strains of L. fermentum, other heterofermentative lactic acid bacteria, or uncultured bacterial consortia from slit-associated pasteurized milk, thereby confirming that low-abundance taxa in milk can negatively affect cheese quality. The likelihood that certain microorganisms in milk cause slit defects can be predicted based on comparisons of the bacteria present in the milk used for cheese manufacture. IMPORTANCE Food production involves numerous control points for microorganisms to ensure quality and safety. These control points (e.g., pasteurization) are difficult to develop for fermented foods wherein some microbial contaminants are also expected to provide positive contributions to the final product and spoilage microbes may constitute only a small proportion of all microorganisms present. We showed that microbial composition assessments with 16S rRNA marker gene DNA sequencing are sufficiently robust to detect very-low-abundance bacterial taxa responsible for a major but sporadic Cheddar cheese spoilage defect. Bacterial composition in the (pasteurized) milk and cheese was associated with slit defect development. The application of Koch’s postulates showed that individual bacterial isolates as well as uncultured bacterial consortia were sufficient to cause slits, even when present in very low numbers. This approach may be useful for detection and control of low-abundance spoilage microorganisms present in other foods.

Effects of megasphaera elsdenii on ruminal pH, ruminal concentrations of organic acids, and bacterial genomes following a grain challenge AbstractUpon arrival in feedlots, cattle normally must be adapted to high-concentrate diets. The microbial population in the rumen of incoming cattle normally is suited to digestion of forages, and when cattle are transitioned onto concentrate diets, opportunistic bacteria that produce lactic acid can proliferate rapidly, leading to excesses of lactic acid in the rumen. High levels of lactic acid in the rumen may cause mild to severe acidosis. Megasphaera elsdenii is a lactate-utilizing bacterium that normally is present in rumens of cattle that have been adapted to high-grain diets, but numbers of the organism are relatively low during the step-up phase. Increasing the numbers of lactate-utilizing bacteria in newly arrived cattle by orally dosing with M. elsdenii may be a useful means of reducing the risk of ruminal acidosis in feedlot cattle. Our objectives were to evaluate ruminal parameters and determine efficacy of increasing ruminal populations of lactateutilizing bacteria in cattle following an abrupt diet change and administration of 10 mL (low dose), 100 mL (medium dose), or 1000 mL (high dose) of a culture containing 1.62 x 108 CFU/mL of live M. elsdenii compared with a control group given a placebo without live Megasphaera.

. Brand names appearing in this publication are for product identification purposes only. No endorsement is intended, nor is criticism implied of similar products not mentioned. K-State Research and Extension is an equal opportunity provider and employer. Beta acid extracts of hops have a modest effect on ruminal metabolism and apparent total tract digestibility by steers fed high-concentrate diets AbstractHops have been used for centuries to control bacterial contamination in beer production. Today, α acids are extracted from hops for use in flavoring beer, leaving residues that are rich in beta acids. Beta acid fractions of hops can selectively inhibit specific ruminal Gram-positive bacteria that are responsible for major digestive disturbances, such as acidosis and bloat, and have a chemical structure similar to that of ionophores used in feedlot production. Use of ionophores improves efficiency of feed utilization and decreases the incidence of digestive disturbances that are a major cause of morbidity and mortality in cattle feeding operations. The objectives of this study were to evaluate the effect of beta acid extracts of hops on ruminal fermentation and diet digestibility in cattle fed high-concentrate diets and determine response to different doses of beta acid extracts of hops.