Members

Principal investigator : Dr Marie Archambault

Telephone :   (450) 773-8521, office 8679 or laboratory 0185

Fax :            (450) 778-8108

E-mail : This email address is being protected from spambots. You need JavaScript enabled to view it.

Ph.D. Students :

• Audrey Charlebois
  Codirection : Dr Mario Jacques
  Since January 2010
• Cindy-Love Tremblay
  Codirection :  Drs Ann Letellier and Jérôme del Castillo
  Transfer to Ph.D. since May 2006

M.Sc Students :

• Michael Beaudry Ferland
  Codirection : Drs Ann Letellier and François Malouin (U. of Sherbrooke) 
  Since September 2008
• F. Carl Uhland
  Codirection : Drs Josée Harel and Patrick Boerlin (U. of Guelph)
  Since September 2008
• Cindy-Love Tremblay
  Codirection :  Drs Ann Letellier and Jérôme del Castillo
  January 2005 to April 2006 (passage direct au Ph.D.)

Financing :

• Ministère de l’Agriculture et des Pêcheries du Québec (MAPAQ)
• Poultry Industry Council (PIC)
• Fédération des producteurs de porcs du Québec (FPPQ)

Bacterial antibioresistance (english version coming soon):

L’arrivée des antibiotiques a été un fait médical marquant de notre siècle due à leur potentiel de guérison des infections bactériennes. Par contre, les cellules bactériennes ont su développer la capacité à se défendre contre un antibiotique, on dit qu'elles acquièrent alors une résistance aux antibiotiques. Au fil du temps, les bactéries pathogènes, soit les bactéries qui causent des maladies, et potentiellement les bactéries de la flore normale, sont devenues résistantes à de nombreux antibiotiques en raison de leur surutilisation ou de leur utilisation inadéquate. La résistance se caractérise principalement par rapport à une souche bactérienne. On dit qu’une souche bactérienne est résistante à un antibiotique lorsqu’elle est capable de se développer en présence d’une concentration élevée de cet antibiotique. La résistance bactérienne menace maintenant notre capacité à traiter les infections bactériennes.

La résistance s'expliquerait principalement par le fait que les gènes bactériens de résistance ont permis aux bactéries de modifier la cible visée par l'antibiotique afin de rendre celle-ci méconnaissable. De plus, les bactéries ont développé la capacité d’expulser l'antibiotique hors de la cellule bactérienne (Manual of Clinical Microbiol. 2004). De plus, les gènes bactériens de résistance peuvent être transférés entre plusieurs bactéries. La transmission de ces gènes via l’eau, la chaîne alimentaire, l’environnement et les zoonoses inquiète la communauté médicale et scientifique ainsi que le publique en général.

Le problème de l’antibiorésistance est mondial. Des bactéries résistantes se retrouvent dans tous les pays et leur profil de résistance dépend grandement de leur exposition aux antibiotiques. Certains scientifiques croient qu’il n’existe aucun moyen efficace de bloquer la résistance aux antibiotiques. Il n’existe pas de vaccin contre la résistance et les alternatives aux antibiotiques sont peu connus donc peu utilisés. On parle alors de probiotiques, de bactériophages, d’acidifiants alimentaire. Voilà pourquoi l’Organisation Mondiale de la Santé et plusieurs autres organismes pronent une utilisation judicieuse des antibiotiques tant en médecine humaine qu’en médecine vétérinaire. La résistance bactérienne pourrait être diminuée en réduisant la pression de sélection de l’antibiotique. Cette pression pourrait être réduite en arrêtant l’utilisation d’un antibiotique pour éviter sa surutilisation ou son utilisation inadéquate. Celle-ci pourrait être également réduite en favorisant une utilisation adéquate (dosage et durée du traitement) des antibiotiques.

Les politiques d’usage des antibiotiques en médecine humaine et vétérinaire de plusieurs pays deviennent progressivement plus restrictives. Le Danemark, la Suède et l’Australie sont particulièrement pro-actifs en matière de réglementation des antibiotiques en médecine vétérinaire. L’usage des antibiotiques en médecine vétérinaire comme promoteur de croissance n’est plus permis dans ces pays. Les impacts de ces décisions sont rapportés dans un de nos travaux afin de comprendre les effets que pourraient avoir une telle réglementation au Québec. Une de nos étude discute des impacts observés par ces pays sur la santé publique, sur l’industrie animale porcine, aviaire et bovine suite à l’arrêt de l’utilisation des antibiotiques comme promoteurs de croissance.

Un premier projet du laboratoire est donc la caractérisation de la résistance antimicrobienne des Enterococcus spp. aviaires et porcins au Québec. Pour ce projet le laboratoire met tout d’abord au point un système d’étude de détection des gènes de la résistance antimicrobienne par hybridation sur colonies, concentration minimale inhibitrice et PCR afin de :

· Caractériser les profils de résistance phénotypiques et génotypiques des Enterococcus feacalis et Enterococcus feacium de poulets, de dindes et de porcs au Québec

· Étudier la présence ou l’absence des intégrons chez ces bactéries

· Rechercher les gènes de résistance au chloramphénicole, à la gentamicine, kanamycine, avilamycine, bacitracine, macrolide-lincosamide-streptogramine B, quinupristine-dalfopristine, pénicilline, vancomycine et tétracyclines chez ces bactéries résistantes

Un deuxième projet du laboratoire sera de développer des alternatives aux antibiotiques en médecine vétérinaire.

Alternatives to the use of antibiotics:

Consumer expectations have changed in recent years in respect to residues and drug free products. The consumer is also aware of the potential risk of establishment of antibiotic resistance in animal and human population. We believe the development of new alternatives to antibiotics in poultry medicine is the way to meet new purchaser and consumer expectations and to enhance their confidence. On the other hand, the incidence of Clostridium perfringens -associated necrotic enteritis in poultry has increased in countries that stopped using antibiotic growth promoters (1). One of our research is therefore necessary and timely because little time is left to build a strategy for alternatives to antibiotics to prevent and control C. perfringens necrotic enteritis in Canada since most antimicrobial growth promoters, controlling the disease until now, may likely be banned in a near future.

C. perfringens in poultry constitutes also a risk for transmission to humans through the food chain (2). The surveillance reports of the Centers for Disease Control and Prevention ranked C. perfringens as one of the most common causes of food-borne disease in the United States (3). Experimental model reproducing the disease have been developed but few tools and strategies are available for prevention and control of C. perfringens in poultry. Vaccination against the pathogen and the use of probiotic and prebiotic products has been suggested, but are not available for practical use in the field at the present time (2). Bacteriophages (phages for short) are viruses that infect bacteria. They are genes wrapped in a protective coat of protein and cannot multiply outside a bacterial cell because they are not alive. If a phage encounters a bacterial cell that is sensitive to it, the phage will enter the bacteria and more phages will be made which kills the bacterium, releasing more phages. The surrounding bacteria are infected as phages spread. It is therefore important to find a phage that is as disease specific as possible for control and prevention purposes. Althought the existence of bacteriophages infecting C. perfringens has been reported, it is surprising to see that little is known on Clostridium phages and that they seem to be very poorly characterized (4,5). A German team has recently described a murein hydrolase from bacteriophage 3626 which lysed all tested C. perfringens stains used in their study, but not other Clostridia and bacteria belonging to other genera (4,5). In the light of this work, Van Immerseel et al (2004) recently suggested that bacteriophage active against C. perfringens isolates could possibly be used for controlling C. perfringens infection in poultry and stated that their usage in vivo needed to be investigated since it has not been done so far. Therefore the first objective of this proposal is to validate the in vitro efficacy of bacteriophage 3626 on C. perfringens isolates from the intestinal tract of Quebec turkeys and chickens. Our second objective is to evaluate its possible use for in vivo control of C. perfringens in an experimental chicken model based on the one described by Hofacre et al. (6,7)

References: 1. Kaldhusdal and Lovland, 2000. World Poultry 16: 50-51; 2. Van Immerseel et al. 2004. Avian Pathology 33:537-549; 3. Olsen et al. 2000. Morb.Mortal.Wkly. Rep. 49:1-62; 4. Zimmer et al. 2002. Appl. Environ. Microbiol. 68: 5311-5317; 5. Zimmer et al. 2002. J. Bacteriol. 184: 4359-4368; 6. Hofacre et al. 1998. Avian Diseases 42:579-584; 7. Hofacre et al. 2003. J. Appl. Poultry Research 12: 60-64

Information on how to provide residues and drug free products in Canada is required to keep our industry competitive. Necrotic enteritis is a common disease found in all poultry areas of the world. It has been estimated that the cost of subclinical necrotic enteritis can be as much as $0.05 per bird and that the disease may be most economically important because it has been shown to impair conversion in broilers. So far, preventive strategies have been antibiotics growth promotants. Many countries have observed a near epidemic proportions of necrotic enteritis (25 to 40%) in broiler flocks after the ban of antimicrobial growth promoters. Clinical and subclinical necrotic enteritis can have a significant economic impact in broiler chickens. This impact is especially important when antibiotic feed additives are not used. Therefore, new methods of prevention of necrotic enteritis must be investigated. It is likely that C. perfringens necrotic enteritis will emerge in broiler chickens if antimicrobial growth promoters were banned in Canada. At the present time, Canada has scientific and economic disadvantages in bacteriophages knowledge as alternatives to antibiotics for C. perfringens prevention and control. This innovative applied research proposal may provide a new tool to help the industry in the control and prevention of necrotic enteritis.

Bacterial culture collection, media and growth conditions: Fecal samples from the intestinal tract of chickens and turkeys from randomly selected Quebec abattoirs were obtained through another research project. A total of 250 C. perfringens isolates will be recovered from these fecal samples. These isolates will be grown at 37°C on phenylethylalcohol (PEA) agar in anaerobic jars using the Anaerocult A system (Merck). The isolates will also be typed by a multiplex PCR described by Songer et al. (1).

Isolation and purification of bacteriophage 3626: Bacteriophage 3626 will be isolated from C. perfringens ATCC 3626 as previously described by Zimmer et al (2,3). In this method, C. perfringens ATCC 3626 will be screened for lysogeny by UV irradiation. Briefly, exponentially growing cells will be exposed to UV light for 5 min. After 3 h of incubation at 37°C in the dark, cultures will be centrifuged and supernatants will be cleared by filtration. Phage activity will be tested by the spot-on-the-lawn method against all 250 C. perfringens strains. The soft-agar technique (2) will be used for the purification and propagation of the bacteriophage. Dilutions of the supernatants displaying lytic activity will be added to molten soft agar inoculated with a log-phase culture of the propagation strain. The mixture will be poured on PEA plates and incubated overnight. Single plaques will be picked and placed into tryptone-yeast extract (TY) medium. After 4 h of incubation at 4°C, the phage-containing solution will be filter sterilized and used for a second round of purification.

Determination of the lytic activity of the bacteriophage 3626: A total of 250 C. perfringens isolates will be tested for sensitivity to the bacteriophage 3626 by two methods. Briefly, cultures will be grown to the early stationary growth phase, 16 to 20h, and cells will be harvested from 1-ml aliquots by centrifugation and resuspended in phosphate-buffered saline to an optical sendity of approximately 1.0. Cell suspensions will be used directly as substrates for the lysis assays and will be mixed with crude bacteriophage 3626 in sterile, uncoated polystyrene microplates. The subsequent decrease in optical density will be monitored with a plate reader at 37°C. The ability of the phage to lyse C. perfringens isolates will be also tested by the drop-on-the-lawn-technique where ten microliters of the prepared phage stock (approx. 10 to 7 PFU/ml) will be placed on the plates inoculated with C. perfringens isolates. The lytic activity will be observed after overnight incubation and is identified by the holes or plaques it forms in layers of sensitive bacteria.

Propagation and purification of 3626 for high-titer stocks of bacteriophages: For high-titer stocks (>10 to 9 PFU/ml), liquid cultures will be used. Cultures will be infected at an optical density at 600 nm of 0.1 at a multiplicity of infection of 1. Afterwards, growth will be monitored photometrically, and following lysis, phages will be harvested by centrifigation and sterile filtration of the culture supernatant. Purification of viruses from high-titer stocks has been described earlier (4). Briefly, phages will be concentrated by polyethylene glycol 8000 precipitation, stepped CsCL density gradient centrifugation, and dialysis (5).

Chicken challenge model: Our model will be based on the ones described by Hofacre et al (6) and by George et al (7). The treatment groups will be 1) nonchallenged control; 2) bacitracin methylene disalicylate, 50g/ton; 3) high-titer stocks of bacteriophage 3626 (>10 to 9 PFU/ml); 4) C. perfringens challenge control group. The treatments will be added to the feed during the entire study at inclusion rates indicated above. Preliminary experiments will be performed to determine optimal water concentration of bacteriohpages. The study will use 40 male broiler chickens that will be housed d 0 to 28 in starting batteries and then in growing batteries. Chicks in all groups will be identified by cage number and will be given water and a cornsoybean meal- and fishmeal-based feed ad libitum to favor the pathogenesis of the infection. The model will consist of one oral inoculation with Eimeria acervulina and Eimeria maxima at 15 d of age followed by serial oral inoculation with C. perfringens (approx. 10 to 8 cfu/bird each day) at 18, 19 and 20 d. The basal diet offered from 0 to 16 d will contain 26% fishmeal. Feed consumption will be measured from 0 to 15 d and 15 to 28 d. All birds will be weighed by cage at 0, 15 and 28 d. Necropsies of all mortalities from 18 d will be conducted to determine the cause of death. At 22d, two birds from each cage will be randomly selected, killed, weighed, and scored for the level of necrotic enteritis intestinal lesions (0= none, 1= mild, 2=moderate, 3=marked/severe). All remaining chicks will be killed, weighed, and scored for lesions at 28 d. The treatments will be analyzed by conventional ANOVA. significance will be determined using the post hoc test of least significance differences (LSD with p<0.05).

Ref: 1. Songer et al. 1996. Clin Microbiol Rev 9:216-234; 2. Zimmer et al. 2002. Appl. Environ. Microbiol. 68: 5311-5317; 3. Zimmer et al. 2002. J. Bacteriol. 184: 4359-4368; 4. Zink et al. 1992. Appl. Environ. Microbiol. 58:296-302; 5. Sambrook et al. 1989. Molecular cloning, Cold Spring Harbor, NY; 6. Hofacre et al. 2003. J. Appl. Poultry Research 12: 60-64; 7. George et al. 1982. Poult. Sci. 61:447-450

This research will benefit our Canadian poultry industry by providing a new alternative to antibiotics in poultry medicine for necrotic enteritis. We expect that bacteriophages will be a new strategy that will effectively reduce or replace the need for antibiotics to control and prevent necrotic enteritis. Our innovative proposal will provide data on the efficacy and practicality of this alternative to antibiotics. Our work may likely lead to the development of new vaccines that may increase bird's resistance to necrotic enteritis. It may also be included in new management techniques and strategies used by the industry. Our research will contribute to meet new consumer expectations of antibiotics-free food by reducing antimicrobial residues in the end product. This will likely improve consumer confidence with respect to value added poultry products. This directly benefits the poultry industry in Canada and may increase profitability. It may also open the door to bacteriophages treatment for other diseases in our Canadian poultry industry. Finally, our research results will also benefit the animal and human population as well as the environment at large by reducing the risk of establishment of antibiotic resistance.