research

CURRENT POSITION

Since July 2000, I started my new position as a Science and Technology Agency (STA) Research Fellow at the National Grassland Research Institute, Japan, working on the identification and physiology of lactic acid bacteria. Currently I'm working on the genetic identification of lactobacilli displaying interesting fermentation and anti-microbial properties.
When I have time I'll update this page with details of that, but you can get a good overview of our research at our lab pages.
For information about bacteriocins and lactic acid bacteria on the web, check out my links section.

PREVIOUS POSITION

From Fev 1998 to Jan 2000, I was appointed research fellow in Kyushu University, with a funding from the Japan Society for the Promotion of Science (JSPS) fellowship program. My project was entitled "Bacteriocin purification by Enterococcus faecium, structure-activity and genetic studies". The project has quite a broad range, but can be divided into sub-projects which were the particular focus of my research during the 2 years I spent in Kyushu:

Purification and sequencing of enterocins:
Purification of enterocins was carried out using ammonium sulfate precipitation, desalting on ODP-90 reverse-phase column, and purification through SP Sepharose HP cation exchange and C2/C18 reverse-phase chromatographies. The antimicrobial was eluted from the C2/C18 column as four individually active fractions, designated A81, B81, C81 and D81. The purification procedure used proved particularly efficient for the bacteriocin in fraction D81, with a yield of 46%, while only 3.8% the bacteriocin in fraction B81 could be collected. MALDI-TOF mass spectrometry of the bacteriocins in fractions B81 and D81 showed respective masses of 4,833.0 and 5,462.2 Da. Amino acid sequencing of the two peptides revealed two class-II bacteriocins whose sequences were similar to those of enterocin A and enterocin B, respectively.

Cloning and sequencing of the structural genes:
Using proper primers, chromosomal fragments of 212 bp and 216 bp enclosing bacteriocin structural genes were PCR-amplified. Cloning of the amplicons and their sequencing revealed two genes with sequences identical to the structural genes of enterocins A and B, respectively.

Transcriptional regulation of bateriocin genes:
The sequencing of fragments enclosing the bacteriocin operons for enterocins A and B, revealed no difference with with those previously published by other authors.

It was clearly established that E. faecium WHE 81 produces bacteriocins respectively identical to enterocins A and B. Our results, combined with data from previous reports, suggest that the two bacteriocins may be widespread among enterococcal strains and may play an important role in controlling the growth of pathogens and other undesirable bacteria in certain fermented food products.

POSITION AT THE INRA

In June 1997, I obtained a fellowship for a post-doctoral position at INRA (National Institute for Agronomic Research) in France, where I worked on anti-Listeria activity of Enterococcus strains and class II bacteriocins. One of the outcomes of my research in this laboratory was the establishing of the first evidence that all Listeria strains show the same relative sensitivities to all class IIa bacteriocins: resistant strains to one class IIa bacteriocin could resist to all class IIa bacteriocins.

Thirty one Listeria strains were tested for sensitivity to four class IIa bacteriocins, namely enterocin A, mesentericin Y105, divercin V41, and pediocin AcH, and to nisin A. Class IIa bacteriocins displayed surprisingly similar antimicrobial patterns ranging from highly susceptible to fully resistant strains, whereas nisin A showed a different pattern where all Listeria strains were inhibited. Particularly, it was observed that the strain Listeria monocytogenes V7 could not be inhibited by any of the class IIa bacteriocins tested. These observations suggest that Listeria strains resistant to the whole range of class IIa bacteriocins may occur in natural environments, which could be of great concern with regard to the use of these peptides as food preservatives.

RESEARCH AT THE ULP

My research on bacteriocins started early 1992 when, as a first year's Ph.D. student, I was asked to explore this means in trying to reduce Listeria counts in cheese. My Ph.D. thesis was in fact entitled "Use of bacteriocin-mediated bacterial antagonism to eliminate Listeria monocytogenes in soft cheeses".
Until that time, I had worked on food irradiation (Masters study), as had all my colleagues of the my colleagues of the Department of Food Science (L. Pasteur University, Strasbourg, France). Therefore, being amongst "the pioneers" in France, I had to initiate an independent research, which started by developing experimental protocols for the search of bacteriocin producing lactic acid bacteria, since indeed I did not have the required bacteria. In addition, since the study was aimed at industrial application, I had to establish an active research program and work within a well-defined project management framework. This involvement in an industry project was an opportunity to demonstrate my ability to take my research to the application level, and to understand of the link between the academe, the industry and the consumer.
My research work dealt with the investigation of new antimicrobial means for the elimination of Listeria monocytogenes in dairy products. This consisted in the detection of lactic acid bacteria capable of producing antibacterial peptides (bacteriocins). Also, the biosynthesis of these bacteriocins was studied in bioreactors, some were purified and their structure and genetic determinants described. After conditions for optimal activity were determined for each peptide, trials were successfully conducted on soft cheeses, but also sausages.
During my final year as Ph.D. student, I was appointed Teaching and Research Associate at L. Pasteur University, a position I held for another year after completing my Ph.D. From September 1996, I also held a research associate position at the same university for nearly one year. These positions allowed me to demonstrate a potential for teaching undergraduate and graduate students, and to continue my research work on bacteriocins.
Many more details can be found at my thesis page.

SIGNIFICANT FINDINGS

Isolation of six bacteriocin producing strains from soft cheese.

Isolation of two bacteriocin producing strains from silage.

Purification and sequencing of pediocin AcH, nisin A, enterocin A and enterocin B produced by cheese-isolated lactic acid bacteria.

First evidence of natural heterologous expression of bacteriocins from lactic acid bacteria: natural expression of pediocin AcH in Lactobacillus plantarum.

A new and patented strategy for the use of bacteriocin-mediated bacterial antagonism to eliminate Listeria monocytogenes in soft cheeses.

Successful introduction of pediocin-AcH producing L. plantarum WHE92 (DSM 9296) in fighting L. monocytogenes in several cheese manufactures in France and Europe (Commercial designation: ALC01, Wiesby).

Elimination of L. monocytogenes in soft cheese using cultures of Enterococcus faecium producing enterocins A and B.

First evidence that all Listeria strains show the same relative sensitivities to all class IIa bacteriocins: resistant strains to one class IIa bacteriocin could resist to all class IIa bacteriocins.

Description of enterocin B operon in E. faecium (not yet published).

Description of a third bacteriocin in E. faecium already producing enterocins A and B (not yet published).

MY VIEW

I had three enriching experiences in various research teams and environments which further developed my scientific and interpersonal skills. During these years, I gained experience, not only in the design and execution of experiments, but also in writing, oral and computer skills, which helped me composing various scientific manuscripts, tabulating and critically analyzing scientific data, as well as preparing well-structured presentations for scientific symposia and meetings. These skills also enabled me to independenty write scientific articles, thereby developing an emerging record of peer-reviewed publications. What I consider to be a major achievement is the publication, in FEMS Microbiol. Rev., of the first comprehensive review on class IIa bacteriocins.