Nucleic acid-based approaches to investigate microbial-related cheese quality defects

peer-reviewedThe microbial profile of cheese is a primary determinant of cheese quality. Microorganisms can contribute to aroma and taste defects, form biogenic amines, cause gas and secondary fermentation defects, and can contribute to cheese pinking and mineral deposition issues. These defects may be as a result of seasonality and the variability in the composition of the milk supplied, variations in cheese processing parameters, as well as the nature and number of the non-starter microorganisms which come from the milk or other environmental sources. Such defects can be responsible for production and product recall costs and thus represent a significant economic burden for the dairy industry worldwide. Traditional non-molecular approaches are often considered biased and have inherently slow turnaround times. Molecular techniques can provide early and rapid detection of defects that result from the presence of specific spoilage microbes and, ultimately, assist in enhancing cheese quality and reducing costs. Here we review the DNA-based methods that are available to detect/quantify spoilage bacteria, and relevant metabolic pathways in cheeses and, in the process, highlight how these strategies can be employed to improve cheese quality and reduce the associated economic burden on cheese processors.This work was funded by the Department of Agriculture, Food and the Marine under the Food Institutional Research Measure. Daniel J. O’Sullivan is in receipt of a Teagasc Walsh Fellowship, Grant Number:2012205

Temporal and spatial differences in microbial composition during the manufacture of a Continental-type cheese

peer-reviewedWe sought to determine if the time, within a production day, that a cheese is manufactured has an influence on the microbial community present within that cheese. To facilitate this, 16S rRNA amplicon sequencing was used to elucidate the microbial community dynamics of brine salted Continental-type cheese in cheeses produced early and late in the production day. Differences in microbial composition of the core and rind of the cheese were also investigated. Throughout ripening, it was apparent that late production day cheeses had a more diverse microbial population than their early day equivalents. Spatial variation between the cheese core and rind was also noted in that cheese rinds were found to initially have a more diverse microbial population but thereafter the opposite was the case. Interestingly, the genera Thermus, Pseudoalteromonas and Bifidobacterium, not routinely associated with a Continental-type cheese produced from pasteurised milk were detected. The significance, if any, of the presence of these genera will require further attention. Ultimately, the use of high throughput sequencing has facilitated a novel and detailed analysis of the temporal and spatial distribution of microbes in this complex cheese system and established that the period during a production cycle at which a cheese is manufactured can influence its microbial composition.This work was funded by the Department of Agriculture, Food and the Marine under the Food Institutional Research Measure through the ‘Cheeseboard 2015’ project. Daniel J. O’Sullivan is in receipt of a Teagasc Walsh Fellowship, Grant Number: 201220

High-throughput DNA sequencing to survey bacterial histidine and tyrosine decarboxylases in raw milk cheeses

peer-reviewedBackground The aim of this study was to employ high-throughput DNA sequencing to assess the incidence of bacteria with biogenic amine (BA; histamine and tyramine) producing potential from among 10 different cheeses varieties. To facilitate this, a diagnostic approach using degenerate PCR primer pairs that were previously designed to amplify segments of the histidine (hdc) and tyrosine (tdc) decarboxylase gene clusters were employed. In contrast to previous studies in which the decarboxylase genes of specific isolates were studied, in this instance amplifications were performed using total metagenomic DNA extracts. Results Amplicons were initially cloned to facilitate Sanger sequencing of individual gene fragments to ensure that a variety of hdc and tdc genes were present. Once this was established, high throughput DNA sequencing of these amplicons was performed to provide a more in-depth analysis of the histamine- and tyramine-producing bacteria present in the cheeses. High-throughput sequencing resulted in generation of a total of 1,563,764 sequencing reads and revealed that Lactobacillus curvatus, Enterococcus faecium and E. faecalis were the dominant species with tyramine producing potential, while Lb. buchneri was found to be the dominant species harbouring histaminogenic potential. Commonly used cheese starter bacteria, including Streptococcus thermophilus and Lb. delbreueckii, were also identified as having biogenic amine producing potential in the cheese studied. Molecular analysis of bacterial communities was then further complemented with HPLC quantification of histamine and tyramine in the sampled cheeses. Conclusions In this study, high-throughput DNA sequencing successfully identified populations capable of amine production in a variety of cheeses. This approach also gave an insight into the broader hdc and tdc complement within the various cheeses. This approach can be used to detect amine producing communities not only in food matrices but also in the production environment itself.This work was funded by the Department of Agriculture, Food and the Marine under the Food Institutional Research Measure through the ‘Cheeseboard 2015’ project. Daniel J. O’Sullivan is in receipt of a Teagasc Walsh Fellowship, Grant Number: 2012205

Sequencing of the Cheese Microbiome and Its Relevance to Industry

peer-reviewedThe microbiota of cheese plays a key role in determining its organoleptic and other physico-chemical properties. It is essential to understand the various contributions, positive or negative, of these microbial components in order to promote the growth of desirable taxa and, thus, characteristics. The recent application of high throughput DNA sequencing (HTS) facilitates an even more accurate identification of these microbes, and their functional properties, and has the potential to reveal those microbes, and associated pathways, responsible for favorable or unfavorable characteristics. This technology also facilitates a detailed analysis of the composition and functional potential of the microbiota of milk, curd, whey, mixed starters, processing environments, and how these contribute to the final cheese microbiota, and associated characteristics. Ultimately, this information can be harnessed by producers to optimize the quality, safety, and commercial value of their products. In this review we highlight a number of key studies in which HTS was employed to study the cheese microbiota, and pay particular attention to those of greatest relevance to industry

Injury rates and injury risk factors among federal bureau of investigation new agent trainees

<p>Abstract</p> <p>Background</p> <p>A one-year prospective examination of injury rates and injury risk factors was conducted in Federal Bureau of Investigation (FBI) new agent training.</p> <p>Methods</p> <p>Injury incidents were obtained from medical records and injury compensation forms. Potential injury risk factors were acquired from a lifestyle questionnaire and existing data at the FBI Academy.</p> <p>Results</p> <p>A total of 426 men and 105 women participated in the project. Thirty-five percent of men and 42% of women experienced one or more injuries during training. The injury incidence rate was 2.5 and 3.2 injuries/1,000 person-days for men and women, respectively (risk ratio (women/men) = 1.3, 95% confidence interval = 0.9-1.7). The activities most commonly associated with injuries (% of total) were defensive tactics training (58%), physical fitness training (20%), physical fitness testing (5%), and firearms training (3%). Among the men, higher injury risk was associated with older age, slower 300-meter sprint time, slower 1.5-mile run time, lower total points on the physical fitness test (PFT), lower self-rated physical activity, lower frequency of aerobic exercise, a prior upper or lower limb injury, and prior foot or knee pain that limited activity. Among the women higher injury risk was associated with slower 300-meter sprint time, slower 1.5-mile run time, lower total points on the PFT, and prior back pain that limited activity.</p> <p>Conclusion</p> <p>The results of this investigation supported those of a previous retrospective investigation emphasizing that lower fitness and self-reported pain limiting activity were associated with higher injury risk among FBI new agents.</p

Assessing the ability of nisin A and derivatives thereof to inhibit gram-negative bacteria from the genus Thermus

peer-reviewNisin is a bacteriocin that is globally employed as a biopreservative in food systems to control gram-positive, and some gram-negative, bacteria. Here we tested the bioactivity of nisin A-producing Lactococcus lactis NZ9700 and producers of bioengineered variants thereof against representatives of the gram-negative genus Thermus, which has been associated with the pink discoloration defect in cheese. Starting with a total of 73 nisin variant-producing Lactococcus lactis, bioactivity against Thermus was assessed via agar diffusion assays, and 22 variants were found to have bioactivity greater than or equal to that of the nisin A-producing control. To determine to what extent this enhanced bioactivity was attributable to an increase in specific activity, minimum inhibitory concentrations were determined using the corresponding purified form of these 22 nisin A derivatives. From these experiments, nisin M17Q and M21F were identified as peptides with enhanced antimicrobial activity against the majority of Thermus target strains tested. In addition, several other peptide variants were found to exhibit enhanced specific activity against a subset of strains

Assessing the ability of nisin A and derivatives thereof to inhibit gram-negative bacteria from the genus Thermus

peer-reviewNisin is a bacteriocin that is globally employed as a biopreservative in food systems to control gram-positive, and some gram-negative, bacteria. Here we tested the bioactivity of nisin A-producing Lactococcus lactis NZ9700 and producers of bioengineered variants thereof against representatives of the gram-negative genus Thermus, which has been associated with the pink discoloration defect in cheese. Starting with a total of 73 nisin variant-producing Lactococcus lactis, bioactivity against Thermus was assessed via agar diffusion assays, and 22 variants were found to have bioactivity greater than or equal to that of the nisin A-producing control. To determine to what extent this enhanced bioactivity was attributable to an increase in specific activity, minimum inhibitory concentrations were determined using the corresponding purified form of these 22 nisin A derivatives. From these experiments, nisin M17Q and M21F were identified as peptides with enhanced antimicrobial activity against the majority of Thermus target strains tested. In addition, several other peptide variants were found to exhibit enhanced specific activity against a subset of strains

Lipidomics Reveals Early Metabolic Changes in Subjects with Schizophrenia: Effects of Atypical Antipsychotics

There is a critical need for mapping early metabolic changes in schizophrenia to capture failures in regulation of biochemical pathways and networks. This information could provide valuable insights about disease mechanisms, trajectory of disease progression, and diagnostic biomarkers. We used a lipidomics platform to measure individual lipid species in 20 drug-naïve patients with a first episode of schizophrenia (FE group), 20 patients with chronic schizophrenia that had not adhered to prescribed medications (RE group), and 29 race-matched control subjects without schizophrenia. Lipid metabolic profiles were evaluated and compared between study groups and within groups before and after treatment with atypical antipsychotics, risperidone and aripiprazole. Finally, we mapped lipid profiles to n3 and n6 fatty acid synthesis pathways to elucidate which enzymes might be affected by disease and treatment. Compared to controls, the FE group showed significant down-regulation of several n3 polyunsaturated fatty acids (PUFAs), including 20:5n3, 22:5n3, and 22:6n3 within the phosphatidylcholine and phosphatidylethanolamine lipid classes. Differences between FE and controls were only observed in the n3 class PUFAs; no differences where noted in n6 class PUFAs. The RE group was not significantly different from controls, although some compositional differences within PUFAs were noted. Drug treatment was able to correct the aberrant PUFA levels noted in FE patients, but changes in re patients were not corrective. Treatment caused increases in both n3 and n6 class lipids. These results supported the hypothesis that phospholipid n3 fatty acid deficits are present early in the course of schizophrenia and tend not to persist throughout its course. These changes in lipid metabolism could indicate a metabolic vulnerability in patients with schizophrenia that occurs early in development of the disease. © 2013 McEvoy et al

Following a foraging fish-finder : diel habitat use of Blainville's beaked whales revealed by echolocation

© The Author(s), 2011. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in PLoS One 6 (2011): e28353, doi:10.1371/journal.pone.0028353.Simultaneous high resolution sampling of predator behavior and habitat characteristics is often difficult to achieve despite its importance in understanding the foraging decisions and habitat use of predators. Here we tap into the biosonar system of Blainville's beaked whales, Mesoplodon densirostris, using sound and orientation recording tags to uncover prey-finding cues available to echolocating predators in the deep-sea. Echolocation sounds indicate where whales search and encounter prey, as well as the altitude of whales above the sea-floor and the density of organisms around them, providing a link between foraging activity and the bio-physical environment. Tagged whales (n = 9) hunted exclusively at depth, investing most of their search time either in the lower part of the deep scattering layer (DSL) or near the sea-floor with little diel change. At least 43% (420/974) of recorded prey-capture attempts were performed within the benthic boundary layer despite a wide range of dive depths, and many dives included both meso- and bentho-pelagic foraging. Blainville's beaked whales only initiate searching when already deep in the descent and encounter prey suitable for capture within 2 min of the start of echolocation, suggesting that these whales are accessing prey in reliable vertical strata. Moreover, these prey resources are sufficiently dense to feed the animals in what is effectively four hours of hunting per day enabling a strategy in which long dives to exploit numerous deep-prey with low nutritional value require protracted recovery periods (average 1.5 h) between dives. This apparent searching efficiency maybe aided by inhabiting steep undersea slopes with access to both the DSL and the sea-floor over small spatial scales. Aggregations of prey in these biotopes are located using biosonar-derived landmarks and represent stable and abundant resources for Blainville's beaked whales in the otherwise food-limited deep-ocean.The work was funded by the Office of Naval Research and the National Ocean Partnership Program (US), by a consortium consisting of the Canary Islands Government, the Spanish Ministry of Environment and the Spanish Ministry of Defense, and by the European environmental funding LIFE-INDEMARES program for the inventory and designation of the Natura 2000 network in marine areas of the Spanish territory, headed by Fundacion Biodiversidad, with additional support from the Cabildo Insular of El Hierro. PA is currently supported by the National Research Project: Cetacean, Oceanography and Biodiversity from La Palma and El Hierro (CGL2009-13112) of the Spanish Ministry of Science and NAS by a Marie Curie fellowship from the 7th European Frame Program. MJ was supported by grants from the Strategic Environmental Research Development Program and from the National Ocean Partnership Program. PTM was supported by frame grants from the National Danish Science Foundation