Clostridium Difficile Associated Infection
Bacteriocins and Lytic Bacteriophages as a Potential Alternative for Antibiotics in Treating C.Difficile-Associated Nosocomial Infection
Tech Area / Field
3 Approved without Funding
State Research Center for Applied Microbiology and Biotechnology, Russia, Moscow reg., Obolensk
- McGill University / Sir Mortimer B. Davis Jewish General Hospital, Canada, QC, Montreal
Project summaryThe objective of the project is to find bacteriocins and lytic bacteriophages active toward highly virulent epidemically important strains of Clostridium difficile, including antibiotic-resistant ones, as well as to use them for preventing and treating C.difficile-associated experimentally induced infection.
C.difficile is a Gram-positive spore-forming anaerobic bacillus. It is a main cause of nosocomial diarrhea associated with antibiotic therapy. The spectrum of C. difficile manifestations span from asymptomatic carriers to death. C.difficile-associated disease (CDAD) is primarily manifested by gastrointestinal manifestations ranging from mild diarrhea to toxic megacolon and bowel perforation with associated peritonitis and sepsis.
Over the past 5 years Clostridium difficile-associated diarrhea is being diagnosed not only with increasing frequency but with greater associated morbidity and mortality (Loo et al., 2005). This increase in CDAD cases as well associated case severity has been noticed in Canada, USA, as well as in United Kingdom, Netherlands. It is therefore not surprising that this area has become the focus of intensive investigation by researchers of the United States of America, Canada, Sweden, Hungary, England, France, Japan, the Netherlands and some other countries. In North America the appearance of a more virulent strain is thought to contribute to the extent and severity CDAD. The disease is under research in Russia as well. In Russia the disease is spread both among adults and children. Since the 80s, the number of cases has being impressively increased due to mainly the wide application of antibiotics in medicine, as well as to the occurrence of virulent strains of C.difficile on this background.
The treatment of C.difficile-associated diarrhea relied on two antibiotics, such as vancomycin and metronidazole. Other agents such as rifampin, fucidic acid, teicoplanin have been tried with varieable success. The both, vancomycin and metronidazole, possess bactericidal action against C.difficile. However these agents are not risk free. Metronidazole has been associated with peripheral neuropathy and neutropenia whereas Vancomycin over utilization may enhance the institutional spread of Vancomycin resistant enterococcus. In case of the uncomplicated disease, the early initiation of either of these antibiotics gives usually a good therapeutic result. Patients usually improve over 1-4 days, and recovers fully within two weeks. But these drugs have some disadvantages and limitations. They fail to restore anti-colonization resistance of patient’s intestine after applying antibiotics to treat the main disease. They do not reduce the number of C.difficile spores a patient is releasing, thereby creating new loci of the infection. And, finally, the relapsing disease is observed in 12-24 % of patients within two months after diagnose of the infection and antibiotic therapy. Because of the important failure and relapse rates a variety of approaches have been tried eg extending duration of Vancomycin treatment with or without tapering, IVIG, combining anti CDAD treatments as well as development of new agents and vaccine., Clearly no regimen has emerged as adequately satisfactory.
Therefore, new means of treatment of C.difficile-associated infections are needed. These ideally should replace or enhance existing therapeutic modalities by improving on CDAD treatment outcomes and minimize or eliminate relapses. Furthermore these agents may have a role in the prevention of CDAD in at risk patients.
Ideally, new therapeutics should be highly effective against C.difficile (to inhibit the growth of vegetative cells and spore formation) without disturbing natural microbial community, killing representatives of normal intestinal microbiota, and decreasing resistance of the intestine to colonization of the pathogen. Moreover, they should stimulate the immune response of the body to C.difficile and some other pathogenic microorganisms.
In this context, of particular interest is research aimed at investigating a possibility to use bacteriocins and specific bacteriophages for treating diarrhea, pseudo-membranous colitis and some other pathologies associated with highly virulent strains of C.difficile.
Bacteriocins are low-molecular-weight peptides (~3-6 kDa), which are being produced on a ribosome by different species of microorganisms. They possess a series of remarkable properties. They are active both toward antibiotic susceptible and resistant Gram-positive and Gram-negative pathogens. Their activities are comparable with those of antibiotics (and are even superior to them in some cases). They do not accumulate in the body and stimulate the immune system, etc.
Between 2001 and 2006, researchers from the State Research Center for Applied Microbiology &Biotechnology, authors of the present project proposal, in conjunction with American scientists have isolated and chemically purified more than 20 structurally unique low-molecular-weight peptides (mainly of Class IIa). The peptides are being produced by representatives of Lactobacillus, Lactococcus, Enterococcus, Paenibacillus, Bacillus, Pseudomonas, and E.coli. These bacteriocins possess wide spectrum of antimicrobial activity and inhibit in vitro the growth of E.coli, Salmonella spp., Klebsiella spp., Citrobacter spp., Enterobacter spp., Proteus spp., Morganella spp., Y.enterocolitica, P.aeruginosa, Campylobacter spp., Helicobacter pylori, Acinetobacter baumannii, Listeria monocytogenes, Staphylococcus aureus, S.epidermitidis, Streptococcus spp., Clostridium spp., and Mycobacterium tuberculosis. They appeared effective against clinical (isolated from animals and humans) antibiotic-resistant strains of S.aureus, P.aeruginosa, E.coli O157:Н7, Klebsiella pneumoniae, Enterobacter cloaceae, Acinetobacter baumannii, and Campylobacter jejuni.
Another alternative for antibiotics are bacteriophages, bacterial viruses, which enter bacterial cells, break their metabolic processes and, finally, lyse bacteria. In the 30s and 40s bacteriophages were widely used for therapeutic purposes worldwide. With the advent of antibiotics, the application of bacteriophages was cut down, although they were still in use in countries of the former USSR and Eastern. In 1999, Ramesh et al. demonstrated the ability of bacteriophage specific for C. difficile to prevent ileocecitis in a hamster model. Our suggestion to use bacteriophages for treating C.difficile-associated infections is based on data provided by his team, as well as on our own results from the research to apply bacteriophages for treating bacterial infections in animals.
So, we plan to find microbial antagonists producing bacteriocins effective against C.difficile, including its spores. Microorganisms of different systematic groups, including the genus of Clostridium, will be screened to select cultures producing bacteriocins. Methods of isolation and purification of selected bacteriocins will be developed. Selected bacteriocins will be characterized, and lytic phages will be isolated. Formulations of bacteriocins and bacteriophages will be tested using animal models.
Tasks to be pursued:
- Make up a collection of clinical high-virulence strains of C.difficile (isolated both from humans and animals); characterize the strains;
- Iisolate, identify and investigate bacteriocins with anti-C.difficile activity in vitro;
- Select and characterize anti-C.difficile bacteriophages;
- Induce C.difficile- associated infection in laboratory animals;
- Assess therapeutic effects of selected bacteriocins (both with narrow and wide spectra of activity), as well as of bacteriophages on experimentally induced C.difficile-associated infections.
A main result from the implementation of the project will be a basis for further developing novel anti-C.difficile. antimicrobials. That is why procedures of identification, isolation and characterization of new bacteriocins and bacteriophages active toward C.difficile are a key step of the project proposal.
We plan to identify and characterize:
- Bacteriophages lytic for C.difficile (a possibility of use them for preventing and (or) treating C.difficile-infection will be studied);
- Bacteriocins with narrow and wide spectra of activity, which would effective against high-virulence strains of C.difficile, including antibiotic-resistant strains;
- Bacteriocins holding promise from the standpoint of their clinical application.
Additionally, we are going to receive
- Data on the spread of strains of C.difficile among hospitalized patients, as well as among farm animals in Russia; data on gene types of the strains;
- Results from the comparative analysis of susceptibilities of C.difficile cultures to antibiotics and bacteriocins;
- Data on the therapeutic efficacy of bacteriocins against experimentally induced C.difficile infection in laboratory animals.
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