----start---- microbial invasiveness 9/8/98 PRESENTATION: Jody: colonization: E.coli, how it gets into urinary tract, how it adheres and colonizes the tract. Our studies are mostly human based. One study she looked at looked at canine urine, which also had proteus mirabilis as well as e.coli in most samples. but the main thing is that it has to adhere and colonize once it enters the tract. if it can colonize the urethra,it can get up to the kidney. they get into the tract via fecal contamination of the urethral opening. GI tract has a lot of bacteria in it; urinary system does not. E. coli = gram neg; has cytoplasmic membrane, and cell wall which contains LPS, then there is also a capsule with fimbriae on it. now, the urothelial cells have receptors on them. there are attractive and repulsive forces b/w cell and bacteria - the fibria are what allows the bacteria to overcome the repulive forces due to their small radius (smaller negative charge) and hydrophobic interaction wth the receptor, as well as the specific bond b/w the p-fimbria and receptor. a mouse study looked at various fimbria - type I fimbria only --> need a large dose of e.coli for it to reach kidney. p fimbria only --> smaller dose needed. so P fimbria are a stronger virulence factor. when both are present,bacteria is more virulent still. another study on e.coli with no fimbria found that if it gets to the bladder it can cause pyelonephritis if there is a reflux situation. the fimbriated e.coli produce endotoxins which can mediate an alpha-adrenergic neurally mediated reflux, also. in the kidney there is a receptor for the fimbriae, so if bacteria get there, they can colonize. -- part two presentation: what happens after attachment - e.coli are sitting on urothelial cells, bound to receptors by fimbria. lipid A part of LPS calls in other molecules which draw in neutrophils - which cause damage to the mucosal surface. the neutrophils are what cause the damage. LPS calls in IL1, IL6, complement, TNF, which draw in the neutrophils. Dr. Schifferle adds: the paper isn't exactly right. they keep saying probably, possibly, perhaps this, pehaps that. so maybe this is more theoretical...this paper was written some time ago by a clinician who was already 3 or 4 yrs behind. fact is,LPS is important but all enterics have LPS and do not always cause this problem. LPS may play a role bu tmore recent studies show that the different fimbriae on the same bacteria with same amt of LPS are what mediate this. the fimbriae induce much more of this IL6 and IL8 response. this is more recent data. other bacterial molecules like hemolysin are there at low concentration and also induce some responses. histamine response and so on. probably they may also and again we say "may" because we do not know even if it isn't reacting with the epithelial cell it will react with neutrophil or mphage and produce inflammatory mediators. so LPS role is questionable. continued presentation- IL8 is a chemoattractant - the others aren't really. complement opsonizes bacteria, c3 activates neutrophil, produces respiratory burst which makes superoxide radicals.some studies showed that if steroids were given to prevent neutrophils from coming to area, it decreased inflammatory response. radicals kill the bacteria, and damage the mucosal lining of the tubules. urothelial epithelial cells have no superoxide dismutase, unlike other cells. so they can't protect themselves from this reaction. article showed studies where superoxide dismutase was given and it prevented some of the inflammatory response. prevention of colonization: the study showed that if you vaccinated with p-fimbria, animal made Ab to it, reducing colonization. also if you gave the receptor orally it could competitively inhibit colonization- bacteria bound to the soluble receptor, instead of to the cells. types of host: secretors and nonsecretors secretors: fewer host cell receptors, more soluble receptors, more compounds on mucosal cells - so less colonization non secretors - more host cell receptors, less soluble receptor, fewer compounds on mucosal cells. so, nonsecretors have increased likelihood of infection, would benefit more from a vaccination. vaccinated subjects might pass on antibody to the children. if it is hereditary, it would be useful to vaccinate nonsecretors to pass on antibody to the kids, because kids under 3 yrs of age who are infected tendto have problems with chronic pyelonephritis and CRF. pyelonephritis tx: antibiotics - give early, give often :) there is no vaccination available at this time the earlier that you give the abx, the better. within 72 hrs of initial infection really decreases the damage caused by the inflammatory response. also, monkey studies showed that animals not treated had renal damage within 48 hrs. so these bacteria act fast. dr. schifferle asks: what about the vaccine? what's a good antigen,what are pros and cons? Betsy remarks if the bacteria evolve so quickly, you would want to make sure to use a well conserved part of fimbria. Dr S says there isn't much problem here - the major subunit is one type and there is not really any antigenic variation. so we can get around tht. but there are other fimbriae which have mechanisms for antigenic variations which are very sophisticated. but enterobacteriaciae don't have that - they have phase variation - a few major fimbriae. the other thing is that - when bug is in urinary tract, growing, is it antigenic? not really. so we do not know if this works. it does cause inflammatory response. when you immunize, you organize an inflammatory response, except if you give something orally- then where does it go? lymphatic system? places where you have inflammatory molecules ready to go. gut is organized to take care of this. even if theygo into epi cell and out into mucosa it is organized, as is the subcutis so you can have intradermal inoculations...you always cause local inflammation. each time, you make this inflammation which is bad, you dno't want inflammations in your patients. if you do notget one, though, it's not a good antigen. so this type of thing, to immunize against these infections is orally - or any other - but orally may induce IgA some of which goes into urine, but not too much. antibody doesn't really go to urine unless there is an inflammation and barriers are breached. but that could be good - that may work. monica asks - does the secretor/nonsecretor thing apply to dogs? we'll get back to that. dr s says- in human medicine a paper came out last year, some company tested a vaccine based on an adhesin, which is something that binds mannose residues. it is on the fimbriae. you could immunize with adhesin molecules - the molecules that bind the receptors - and these may be more conserved b/c they have to bind the same receptor. so you find a few major adhesins,...that could work. in dogs, it's slow, but this could still work if similar adhesins are used. once technology is deployed, we can follow up with it. ok, back to secretor concept...we don'tknow if it applies to dogs or not. we know about mice better than dogs. also humans. it's probably there in dogs, no reason it isn't, but we do not know for sure. some breeds might have more problems than others,and that kind of thing. but usually it's something else,like dobes have intrapelvic bladders that cause stagnation, and that kind of thing. or dogs with disk disease and atonic bladders.... what about mice - they usually have vesico-ureteral reflux per the paper. do dogs? well, it isn't normal. it is seen on cystograms sometimes, it is abnormal. what is it? usually, you can imagine it is like antiperistalsis. it exists, you can induce it or find it in patients with some problems... [digression into neurophysiology] question: assuming this vaccine worked, how common or serious a problem is this? would it require vaccination instead of treatment? is there some specific population more prone to this? well, nonsecretor women who get cystitis every time they have sex. and their daughters. in veterinary medicine, we're moving away from the idea of vaccines - but there is more and more push - due to abx resistance.we're finding new ones,more and more (new what? vax or abx?). but we're looking at WHO doctors insisting that we restrict antibiotic use from all animals, using them only in humans. whoah!!! that's scary, because then we are really screwed. what about the superoxide dismutase? it filters into the urine really well. so it works out well. but it didn't prevent infection completely. clinically many UTIs are seen and recently one young puppy was seen, well taken care of. vaccine might be useful in a puppy like this to prevent future problems. especially against proteus, which makes a urease that is conducive to stone formation. pyelonephritis - to start tx w/i 36 hrs to prevent permanent damage is a nice theory, but when do people bring them in? they stop eating, they have vomiting...no lower urinary signs....you may pick an abx blindly andjust get lucky...dogs need 4-6 wks of treatment for pyelonephritis, too. people only 5 days. it's due to the different etiology, maybe.... --part 3. norfloxacin: a study using 6 dogs with UTi was done. 5 dogs had regular UTI and one was nosocomial. two had prostatitis too. C&S was done, of urine and prostatic fluid. 8 isolates were found, ,6 were gram neg. 5/6 of them were txd with standard therapy unsuccessfully. all signs are on p 714 table 1. all dogs were various age, breed. c&s found all bugs sensitive to norfloxacin. it took 3 wks or 4 wks (with prostatitis) to treat. after 1 wk post treatment, reevaluation occured, and also at 1 month for prostatitis cases. table 2 shows all isolates. results were - all bacteria were multidrug resistant but were sensitive to norfloxacin. all 6 were cured after treatment. the two with prostatitis also had reduced size of prostate gland after treatment. gram negs are most common UTI pathogens; 6/8 in this study had gram neg infxns. the dogs with prostatitis had bugs sensitive only to norfloxacin and an aminoglycoside. norfloxacin has low protein binding, is basic,can pass blood/prostate fluid barrier and get trapped in the acidic prostate. norfloxacin is a fluoroquinolone. these drugs are used as broad spectrum antimicrobials. it's a DNA gyrase inhibitor, so it is bactericidal. the mammalian gyrase is different from the bacterial gyrase, though, so mammalian toxicity isn't as great as bacterial toxicity. activity against obligate anaerobes is poor. it is orally abosrbed, has long half life in serum, excretion is renal, urine gets concentrated with it within 24 hrs. urine is 12 times more concentrated than serum. also therapeutic success has been seen with respiratory and skin and other infections. resistance rare, not a big problem at this time. low toxicity and well tolerated by the host. flip side- quinolones in general can be really neurotoxic and can cause convulsions at really high doses. vomiting and diarrhea seen rarely . in high, prolonged dosing regiments, in growing animals, there are cartilage erosions which occur, causing permanent lameness. contraindicated in pregnant or growing animals. in summary, the study showed high success treating MDR bacterial UTI with norfloxacin, but suggests using other antibiotics when possible to avoid potential development of antibiotic resistance. it's a good drug but you can't use it for everything. ---part 4 swarming and pathogenicity of proteus mirabilis in the urinary tract dog had fever, vomiting, lethargy and a bladder stone - removed by cystotomy. did c&s too. culture positive for proteus mirabilis - unusual in dog UTI. back in august biff was hospitalized for dysuria and had a urinary catheter placed for one to two weeks. that was probably the source of the problem - proteus can show up after two days. proteus can cause urinary stone formation as well as acute pyelonephritis. proteus can change around - can be swimmer cell or swarmer cell. depends on environmental cues, synthesis of the flagellar protein, and multicellular interactions that result in diferentiation. swarmer cells are internalized by host - but also swimmer cells can. when nswarmer cells are put into culture they may revert to swimmer cells and then be internalized. but mortality rates are reduced when mice are infected with nonswarming mutants. proteus secretes a urease, which hydrolyzes urea to ammonia, producing alkalinity and causing struvite and calcium phosphate crystal formation. the proteus is caught in the stone, so organism isn't cleared - stone can also be reservoir for other bacterial species but that didn't happen here. other research - virulence factors of proteus mirabilis - urease, flagella - also hemolysin, fimbria of four types, an AA deaminase, an IgA degrading protease. the fimbria are of four distinct types - this is useful to the organism. it can survive in various niches because of this variation. but they also protect bacteria from phagocytes by enhancing adherence and enabling invasion factors to be expressed to prevent killing by phagocytes. also some fimbria mutate to appear more like self, reducing phagocytosis likelihood. also, there may be overexpression of invasive forms of fimbria ... the expression of the fimbria is regulated by the swarmer cell regulon, in which there is a hierarchy of factor expression (?) colonization: starts in GI tract, low incidence in UTI. there are two main fimbria which promote colonization. proteus lacks siderophores but makes the AA deaminase which forms alpha ketoacids, binds iron, and allows it to grow in iron limiting environment. also cleaves IgA with IgA degrading protease. is the swarmer cell important for infection? we think so. it undergoes differeniation into swarmer cells, makes a lot of flagella, and swarms to kidney, we think. genes encoding flaggelin, urease, andhemolysin increase activity in swarmer cel configuration. as discussed, IgA protease cleaves IgA - yet this bug also has multiple flagellin encoding genes, so we think it can vary flagellar epitopes. why was biff experiencing bloody urine? hemolysin and urease. proteus mirabilis - very virulent and well adapted for UT life. can colonize urothelium, swarm up to kidneys, bind renal epithelium, hydrolyze urea, increase pH, promote struvite formation.. also may invade tubular epi and damage kidney, getting into blood.this may cause the lethargy and fever. tx: antibiotics, immediately - perhaps norfloxacin. Q & A FIRST: Budsberg et al., Norfloxacin therapy....JAAHA 25:713-716, 1989 1. what combination of factors were required for the micro lab to label all the bacterial spp mentioned in table two as sensitive to norfloxacin? are gram + bacteria and pseudomonas aeruginosa typically sensitive to norfloxacin? norfloxacin is usually effective against a wide variety of gram pos and gram neg aerobic bacteria. To show sensitivity, they had to grow the pathogens in culture,then kill them with the drug. 2. what are the problems of using fluoroquinolones in animals? ?? resistance? dosing schedules? no idea. ok: schifferle says resistance to fluoroquinolones is a problem; a chromosomally encoded resistance occurs. but the big problem now is the MDR - the pump - which pumps antibiotics out of the bacteria. there is a big group of drugs affected by this. this resistance is seen moreand more in hospitals where there are nosocomial infections...some strains of bacteria have this resistance and fluorouquinolones can't be used. still, they are good drugs in human medicine. it seems in europe enrofloxacin and other veterinary drugs are used in poultry industry- and lots of campy is resistant now, and some salmonella now too. obviously, where we have hospitals and drugs are used, we develop resistant strains, and even if they have low pathogenicity they can cause problems in sick animals. can you vaccinate against the pump? good question! what is known for the pump - you have to target somethign that is surface exposed. there is a protein that is exposed...wanna do some research on it? how well is that protein conserved? dunno. protein is also used to extrude hemolysins by gram negatives. also recognition domain for bacteriophages, and for some other thing. phage therapy has come back...was an old story in the 60s...virology grew out of this studying of bacteriophages. that knowledge grew into virology... can you use them to tx disease? there were nasal sprays containing bacteriophages used to treat infections... dr s thinks bacteriophages as treatment are crap. for people trying to get research money they come up with these ideas but they will not work b/c they have specific ligand functions and you can't make that many specific phages - and if you do, the receptors will mutate anyway. for the moment, a few people get money for it, but it's not that useful. the question is will retroviruses be used as vaccines??? we'll see. MDR is such a stumbling block for our antibiotics...but if we stay a step ahead like we have been, we're ok...we have not totally run out of antibiotics yet. lots of researchers want to scare the public so they can get grant money. another problem with fluoroquinolones is that they do not cover anaerobes. you could have an anaerobic infection in the bladder or prostate, easily. it's possible that in this study the first treatment got rid of the anaerobes and then the norfloxacin got rid of the aerobes. also,this urine was cultured without looking for anaerobes. they only looked for aerobes. they didn't do a gram stain,either, which is really useful for looking for anaerobes - if you see small fusiform gram negs....or if you see no growth at all...suspect anaerobes. if you see pus in the urine, you can look at the cells - you might see that there is an inflammatory response and then you can do gas chromatography, and can test for anaerobes. 3. were gentamicin and erythromycin good choices for prostatitis in dog 6? why? No. The pathogen was resistant to these drugs. before they found that out- they used gentamicin b/c it was broad spectrum, but it isn't a good idea to use in UTI dogs b/c it is an aminoglycoside that doesn't cross into tissues. erythromycin can be enriched in some tissue but covers only gram positives. prostate infections are usually gram negative. SECOND: Roberts, J.A. Etiology and pathophys. of pyelonephritis, Am.J.Kidney Disease, 17:1-9, 1991 1. as per p 2, are capsule and LPS really anti-adhesive molecules? Well, they seem to be "repulsive forces," but can be overcome by the bug. if fimbria aren't there,they will repel the bacteria, but fimbria allow bacteria to bind to the cell. Dr Schifferle adds: e.coli have or do not have fimbria - but e.coli are living cells which adapt to their environment very quickly, so sometimes they produce capsule instead of fimbriae, or fimbriae instead of capsule, or both or neither or whatever. these expressions are regulated by the bacteria [?]so when you have fimbria, you usually don't also have capsule - because fimbrial expression means colonization of epithelial surfaces, while capsule usually means you have gotten through epithelium and you want to keep molecules like complement away. another thing which is -not a lot of literature on pets, but very little in cats, or none - in dogs, there are some studies which hopefully didn't confuse you - usually you look at the studies, one is european, one is american,they say opposite things - in dogs, you can have proteus infections frequently. most often it is e.coli but in dogs also proteus causes an obstructive type of phenomenon. a neural problem, bacteria beinng able to not have fimbria to colonize areas like perineum but can colonize better not even the urethra but maybe the ureter. so also there is a stage b/w intestinal colonization and uti colonization which is skin/perianal colonization. so this is very important, these types of colonizations will necessitate some kind of mechanism. that may explain why is more frequent when you havea UTI to have e.coli, but proteus may have some advantage in some situation,where you havev obstructive problems or something. some study found - 2 or 3 maybe - which evaluated uti agents for frequency - one found proteus and the other found e.coli. it's probably also that - the etiology of UTI may be different in animals and people. in humans, females often develop UTI after sexual activity, but in animals you have a lot of UTIs linked to some other chronic problem, especially the recurrent ones. so different etiology may explain different microbial population. The disaccharide-fimbria interaction is important b/c it determines specificity - what kind of receptor is on host and what fimbria is on bacteria. in humans there are differences among individuals. 2. describe the most important differences b/w a UTI model in mice and clinical UTI in dogs. Mice urothelial cells have the P-fibria receptor. 3. How does E. Coli cause inflammation in bladder or ureter? E. Coli adhere ad release endotoxin, which has proinflammatory effects, and also affects a-adrenergic nerves in the muscle, causing dilatation and physiological obstruction. also adhesion brings bugs closer to cell, increasing effect of LPS, causing formation of acute phase reactants. also bacteria act as chemoattractins bringing in IL and lymphokines 4. how would you explain that decomplementation with cobra venom prevented (completely?) the inflammatory response? without complement, bacteria are not opsonized, neutrophils are not acting as active phagocytes and therefore are not creating all the radicals, etc which normally cause the tissue damage. 5. why is it better for the microbial pathogen that its adhesins are not always expressed? Imagine what could regulate the expression of various adhesins? if they were always expressed, host would develop Ab to them? THIRD: Mobley, HLT, and Belas, R. Swarming and Pathogenicity of Proteus Mirabilis in the urinary tract. Trnds Microbiol., 3:280-285, 1995 1. comment on the authors' questions, last page 2. what selective advantage is there for a pathogen to express many different type of fimbriae? - depends on environment it is in at the time. if it can adapt to environment by making a different adhesin, that is useful. may be different regulations of expression. 3. suggest various mechanisms used by fimbriae to protect bacteria against the killing effect of phagocytes. - when they bind epi cells, they promote expression of invasion factors - these allow invasion into epi cells. if they bind epi cells they may induce signal transduction, causing epi cell to take in the bacteria,allowing them to hide in there safely. also - lectinophagocytosis can occur - lectin is a molecule that binds sugar residues, so the fimbria is a lectin, it binds and is taken up by some mechanism (isn't this the same?) it's different from complement induced phagocytosis - killing of bacteria doesn't occur, it's just taken up and now it is in a little house ;) it sneaks in through the back door, without setting off the burglar alarm, so it doesn't cause killing of the bacteria, it doesn't undergo lysosome/phagosome fusion, etc. or, can bind complement without activating the complement cascade - that hasn't been described, but is conceptually a concept (eh?) dr s says: so, about the swimmer/swarmer thing - it's not well understood. evidence for flagellar antigenic variation is very weak. IgA degrading protease is also unclear - may be a different protease that happens to degrade IgA also. usually IgA is a secondary response so. you could argue that maybe it is important with chronicity. two fimbria seem important. swarmer phenotype makes more virulence factors. this is true. but we aren't sure of targets and how things function. one other thing some years ago... only 11% of pseudomonas were sensitive to enrofloxacin, while 91% were sensitive to cipro actinobacter - 20% and 12% sensitivity so resistance occurs! 80-90% of enterobacteriaciae are still sensitive to fluoroquinolones, but resistance is sneaking in. ---end----