Talks

Antibiotic resistance in Gram-negative bacilli: a major public health issue - T. Naas

The fight against multi-resistant bacteria is a public health emergency, particularly with the global emergence of potentially pan-resistant carbapenemase-producing enterobacterales (CPEs). b-lactams, due to their good tolerance and clinical efficacy, are the most widely prescribed antibiotics for treating bacterial infections. However, their use is threatened by the worldwide dissemination of β-lactamases (βL) capable of hydrolyzing them, especially in Gram-negative bacteria GNBs. The dissemination of these βLs among Enterobacterales is of particular concern because these bacteria are the primary cause of nosocomial and community infections, and carbapenems are the treatment of choice for severe infections (bacteremia) caused by these germs in hospital. In high prevalence countries, many strains have also been described in the environment, in farm animals, in pets, but also in animal and human food. These observations highlight the need for strategies to combat these BMR bacteria in all hospital, community and environmental compartments. Until novel antibiotics are available to treat efficiently infections with these bacteria, isolation of patients carrying or being infected with bacteria in hospital settings is mandatory to avoid spread to the most fragile patients. This can only be achieved with the use of rapid and specific diagnostics tools.

Microfluidics for diagnostics of viral and bacterial infections P. Garstecki

TBD

Droplet Crystals, Digital PCR & COVID-19 - R. Dangla and H. Péré 

The commercialization of digital PCR platforms has sparked a revolution in quantitative nucleic acid detection over the past decade, dramatically increasing the sensitivity and precision of PCR tests. Applications of digital PCR range from the analysis of cancer biomarkers in liquid biopsies to GMO detection in food samples, including infectious diseases.
Microfluidics is the enabling science and technology behind digital PCR. Here, we focus on the Crystal Digital™ PCR workflow and the microfluidic technology developed by Stilla Technologies, which relies on confinement gradients to partition the samples into droplet crystals prior to PCR amplification and readout.
The key advantages of the Crystal Digital PCR workflow for infectious diseases are its ease-of-use, its sensitivity, its robustness and its multiplexing capability, with up to 6 detection channels available.
One application developed in the context of the COVID-19 pandemic is hig-sensitivity group testing by digital PCR. We present the results from a collaboration between Stilla Technologies, Ecole Polytechnique and Hopital Bichat, demonstrating how group testing by digital PCR can combine a reduction in cost and reduction in use of reagents while maintaining a sensitivity comparable to individual RT-PCR testing.

Background. Coronavirus disease 2019 (COVID-19) is a global public health problem that has already caused more than 662,000 deaths worldwide. Although the clinical manifestations of COVID-19 are dominated by respiratory symptoms, some patients present other severe damage such as cardiovascular, renal and liver injury or/and multiple organ failure, suggesting a spread of the SARS-CoV-2 in blood. Recent ultrasensitive polymerase chain reaction (PCR) technology now allows absolute quantification of nucleic acids in plasma. We herein intended to use the droplet-based digital PCR technology to obtain sensitive detection and precise quantification of plasma SARS-CoV-2 viral load (SARS-CoV-2 RNAaemia) in hospitalized COVID-19 patients.
Methods. Fifty-eight consecutive COVID-19 patients with pneumonia 8 to 12 days after onset of symptoms and 12 healthy controls were analyzed. Disease severity was categorized as mild-to-moderate in 17 patients, severe in 16 patients and critical in 26 patients. Plasma SARS-CoV-2 RNAaemia was quantified by droplet digital Crystal Digital PCR™ next-generation technology (Stilla Technologies, Villejuif, France).
Results. Overall, SARS-CoV-2 RNAaemia was detected in 43 (74.1%) patients. Prevalence of positive SARS-CoV-2 RNAaemia correlated with disease severity, ranging from 53% in mild-to-moderate patients to 88% in critically ill patients (p=0.036). Levels of SARS-CoV-2 RNAaemia were associated with severity (p=0.035). Among nine patients who experienced clinical deterioration during follow-up, eight had positive SARS-CoV-2 RNAaemia at baseline while only one critical patient with undetectable SARS-CoV-2 RNAaemia at the time of analysis died at day 27.
Conclusion. SARS-CoV-2 RNAaemia measured by droplet-based digital PCR constitutes a promising prognosis biomarker in COVID-19 patients.

The fungus Candida albicans on a chip C. Villard

Candida albicans is an opportunistic human pathogen of the gastrointestinal tract.  Its pathogenicity under specific conditions, such as the immune deficiency of the host, involves a transition from the yeast form to the invasive filamentous (hyphal) phenotype.

We have focused during the last years on the growth modalities of individual C. Albicans hyphae. This work relies on the development of dedicated microfluidic devices allowing yeast positioning, hyphal guidance and 3D confinement, and more generally the control of environmental parameters. I will mainly focus in this talk on the reversible transitions between straight, sinusoidal and helicoidal growth of C. Albicans hyphae as a function of adhesion and confinement. 

Mechanism and impact of bacterial confinement during Neisseria meningitidis vascular colonization - D. Bonazzi

Neisseria meningitidis (Nm) is a human specific pathogenic bacterium. The process of infection depends on the ability of bacteria to adhere to human endothelial cells and proliferate inside blood vessels in the form of multicellular aggregates, leading to vascular damage. Recent work unraveled the unique viscous liquid behavior of these aggregates, that allows adaptation to the geometry of the capillary network and efficient bacterial colonization of the vascular space, therefore being a key element of disease progression (Bonazzi et al, Cell, 2018). However, how bacteria react once they fill up the entire vessel lumen and get confined remains unknown. In particular, we are interested in investigating whether bacterial growth in a confined space leads to the build up of large mechanical pressure and their impact on vessel function (Delarue et al, Nat Phys, 2016). Here, we  combine novel microfluidic systems, genetic tools and live cell imaging to investigate the biological and physical consequences of mechanical constraints on bacterial aggregates and their potential role in the context of infection. 

Cells-in-Drops: microbes, parasites and artificial cells - J.C. Baret

Single cells are essential tools in microfluidic screening technologies. They are widely used as a direct mean to link genotype and phenotype, this link being required to process simultaneously functional molecules (typically enzymes) and information-bearing molecules (typically DNA). In return, the microfluidic technology brings key capacities to analyze single cells quantitatively and dissect functional and regulatory mechanisms at the single cell level. We will discuss experimental approaches coupling cell manipulation (bacteria, yeast, parasites) and droplet-based microfluidic technologies and we will open the discussion on the potential of microfluidics for the de novo construction of artificial cells, a promising avenue for bio-micro-technologies.

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