Christian Laflamme

Assessment of bacterial endospore viability with fluorescent dyes.

Aim:  To validate three fluorescence viability assays designed primarily for vegetative cells on pure Bacillus endospores.

Detection of Legionella spp. by fluorescent in situ hybridization in dental unit waterlines

Aims:  To confirm the presence of viable Legionella spp. in dental unit waterlines (DUWL) using fluorescent in situ hybridization (FISH) and compare this method with culture approach and also to validate the utility of an enrichment to increase FISH sensitivity.

Evaluation of the plasmid copy number in B. cereus spores, during germination, bacterial growth and sporulation using real-time PCR.

Only a small number of studies have measured the plasmid copy number (PCN) variation during bacterial growth. Besides, information about the PCN in spores is still rare. In this work, we utilized a real-time PCR assay to evaluate the PCN of four different plasmids in Bacillus cereus. The PCN was measured in spores as well as during germination, active bacterial growth, and sporulation. Plasmid stability was also evaluated to ensure that plasmid loss does not affect the accuracy of the PCN measurement. We demonstrated that the PCN of low and high copy number plasmids varies with growth phase as well as culture media over B. cereus life cycle. The PCN was minimum during the germination and maximum during the stationary growth phase for all plasmids tested. We also demonstrated that the use of antibiotic in the culture media is not enough to ensure stable inheritance in spores of plasmids carrying antibiotic resistance genes. Moreover, we revealed that the PCN in spores is related to the PCN during endospores formation. Therefore, the plasmid partitioning during sporulation is not influenced by the unequal-size of the forespores and the mother cells, even for a plasmid distributed randomly.

Permeabilization and hybridization protocols for rapid detection of Bacillus spores using fluorescence in situ hybridization.

BACKGROUND Fluorescence in situ hybridization (FISH) is not adapted for the detection of bacterial spores because of their resistance to conventional permeabilization treatments. Since spore-forming bacteria have important ecological, economical, and medical roles, their in situ detection needs to be improved. The aim of this study was to develop rapid and effective protocols to permeabilize Bacillus spores in order to apply the FISH technique. METHODS Permeabilization protocols were developed for three species of Bacillus spores. Hybridization was performed using universal and specific probes. Surface structural analysis of the permeabilization treatments was performed using scanning electron microscopy. RESULTS With the proposed protocols, Bacillus spores can be labeled in less than 1 h. The scanning microscopy showed some visible structural differences between the permeabilized spores compared to intact ones. CONCLUSION For the first time, rapid and effective protocols to detect Bacillus spores by FISH were developed and can be applied to study Bacillus spores using in situ labeling within 1 h. Previously published in situ hybridization protocols have never reached or been close to the currently described rapidity. This work will contribute to the possibility of near real time detection of biological threats that may be present as spores.

Flow Cytometry Sorting Protocol of Bacillus Spore Using Ultraviolet Laser and Autofluorescence as Main Sorting Criterion

The ultraviolet (UV) Fluorescent Aerodynamic Particle Sizer (FLAPS), a flow cytometer-like apparatus was developed by the Canadian Department of National Defence for real-time detection of autofluorescence of biological aerosol particles such as bacterial spores. The direct relation between autofluorescence intensity and viability has recently been reported and viable spore are more autofluorescent in UV (Laflamme, Frontiers in Bioscience). The goal of this manuscript is to describe a flow cytometry sorting protocol based on UV-induced autofluorescence. An EPICS® ELITE ESP flow cytometer equipped with a UV laser and cell sorter was used to mimic the optical properties of FLAPS and to study the two extremes of a spore population according to its autofluorescence (lower level of autofluorescence (LLA) and higher level of autofluorescence (HLA) spores). Bacillus subtilis var niger was used as a surrogate for Bacillus anthracis spores and sorted using autofluorescence intensity as the main criterion. The protocol developed in our laboratory to sort Bacillus spores according to their autofluorescence properties is described. Purity of each sorted population was greater than 95%. Using autofluorescence as the main criterion, we demonstrate that it is possible to separate two distinct spore populations.

In situ detection of antibiotic-resistance elements in single Bacillus cereus spores

Rapid detection of Bacillus spores is a challenging task in food and defense industries. In situ labeling of spores would be advantageous for detection by automated systems based on single-cell analysis. Determination of antibiotic-resistance genes in bacterial spores using in situ labeling has never been developed. Most of the in situ detection schemes employ techniques such as fluorescence in situ hybridization (FISH) that target the naturally amplified ribosomal RNA (rRNA). However, the majority of antibiotic-resistance genes has a plasmidic or chromosomal origin and is present in low copy numbers in the cell. The main challenge in the development of low-target in situ detection in spores is the permeabilization procedure and the signal amplification required for detection. This study presents permeabilization and in situ signal amplification protocols, using Bacillus cereus spores as a model, in order to detect antibiotic-resistance genes. The permeabilization protocol was designed based on the different layers of the Bacillus spore. Catalyzed reporter deposition (CARD)-FISH and in situ polymerase chain reaction (PCR) were used as signal amplification techniques. B. cereus was transformed with the high copy number pC194 and low copy number pMTL500Eres plasmids in order to induce resistance to chloramphenicol and erythromycin, respectively. In addition, a rifampicin-resistant B. cereus strain, conferred by a single-nucleotide polymorphism (SNP) in the chromosome, was used. Using CARD-FISH, only the high copy number plasmid pC194 was detected. On the other hand, in situ PCR gave positive results in all tested instances. This study demonstrated that it was feasible to detect antibiotic-resistance genes in Bacillus spores using in situ techniques. In addition, in situ PCR has been shown to be more sensitive and more applicable than CARD-FISH in detecting low copy targets.