February 25, 2010 at 4:04 am #2803
More than 90 percent of all infections begin at a mucous membrane site (oral, nasal, upper or lower respiratory, ocular, intestinal or urogenital). The Fischetti lab is working to understand the earliest events that occur when gram-positive bacteria interact with human tissues and cause disease. Its research is aimed at interfering with these events by: developing vaccines to induce a mucosal immune response; blocking the attachment of surface protein in the bacterial cell wall to prevent infection; and using phage lytic enzymes to both remove colonizing pathogenic bacteria to prevent infection and treat established infections.
Dr. Fischetti works with gram-positive bacteria, such as streptococci, that do not contain a second cell membrane outside of the cell wall. In the fight against infectious disease, Dr. Fischetti investigates two nonantibiotic treatment strategies. This two-pronged approach involves blocking bacteria from attaching to cells and exploring the use of phage lytic enzymes to remove pathogenic bacteria once they have colonized in the host.
To infect their host, bacteria use their surface molecules to attach and invade human tissues, particularly those that line the nose and throat. Knowledge of the process bacteria use to anchor these molecules in their cell wall could lead to strategies to prevent infection. The M protein is a surface protein that is the major virulence factor of group A streptococci because of its ability to impede attack by human white blood cells. Analysis of this molecule by Dr. Fischettis lab shows that the region used to attach the M protein to the cell surface is highly conserved in gram-positive bacteria, indicating that the mechanism for anchoring surface proteins in bacteria is also conserved. Since bacteria cannot cause infection without their surface proteins, a molecule that blocks surface protein attachment will be broadly applicable to different gram-positive bacteria.
Dr. Fischettis lab has also shown that the M protein can be used to deliver the molecules to the surface of gram-positive bacteria to be used as a vaccine. A vaccine that employs this approach could be used against a variety of harmful pathogens and is currently being tested in clinical trials. Dr. Fischetti has also identified a membrane-associated enzyme responsible for cleaving the highly conserved anchor region of surface proteins. Inhibition of this enzyme prevents both cell wall assembly and the proper attachment of most surface proteins, resulting in nearly naked bacteria. Studies are under way to further define the role of this enzyme in cell wall assembly and the protein attachment process to identify inhibitors that may be used as a new class of antibiotic.
As new antibiotics are proving futile against resistant strains of bacteria, the Fischetti lab is investigating the efficacy of lytic enzymes, which are found exclusively in viruses called bacteriophages (or phages), viruses that infect bacteria.
Dr. Fischettis lab has recombinantly produced lysins that will kill the major gram-positive pathogens Streptococcus pyogenes, Streptococcus pneumoniae, Staphylococcus aureus, Enterococcus faecalis and Bacillus anthracis and has used these proteins to destroy their respective bacteria in animal models of disease. The enzymes are extremely potent; only very small amounts are needed to destroy millions of organisms within seconds of contact. They are also highly specific and unlike antibiotics, only kill the disease-causing bacteria without harming the beneficial bacteria. Dr. Fischettis studies have shown that when small amounts of the enzymes are administered to mice that have intentionally been infected with these bacteria, the disease-causing bacteria are rapidly destroyed. In an animal model of pneumococcus pneumonia, Dr. Fischetti has shown that systemic administration of the phage enzyme Cpl-1 can rescue mice infected with the pathogen and completely reverse lung tissue damage if given within 24 hours post-infection. Fischetti and his colleagues showed that when the enzyme is delivered to the brain of mice with pneumococcal meningitis, it effectively removes the organisms from the site. The lab has also shown that by removing colonizing S. pneumoniae from the nose of mice, they could completely prevent secondary ear infections triggered by influenza.
Using lytic enzymes as a tool, Dr. Fischettis lab developed a method of drilling through the thick cell walls of gram-positive bacteria while keeping them intact. The technique enabled the Fischetti lab to access the bacterial cytoplasm with labeled antibodies to study intracellular molecules that were previously inaccessible.
Dr. Fischetti grew up in New York City, receiving his B.S. in bacteriology from Wagner College in 1962 and his M.S. in microbiology from Long Island University in 1967. He received his Ph.D. in microbiology from New York University in 1970. Dr. Fischetti came to Rockefeller as a postdoc in 1970 and became assistant professor in 1973, associate professor in 1978 and professor in 1990. In 1987 Dr. Fischetti received a 10-year National Institutes of Health MERIT Award that was renewed in 1997.
April 6, 2010 at 5:07 pm #3366
April 11, 2010 at 9:50 pm #2804
The following was excerpted and edited for clarity from the on-camera interview.
Question: Your lab is focused on bacterial pathogenesis and immunology. Tell us about your current research.
Answer: Right now were doing several things. One is to look at phage lytic enzymes as a means to control infection or prevent infection. Were also looking at vaccine development for Group A strep and trying to understand how molecules attach on the surface of gram-positive bacteria; to inhibit their presentation on the surface. Naked bacteria can cause infection, and that would be a novel way to control bacterial infections. And also, were looking at novel antibiotics and novel pathways to identify new antibiotics. So we do a variety of things.
Question: Professor, youve researched streptococcus for more than four decades, which seems like an amazing commitment. Is that because thats an amazing microbe? And how is your understanding about this microbe evolved?
Answer: I started here as a technician in this laboratory over 40 years ago. And the projects were streptococcal-related, and I stayed with the project because its, its an organism that 40 years later still causes serious disease and we have no vaccines or good ways of preventing infection from Group A strep. We can treat it once infections occur, but its very difficult to prevent these infections. And thats really been the thrust of this lab, is to try to prevent infection
.Weve been interested in developing these phage lytic enzymes that will prevent infection; that is, to decolonize people of their pathogenic bacteria at least 50% of humans carry pathogenic bacteria in their upper, in their upper respiratory tract
For instance, Group A strep, pneumococci, and staphylococci are colonized in the upper respiratory tract of humans and thats their only reservoir in most instances. And those organisms will cause infection in the individual that is carrying those organisms. If you could eliminate them in some way, safely, you could eliminate a lot of disease. And, right now, there is no good way of eliminating these organisms specifically, and weve developed a method by which we can specifically eliminate these organisms from mucous membrane surfaces.
Question: What is a biofilm, and how might they, how might they present problems to humans?
Answer: Basically any bug can form a biofilm. A biofilm is just an accumulation of organisms at a particular site. These organisms secrete polysaccharides that cover them in a slime. There are channels in that slime. They can communicate with each other in that slimy surface and that is basically the biofilm. They dont tend to grow very much once theyve established the biofilm, which is one of the reasons why theyre difficult to treat.
Question: Studies from the CDC say that up to 80% of chronic infections involve biofilms. Whats the connection between bacterial biofilms and chronic health conditions?
Answer: They are very difficult to eradicate because they dont grow rapidly, and most antibiotics kill organisms that are growing and they will not kill organisms that are not growing. So these organisms can fester in a particular area. For example, endocarditis is (caused by) a biofilm of organisms on the heart valve.
in diabetic foot ulcers, where organisms will colonize the ulcer, its difficult to eradicate those organisms. So, these are chronic conditions where organisms have become intractable and unable to be eradicated with just simple antibiotic treatment. They just dont grow very well; the biofilms really cover them and prevent the antibiotics from getting in properly to give them a high enough dose to kill them. So, in a sense, (this is) a protective way in which these organisms can survive for long periods of time in a particular area of the body.
Question: Are there any standards to characterize either mono- or polymicrobial biofilms?
Answer: Well, as far as I know, the only way you can do that is to take the biofilm, break it up into its individual components and measure what organisms are in there and what percentages of those organisms make up the biofilm itself. Unless you can culture the organism, you wont know what the comp-composition of that biofilm happens to be. And since about 90% of the organisms in our bodies are unculturable, we can have biofilms that are representative of organisms that will not grow under normal conditions, and theyll be underrepresented when we look at the compositions. So they could be doing something to that biofilm, but we wouldnt have any idea what its doing, since we cant isolate that organism.
Question: Studies from the 90s, or actually even the 80s report that certain antibiotics can actually cause microbes of all different types to change form. Does that imply that certain meds — whether it be antibiotics or other — can drive some microbes into a state of dormancy, only to have them reemerge, months or years later? Are these called cyst forms?
Answer: As I mentioned earlier, organisms are more susceptible to antibiotics if theyre actively growing. If their metabolism slows down significantly, then theyre more resistant to the antibiotics. So what youre doing is, when youre treating with certain antibiotics, youre killing the organisms that are growing. You select for mutants that are slow growers — and those are the guys that will be the persisters because youre just selecting.
Genetically, those organisms grow much more slowly and therefore theyre hiding out. There has always been a suggestion that when you treat gram-positives with penicillin, which causes an effect directly on the cell wall, that the cell wall falls off and you now have some resistant organisms — because they are called, (pause) cell wall-less organisms or L-forms. And then these L-forms can survive in the presence of an antibiotic because they dont have a cell wall. How much latent infection that actually causes has been con- very controversial for decades.
Question: How might microbes of all different kinds work together? For example, I think some of Dr. Bill Costertons early work suggested that gram-negative and gram-positive bugs might kind of work to cooperate to help each other survive. Can you comment on that?
Answer: Well, I think all bacteria work together and in combination even with bacteriophage. I mean, you cant forget that bacteriophage play a major role in all of this. We tend to ignore a bacteriophage, because they dont cause human disease, but they really have a lot of control over the bacteria that they infect. And so youre really dealing with gram-positives and gram-negatives. They produce a variety of different, secretory compounds that either will, will control their environment or stimulate the environment for certain purposes. I mean, theyre all there to survive and its a matter of what they produce and how they can live together in this environment — and its these molecules that allow them to interact with each other and live in a particular environment. So thats absolutely true that they work together to survive in the body or anywhere else — in the soil if theyre soil organisms or on plants or what have you. Theyre all working together to survive.
Question: Are there any bacteria that are generally regarded as being the most sophisticated? For example, MRSA has demonstrated its ability to employ quorum sensing to detect neutrophils and perforate them with Phenol-soluble modulins. Are there myriad examples of clever defensive strategies and how does that gel with newer synthetic methods to eradicate them?
Answer: Well, each pathogen has its own unique set of traits that allows it to cause infection. They either have surface molecules that allow them to attach to a specific site in the body and then they secrete molecules that allow them to invade. And then, once they invade, they have other molecules that prevent them from being killed by white blood cells, and a hole they just get through, a whole myriad of changes, and at different sites, and different periods in the infection cycle that allows them to survive. E. coli, for example, has sophisticated injection devices where they can inject molecules into a cell that theyve attached to; control the cell, allow it to be taken in .and in very, very sophisticated ways in which these bacteria have been able to subvert humans and cause infection.
What we have to do is learn how these things work and then try to circumvent those problems by killing them in a specific way to get around these very sophisticated methods. Were using enzymes to actually slam em on the head — basically, it doesnt make a difference what they have that allows them to cause infection if we punch a hole in their cell wall and they explode. Theyre dead, and they cant go any further than that.
Question: How does Strep pneumonia, pneumoniae cause something like meningitis or septic arthritis or even endocarditis? Can you maybe pick one of those conditions, and kind of walk us through the disease process?
Answer: Well, endocarditis is just a good example of that. Endocarditis is an infection of the heart valve and it usually occurs when a person has a defect in the heart valve. It could be rheumatic heart disease, where the heart valve has been damaged because of a strep infection. So these defective valves can become colonized by any organism that enters your blood. One of the organisms that can enter your blood is Pneumococcus, because its found in the nasal cavity, and under certain conditions it can get into the blood and will colonize the heart valve. Once it vegetates on the heart valve, it then becomes very difficult to eradicate it — because, again, its a biofilm — and very difficult to eliminate.
Question: Tell us a little bit more about your research. Its sounding like there are really there are two distinct systems here were talking about in terms of phages, or not?
Answer: Phage is one system and phage enzymes is a whole different system. Theyre both unique. Phage, we eat, drink phage all the time. As Ive mentioned, every gram, every gram of soil has about a hundred million phage in it. Every cc of water has about the same number of phage, so we are inundated by phage all the time, and we eat, drink phage constantly. So, if you use phage for a therapeutic, I dont think it would cause any detrimental effects in individuals. The real question comes in: will the FDA approve a cocktail of viruses or phage that will not be exactly the same from lot to lot?
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