Sensitization of heat-treated Listeria monocytogenes to added lysozyme in milk.

Listeria monocytogenes was highly resistant to hen egg white lysozyme in whole milk but was sensitive in media and in phosphate buffer. Methods to sensitize the pathogen to lysozyme in milk were investigated. Treatment of whole milk by cation exchange to remove minerals, particularly Ca2+ and Mg2+, slightly promoted inactivation of L. monocytogenes by lysozyme at 4 degrees C over a period of 6 days. Heat treatment (62.5 degrees C for 15 s) strongly sensitized L. monocytogenes to lysozyme in demineralized milk and in MES [2-(N-morpholino)ethanesulfonic acid] buffer. Addition of Ca2+ or Mg2+ to the demineralized milk restored resistance to lysozyme. Cells were more rapidly heat inactivated at 55 degrees C in demineralized milk containing lysozyme, and addition of Ca2+ to the demineralized milk restored the resistance to heat. The results indicate that minerals or mineral-associated components protect L. monocytogenes from inactivation by lysozyme and heat in milk, probably by increasing cell surface stability. The heat treatment of foods containing added lysozyme can probably play a significant role in producing microbiologically safe foods.     

Real-Time PCR Assays for the Specific Identification of Probiotic Strains Lactobacillus gasseri BNR17 and Lactobacillus reuteri LRC (NCIMB 30242)

The broad spectrum of health benefits attributed to probiotics has contributed to a rapid increase in the value of the probiotic market. Probiotic health benefits can be strain specific. Thus, strain-level identification of probiotic strains is of paramount importance to ensure probiotic efficacy. Both Lactobacillus gasseri BNR17 and Lactobacillus reuteri LRC (NCIMB 30242) strains have clinically proven health benefits; however, no assays were developed to enable strain-level identification of either of these strains. The objective of this study is to develop strain-specific PCR-based methods for Lactobacillus gasseri BNR17 and Lactobacillus reuteri LRC strains, and to validate these assays according to the guidelines for validating qualitative real-time PCR assays. Using RAST (Rapid Annotation using Subsystem Technology), unique sequence regions were identified in the genome sequences of both strains. Probe-based assays were designed and validated for specificity, sensitivity, efficiency, repeatability, and reproducibility. Both assays were specific to target strain with 100% true positive and 0% false positive rates. Reaction efficiency for both assays was in the range of 90 to 108% with R square values > 0.99. Repeatability and reproducibility were evaluated using five samples at three DNA concentrations each and relative standard deviation was < 4% for repeatability and < 8% for reproducibility. Both of the assays developed and validated in this study for the specific identification of Lactobacillus gasseri BNR17 and Lactobacillus reuteri LRC strains are specific, sensitive, and precise. These assays can be applied to evaluate and ensure compliance in probiotic products.

     

Effects of seven potential probiotic strains on specific immune responses in healthy adults: a double-blind, randomized, controlled trial

This pilot study investigated the immunomodulatory properties of seven probiotic strains. Eighty-three healthy volunteers aged 18-62 years consumed 2 x 10(10) CFU of bacteria or a placebo (maltodextrin) over 3 weeks (D0-D21). Subjects received an oral cholera vaccine at D7 and at D14; blood and saliva samples were collected at D0, D21 and D28. Serum samples were analyzed for specific IgA, IgG and IgM, and saliva samples were analyzed for specific IgA only, by ELISA. Statistical analyses were based on Wilcoxon's signed-rank test (intragroup analyses) and exact median t-test (intergroup analyses). Salivary analysis showed no difference in specific IgA concentrations between groups. Serum analysis indicated an effect of some of the tested strains on specific humoral responses. Between D0 and D21, IgG increased in two probiotic groups, for example, Bifidobacterium lactis Bl-04 and Lactobacillus acidophilus La-14, compared with controls (P=0.01). Trends toward significant changes in immunoglobulin serum concentrations compared with controls (P<0.1) were found for six out of the seven probiotic strains. In conclusion, some strains of probiotics demonstrated a faster immune response measured with serum immunoglobulin indicators, especially IgG, although overall vaccination was not influenced. Specific strains of probiotics may thus act as adjuvants to the humoral immune response following oral vaccination.     

A fast and simple genetic survey reveals the spread of poplar hybrids at a natural Elbe river site

It is known that various poplar species and cultivated poplar hybrids have the potential to interbreed and produce fertile offspring. Conservation strategies for the genetic resources of the endangered Eurasian black poplar (Populus nigra L.) thus rely on a monitoring which enables the identification and verification of the pure species status. At the same time, the risk of hybrid dispersal and introgressive gene flow has to be estimated. In the present study a combination of two molecular markers, one from chloroplast DNA and the other from nuclear DNA, was applied to evaluate a large P. nigra population on the Elbe River. Hybrid clones of P. × canadensis are scattered within this population and also occur as plantations in the surrounding landscape. By means of the DNA markers the taxonomic status of 208 adult trees in the population and 140 young poplars along the riverbank was monitored. From the analysed young poplars, almost 20 percent were found to exhibit at least one of the two P. deltoides or P. × canadensis diagnostic alleles or genotypes, respectively. Possible vegetative spreads of F1 hybrids and precedent mating scenarios are discussed. Most interestingly we found clear evidence for a small number of backcross hybrids where P.  × canadensis acted as pollen donor. This case had long been debated and thought to be less probable, so far.     

Do we need livestock grazing to promote <Emphasis Type="Italic">Polylepis australis</Emphasis> tree recruitment in the Central Argentinean Mountains?

South American Polylepis mountain forests are endangered due to centuries of logging, fire clearance, and intensive livestock grazing. Polylepis australis (Rosaceae) dominates the tree canopy in the Central Argentinean Mountains, where livestock have been excluded or reduced to very low densities over wide areas. Lowered plant and bird diversity in these abandoned grazing areas suggest that some livestock grazing may be beneficial for conservation purposes. Our study aimed to determine whether this may also be true for forest regeneration. In 300 plots of 1 m² distributed in sub-regions of high to moderate grazing pressure as well as grazing exclusion, we recorded the presence of P. australis seedlings and grazing indicators. Topographical parameters as well as vegetation and soil characteristics were also assessed. Seedling frequencies were highest in the region with moderate grazing, intermediate with grazing exclusion, and the lowest with high grazing pressures. Logistic regression models revealed that seedling occurrence was highest close to seeder trees, with intermediate litter coverage and at intermediate altitudes above sea level. Since grazing ultimately affects seeder tree occurrence through browsing and reduces litter cover and predominates in higher areas, we conclude that grazing is an important driving factor for P. australis regeneration. We confirm previous studies determining high grazing pressure to be detrimental for P. australis regeneration, but also provide data suggesting that its complete exclusion may not be recommendable either. Management should aim to alternate between a moderate grazing intensity to promote seedling recruitment, with subsequent exclusion to accelerate the growth of the established seedlings.     

Acid adaptation sensitizes Salmonella typhimurium to hypochlorous acid.

Acid adaptation of Salmonella typhimurium at a pH of 5.0 to 5.8 for one to two cell doublings resulted in marked sensitization of the pathogen to halogen-based sanitizers including chlorine (hypochlorous acid) and iodine. Acid-adapted S. typhimurium was more resistant to an anionic acid sanitizer than was its nonadapted counterpart. A nonselective plating medium of tryptose phosphate agar plus 1% pyruvate was used throughout the study to help recover chemically stressed cells. Mechanisms of HOCl-mediated inactivation of acid-adapted and nonadapted salmonellae were investigated. Hypochlorous acid oxidized a higher percentage of cell surface sulfhydryl groups in acid-adapted cells than in nonadapted cells, and sulfhydryl oxidation was correlated with cell inactivation. HOCl caused severe metabolic disruptions in acid-adapted and nonadapted S. typhimurium, such as respiratory loss and inability to restore the adenylate energy charge from a nutrient-starved state. Sensitization of S. typhimurium to hypochlorous acid by acid adaptation also involved increased permeability of the cell surface because nonadapted cells treated with EDTA became sensitized. The results of this study establish that acid-adapted S. typhimurium cells are highly sensitized to HOCl oxidation and that inactivation by HOCl involves changes in membrane permeability, inability to maintain or restore energy charge, and probably oxidation of essential cellular components. This study provides a basis for improved practical technologies to inactivate Salmonella and implies that acid pretreatment of food plant environments may increase the efficacy of halogen sanitizers.     

Fully human,HLA-DR-specific monoclonal antibodies efficiently induce programmed death of malignant lymphoid cells

The Human Combinatorial Antibody Library (HuCAL) was screened for antibodies specific to human leukocyte antigen-DR (HLA-DR) that induce programmed death of lymphoma/leukemia cells expressing the target antigen. The active Fab fragments were affinity-matured, and engineered to IgG(4) antibodies of sub-nanomolar affinity. The antibodies exhibited potent in vitro tumoricidal activity on several lymphoma and leukemia cell lines and on chronic lymphocytic leukemia patient samples. They were also active in vivo in xenograft models of non-Hodgkin lymphoma. Cell death occurred rapidly, without the need for exogenous immunological effector mechanisms, and was selective to activated/tumor-transformed cells. Although the expression of HLA-DR on normal hematopoietic cells is a potential safety concern, the antibodies caused no long-lasting hematological toxicity in primates, in vivo. Such monoclonal antibodies offer the potential for a novel therapeutic approach to lymphoid malignancies.     

Wide spread invasion without sexual reproduction? A case study on European willows in Patagonia,Argentina

Willows of the Salix alba–Salix fragilis complex, native to western Eurasia, represent typical invaders of floodplain ecosystems worldwide. Introduced to South America by European settlers probably at the end of the nineteenth century, their distribution has increased significantly along the rivers in Northern Patagonia. This case study carried out mainly in the area around Lake Nahuel Huapi aims to analyze clonal structures and their spatial distribution using molecular markers as well as to relate the observed patterns to settlement history and life history traits of this species complex. Leaf material from 171 trees was collected along selected river floodplains in Northern Patagonia and genotypes were determined at six microsatellite loci. Including 62 reference samples of the S. alba–S. fragilis complex from German rivers, Probability of Identity (P _ID) was calculated and a Principal Coordinate Analysis (PCoA) conducted. From the altogether thirteen different genotypes detected, one dominant genotype (female) formed monoclonal stands along most of the studied river stretches. The maximum linear distance between the most remote ramets of this clone was 790 km. Evidence arose that the colonizing process so far is exclusively based on vegetative propagation in the focal study area and is obviously attributable to the pronounced brittleness of the hybrid parent S. fragilis. However, outside this area the occurrence of male trees and a diversity of genotypes indicate that evolutionary processes by recombination are involved within the willow complex. Therefore, an increase in genotypes can be assumed when male individuals and therefore sexual reproduction would appear in the area around Lake Nuhuel Huapi. This could be a crucial point for the long-term invasion success of the taxa when climatic and other environmental conditions will change in Southern Argentina.     

Lactobacillus acidophilus modulates intestinal pain and induces opioid and cannabinoid receptors

Abdominal pain is common in the general population and, in patients with irritable bowel syndrome, is attributed to visceral hypersensitivity. We found that oral administration of specific Lactobacillus strains induced the expression of mu-opioid and cannabinoid receptors in intestinal epithelial cells, and mediated analgesic functions in the gut-similar to the effects of morphine. These results suggest that the microbiology of the intestinal tract influences our visceral perception, and suggest new approaches for the treatment of abdominal pain and irritable bowel syndrome.     

Acid adaptation promotes survival of Salmonella spp. in cheese.

Salmonella typhimurium was adapted to acid by exposure to hydrochloric acid at pH 5.8 for one to two doublings. Acid-adapted cells had increased resistance to inactivation by organic acids commonly present in cheese, including lactic, propionic, and acetic acids. Recovery of cells during the treatment with organic acids was increased 1,000-fold by inclusion of 0.1% sodium pyruvate in the recovery medium. Acid-adapted S. typhimurium cells survived better than nonadapted cells during a milk fermentation by a lactic acid culture. Acid-adapted cells also showed enhanced survival over a period of two months in cheddar, Swiss, and mozzarella cheeses kept at 5 degrees C. Acid adaptation was found in Salmonella spp., including Salmonella enteritidis, Salmonella choleraesuis subsp. choleraesuis serotype heidelberg, and Salmonella choleraesuis subsp. choleraesuis serotype javiana, associated with food poisoning. These observations support the theory that acid adaptation is an important survival mechanism enabling Salmonella spp. to persist in fermented dairy products and possibly other acidic food products.