Smaltis’ microbiology platform has the tools to characterize and quantify microorganisms, whether they are bacteria, yeasts or molds. We work with isolated strains, nucleic acid solutions or more complex matrices such as stool, biopsies, soil or surface samples.
Among the many methods available to characterize and screen microbial organisms, here are examples of tools we use that can be employed under aerobic or anaerobic conditions.
Accurate identification of microorganisms, whether bacteria, yeast or molds, is an essential prerequisite for many analyses. Smaltis offers identification solutions through different molecular biology techniques, the methods used being dependent on the germs to be identified.
MALDI-TOF de Bruker Mass Spectrometry
The mass spectrometry is an analytical method allowing the identification of the gender and the species of microorganisms in just a few minutes. This fast method, dependent on databases, is well adapted to the identification of most bacteria responsible for infections in humans or animals.
Sanger-Sequencing is useful for the accurate identification of unknown and/or non-cultivable strains for which mass spectrometry is not adapted (e.g. Yeast and Mold strains). Depending on the microorganism, we target species-specific genes that we amplify by PCR and sequence. The identification is then performed by comparing the targeted sequences with international databases.
Next-Generation Sequencing (NGS)
This method can be used either from an isolated microorganism or from a microbiome.
For an isolated microorganism, the technology used (whole genome sequencing) will consist in sequencing the whole genome allowing both the identification of the species and obtaining its genetic identity card.
For a microbiota (all microorganisms populating a microbiome), the technique used will consist in sequencing all the alleles coding for the 16S ribosomal subunits (16S RNA), the best known genetic markers for identifying and classifying bacterial genera. For molds, the sequencing focuses on the ITS1 and ITS2 regions. In addition, for species identification, sequencing can be performed on the entire microbial genome, using the shotgun technique.
Antibiotic resistance profile
Thanks to its expertise in the evaluation of the bacterial resistance phenotype to antibiotics, Smaltis can determine the resistance profile of strains of interest.
After the identification, the accurate characterisation of micro-organisms is another essential step. Indeed, the features of the strains used in your projects will determine the results and their interpretation. To better understand the strains you study, Smaltis proposes to determine their phenotypic characteristics (e.g. Morphological, Physiological, Metabolic).
Determination of Morphological caracteristics
The study of a micro-organism’s morphology allows to check the purity of a strain. It is based on the study of colonies and cells’ morphology.
Smaltis accompanies you in:
– The morphological study of colonies, a macroscopic study (bare eye) allowing to observe the colony’s characteristics namely:
. The shape (round, whole, wavy, thready, …)
. The size
. The colour
. The aspect (sticky, thread…)
. The smell
– The morphological study of cells, a microscopic study that can be performed in the fresh state or after cell staining the cells. It allows to observe:
. The cell form (spherical (cocci), cylindrical (bacillus), spiral (spirilla), coiled (spirochete) or filamentous)
. The gathering mode (in chain like Streptococcus, in group like Staphylococcus…)
. The size
. The presence of spore
. The motility (ability to move), by swimming, swarming or twitching
Determination of Physiological and Metabolic characteristics
Because each micro-organism has its own biochemical characteristics, Smaltis offers you to identify them for each of species by:
. Culture on specific growth media
. The performance of various biochemical tests (e.g. API galleries)
. The realisation of growth curves according to different parameters (temperature, oxygen, pH)
. The assessment of their propensity to make biofilm in a microplate
These services can be done with yeast and/or mold strains.
Molecular genotyping is the method used for epidemiological monitoring of pathogenic microorganisms, the production quality controls or the identification of contamination sources. Thanks to various methods, Smaltis enables you to determine the genotype of your strains and thus to verify their clonality (to verify whether or not you are dealing with the same strain).
Genotyping by MLVA (Multi-Locus Variable number tandem repeat Analysis) is based upon the amplification by PCR (polymerase chain reaction) of various VNTRs (Variable Number of Tandem Repeats) scattered on the bacterial genome, using specific primers. The determination of the molecular size of different amplicons by electrophoresis allows to identify the number of repetitions at a specific locus. By this manner, these results reflect the number of repeated units in the amplified region as the length of repeated units is known. In some species, this method significantly increases the level of bacterial genotyping. Typing results in a numeric code, which includes the number of patterns at each locus.
Smaltis can also offer you the sequencing of these VNTRs, which is called MLST (Multiple Locus Sequence Typing) genotyping.
Pulsed Field Gel Electrophoresis (PFGE)
Genotyping by PFGE (Pulsed Field Gel Electrophoresis) allows to analyze the macrorestriction profiles of total DNA by pulsed field gel electrophoresis. The result is a migration profile, which defines a pulsotype characteristic of a bacterial isolate. PFGE is a standard method of typing for numerous bacterial species due to its highly discriminating potential.
Virulence factors measurement
The study of the activity and/or production of virulence factors is a key element enabling to understand bacterial pathogenicity. In this context, Smaltis offers you to characterize your strains by:
– Studying the activity of structural virulence factors, and more specifically the activity of determinants involved in bacterial adhesion and motility. These determinants, such as pili or flagella, may be observed in specific environments.
– Studying the activity and/or production of secreted factors such as toxins, siderophores or enzymes involved in host colonization or infection. These secreted factors may be either enhanced through appropriate agar media, or measured out in accordance with adapted methods.
Assessment of cytotoxicity
Cytotoxicity assessment consists in measuring the ability of a bacterium to kill cells. This quantification can be performed on different eukaryotic cell lines.
On red blood cells in particular, this evaluation allows to analyze the hemolytic capacity of a bacterium.
The death of red blood cells can be measured by the release of hemoglobin, using the Red Blood Cell Lysis technique, which consists of bringing red blood cells into contact with bacteria, and quantifying the released hemoglobin by spectrophotometric assay. This determination can be done in end-point, i.e. with a measurement at a defined time, or in kinetic, allowing measurements at different times.
On the other hand, the evaluation of the hemolytic capacity of bacteria can be done by culturing the strains on blood agar plates. In case of hemolytic activity, the bacteria will form colonies surrounded by a clear zone, as the lysis of red blood cells makes the medium transparent. This transparency is complete if hemolysis is complete. In case of partial hemolysis, a slight haze is observed.
For other cell types, cell death can be quantified by measuring the Lactate Dehydrogenase (LDH) released into the culture medium after cell destruction, or by measuring MTT cleavage.