In vitro pharmacoynamic evaluation of anti-infectious agents

The BSL2 lab in addition to the in vivo part includes an in vitro room (MSW 135) where are realized in vitro PK-PD experiments with different bacteria and antibiotics. This in vitro room is equipped with 2 microbiological safety cabinets (Herasafe KS 12 and MS 2020 1.2 Maxisafe, Thermo Scientific, France)  and several incubators to carried out antibiotic resistance assessments in static concentrations of antibiotics: MIC, checkerboard, time-kill curves on different multidrug resistant pathogens species as Pseudomonas aeruginosa, Enterobacterales, Staphylococcus aureus and Mycobacterium abscessus.

Microbiological safety cabinet in in vitro BSL2 lab

Recently, we have developed two In-house Hollow Fiber , this allow to study the in vitro efficacy of antibiotics by mimicking the pharmacokinetic of the antibiotic in a patient. Our system is able to fit with model including up to two antibiotics with different half-lives.

Dedicated incubator for Hollow fiber experiments	Hollow fiber system mounted for two drugs

The room is also equipped with CO₂ incubator (Hera Cell) for in vitro intracellular model of infections (Pseudomonas aeruginosa, Mycobacterium abscessus).

CO2 incubator dedicated to intracellular model of infection

 

The BSL2 lab is now equipped with automated station to prepare and analyze microbiological experimentations with a pipetting robot (Assist Plus, Integra Bioscience) and to plate and count automatically bacterial colonies (Spiral Pro and Scan300, Interscience).

Equipment for automation of microbiological experimentations with a pipetting robot (on the left), a SPIRAL automatic plater (center) and an automatic colony counter (on the right).

A plate reader (Mithras LB940, Berthold) able to measure optical density, fluorescence and luminescence is also fully dedicated to experimentation with pathogens in the BSL2 laboratory.

The lab is also equipped with a molecular biology station to characterize resistance mechanisms in pathogen (even on multi-drug resistant clinical isolates) as enterobacterales, Pseudomonas aeruginosa, Acinetobacter baumannii, Achromobacter xylosoxidans and Mycobacterium abscessus by different methods:

• Genome engineering
• Site-Directed Mutagenesis: Construction of bacterial gene-inactivating plasmids, Construction of mutants (Knock out, Knock In, Mutational insertion even on MultiDrug resistant clinical isolates).
• Construction of reporter gene: It allows to follow expression of a gene of interest by fluorescence or luminescence.
• Two PCR (Polymerase Chain Reaction) machines (Thermocycler, SensOQuest, Germany and a Biorad icycler, France) are now available to amplify DNA fragment needed to construct vectors and to check if bacteria are modified in the genome.

Thermocyclers SensOQuest (on the left) and Bio-Rad icycler (on the right)

• Gel electrophoresis and a system safe illuminator are also available to separate DNA fragments on agarose gel and check the fragment size and to visualize DNA band on ge

Gel electrophoresis system Mupid-One (on the left) and Thermo Safe Illuminator (on the right)

• Quantification of gene expression: Resistance genes expression analysis is fundamental to understand dynamic changes in bacterial population in the contact of antibiotic. We have acquired a Biorad CFX96 Touch to develop the gene expression analysis by RT-qPCR on many genes involved in the resistance development in various species as Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae and Acinetobacter baumannii.

• We have access to Sanger Sequencing (laboratoire de génétique, CHU de Poitiers): Amplification sequencing of DNA fragments allows studying the identification of mutations conferring for example resistance phenotypes during antibiotic exposure.
• Access to WGS/NGS platform: New generation sequencing/Ultra deep sequencing of DNA fragments allows to analyze precisely the microbial communities globally (microbiome) or each respective subpopulation to characterize the emergence of antibiotic resistance or presence of bacterial virulence genes. This platform included three Illumina sequencer systems (MiSeq, NextSeq550 and iSeq100), two Nanopore sequencing systems (MinIon, associated to the MinIT) and powerful bioinformatic resources to analyze generated data.

Illumina Sequencing System Mi Seq	Illumina Sequencing System NextSeq550	Nanopore Minlon connected to MinIT

 

 

The BSL2 and molecular biology labs are running according to quality management rules and were awarded ISO 9001 certification in August 2019 (renewal october 2020).