Fast and on-site animal species identification in processed meat via centrifugal microfluidics and isothermal amplification.

We present a novel centrifugal microfluidic approach to rapidly identify animal species in meat products. The workflow requires a centrifugal cartridge for DNA extraction and for preparation of a recombinant polymerase amplification (RPA) reaction, a programmable centrifuge for processing the cartridge and an isothermal reader to perform the RPA. Liquid reagents are pre-stored on the cartridge and the meat sample can be added directly without any pre-treatment. With this system, we are able to identify six different animal species in a single run within one hour. In pork salami containing horse, turkey, sheep, chicken and beef meat, it was possible to identify species levels as low as 0.01%. In beef salami and cooked pork sausages 0.1% of foreign meat could be detected. This novel workflow enables rapid and sensitive species identification in processed meat at the point of need.

Surrogate for Electron Beam Inactivation of Salmonella on Pumpkin Seeds and Flax Seeds.

ABSTRACT: This study aimed to identify a suitable nonpathogenic surrogate for industrial validation of irradiation process by high-energy electron beam (5 MeV) of dried seeds. Pumpkin seeds (Cucurbita pepo var. styriaca) and golden flax seeds (Linum usitatissimum) were contaminated with a five-strain Salmonella cocktail comprising five serovars or a two-strain Escherichia coli cocktail comprising pathogenic strains, including E. coli O157:H7. Comparison of log survival fractions of the E. coli and Salmonella cocktails revealed that on both types of seeds, the Salmonella cocktail exhibited higher tolerance against high-energy electron beam at doses of 4 kGy than the E. coli cocktail, with a log survival fraction of -4.1 ± 0.7 compared with -6.0 ± 0.2 on pumpkin seeds and -4.7 ± 0.7 compared with reduction from 1.8 × 108 CFU/g to below the limit of detection (1 × 102 CFU/g) on flax seeds. For surrogate selection, the Salmonella cocktail and the strains E. coli DSM 18039 (strain MG1655) and Enterococcus faecium NCCB 86023 (strain NRRL B-2354) were subjected to electron beam processing at doses of 2 to 6 kGy. The calculated D10-values of the Salmonella cocktail were not significantly different (P > 0.05) from those of E. coli DSM 18039, i.e., 1.07 ± 0.10 kGy compared with 1.20 ± 0.07 kGy on pumpkin seeds and 0.88 ± 0.04 kGy compared with 1.07 ± 0.03 kGy on flax seeds. E. faecium NCCB 86023 exhibited significantly higher tolerance on pumpkin seeds (3.07 ± 0.18 kGy) and on flax seeds (2.22 ± 0.29 kGy), ∼3 log and 2 log higher than the Salmonella cocktail, respectively. Hence, the nonpathogenic E. coli DSM 18039 is suggested to serve as a surrogate for Salmonella in industrial validation trials. Because on both types of seeds E. faecium NCCB 86023 showed significantly higher tolerance against electron beam than the Salmonella cocktail, this nonpathogenic strain could serve as a process control indicator for the decontamination of dried seeds by electron beam.

Streptococcus pyogenes Escapes Killing from Extracellular Histones through Plasminogen Binding and Activation by Streptokinase.

Histones are small basic proteins and highly conserved among eukaryotes. Their main function is binding, packaging and organizing of DNA in the nucleus, but extracellular histones are also potent antimicrobial proteins. Here we found that Streptococcus pyogenes - an important human pathogen - protects itself from histone-killing by the acquisition of plasminogen. Plasminogen, bound to the streptococcal surface, efficiently prevents histone-mediated killing. Moreover, the streptokinase/plasminogen complex degrades all classes of histones and abrogates their antibacterial and hemolytic effects. This novel streptokinase-mediated virulence mechanism may contribute to the escape of S. pyogenes from the human innate immune system.

High intravascular tissue factor-but not extracellular microvesicles-in septic patients is associated with a high SAPS II score.

BACKGROUND: Sepsis is associated with coagulation abnormalities, and a high content of intravascular tissue factor (TF) may contribute to the development of multisystem organ failure. Circulating microvesicles (MVs) are increased during sepsis and characterized by their phosphatidylserine content. It is unclear whether MVs-as a part of the host response to the infection-are beneficial or rather contribute to systemic complications in sepsis. In the present prospective clinical pilot study, we investigated whether plasma TF and MVs are associated with the risk of multiple organ failure and mortality. METHODS: Thirty patients diagnosed with sepsis, severe sepsis, or septic shock were enrolled and classified as 19 survivors and 11 non-survivors. Blood samples were collected on the day of admission and then daily for up to 2 weeks. MVs and TF were quantified in plasma by ELISA. RESULTS: Non-survivors had significantly higher TF concentrations on day 3 compared to survivors. Logistic regression analysis revealed that patients with high amounts of TF had significantly increased risk for severity of disease, according to high Simplified Acute Physiology Score II (SAPS II) scores (odds ratio 18.7). In contrast, a higher content of phosphatidylserine-rich MVs were apparently associated with a lower risk for mortality and multiple organ failure, although this was only a trend and the odds ratios were not significant. CONCLUSIONS: This study showed that a high amount of TF in septic patients is significantly associated with increased risk for disease severity, according to a high SAPS II score. Quantification of total MVs in plasma, independent from their cell origin, might be indicative for the outcome of patients in sepsis.

Streptococcus pyogenes triggers activation of the human contact system by streptokinase.

Severe invasive infectious diseases remain a major and life-threatening health problem. In serious cases, a systemic activation of the coagulation cascade is a critical complication that is associated with high mortality rates. We report here that streptokinase, a group A streptococcal plasminogen activator, triggers the activation of the human contact system. Activation of contact system factors at the surface of the Streptococcus pyogenes serotype M49 is dependent on streptokinase and plasminogen. Our results also show that secreted streptokinase is an efficient contact system activator, independent from a contact surface. This results in the processing of high-molecular-weight kininogen and the release of bradykinin, a potent vascular mediator. We further investigated whether the ability of 50 different clinical S. pyogenes isolates to activate the contact system is associated with an invasive phenotype. The data reveal that isolates from invasive infections trigger an activation of the contact system more potently than strains isolated from noninvasive infections. The present study gives new insights into the mechanisms by which S. pyogenes triggers the human contact system and stresses the function of soluble and surface located plasmin exploited as a group A streptococcal virulence factor through the action of streptokinase.