PCR standard curve quantification in an extensive wastewater surveillance program: results from the Dutch SARS-CoV-2 wastewater surveillance.

Since the start of the COVID-19 pandemic in 2020, wastewater surveillance programs were established, or upscaled, in many countries around the world and have proven to be a cost-effective way of monitoring infectious disease pathogens. Many of these programs use RT-qPCR, and quantify the viral concentrations in samples based on standard curves, by including preparations of a reference material with known nucleic acid or virus concentrations in the RT-qPCR analyses. In high-throughput monitoring programs it is possible to combine data from multiple previous runs, circumventing the need for duplication and resulting in decreased costs and prolonged periods during which the reference material is obtained from the same batch.

Inactivation of the Mla system and outer-membrane phospholipase A results in disrupted outer-membrane lipid asymmetry and hypervesiculation in .

is the causative agent of a respiratory infection known as whooping cough. With the goal of improving the production of outer-membrane vesicles (OMVs), we studied here the mechanisms that are involved in maintaining lipid asymmetry in the outer membrane of this organism. We identified homologues of the phospholipid (PL)-transport systems Mla and Pqi and of outer-membrane phospholipase A (OMPLA).

Regulated Expression of Allows for Reduction of Endotoxicity in .

The Gram-negative bacterium is the causative agent of a respiratory infection known as whooping cough. Previously developed whole-cell pertussis vaccines were effective, but appeared to be too reactogenic mainly due to the presence of lipopolysaccharide (LPS, also known as endotoxin) in the outer membrane (OM). Here, we investigated the possibility of reducing endotoxicity by modulating the LPS levels.

Conditional growth defect of Bordetella pertussis and Bordetella bronchiseptica ferric uptake regulator (fur) mutants.

Outer-membrane vesicles (OMVs) are promising tools in the development of novel vaccines against the respiratory pathogens Bordetella pertussis and Bordetella bronchiseptica. Unfortunately, vesiculation by bordetellae is too low for cost-effective vaccine production. In other bacteria, iron limitation or inactivation of the fur gene has been shown to increase OMV production, presumably by downregulation of the mla genes, which encode machinery for maintenance of lipid asymmetry in the outer membrane.

Pal depletion results in hypervesiculation and affects cell morphology and outer-membrane lipid asymmetry in bordetellae.

Current vaccines against Bordetella pertussis do not prevent colonization and transmission of the bacteria, and vaccine-induced immunity wanes rapidly. Besides, efficacy of vaccines for Bordetella bronchiseptica remains unclear. Novel vaccines could be based on outer-membrane vesicles (OMVs), but vesiculation of bordetellae needs to be increased for cost-effective vaccine production.

PMAP-36 reduces the innate immune response induced by -derived outer membrane vesicles.

Host defense peptides (HDPs), such as cathelicidins, are small, cationic, amphipathic peptides and represent an important part of the innate immune system. Most cathelicidins, including the porcine PMAP-36, are membrane active and disrupt the bacterial membrane. For example, a chicken cathelicidin, CATH-2, has been previously shown to disrupt both membranes and to release, at sub-lethal concentrations, outer membrane vesicles (OMVs).

Heat shock enhances outer-membrane vesicle release in spp.

Pertussis, also known as whooping cough, is caused by the Gram-negative bacterium , an obligate human pathogen. Despite high vaccination rates in high-income countries, resurgence of pertussis cases is an occurring problem that urges the necessity of developing an improved vaccine. Likewise, the efficacy of vaccines for , which causes similar disease in pigs and companion animals, is debatable.