[speaker002:] Okay,le let's get started! We're, we're going to conclude now the discussion of the various ways in which, bacteria, in particular er... lead to the production of infective disease... and really to centre round, centres round the discussion of the pathogenesis, that is the mechanism by which the disease is produced... and lunanspach. And I hope to run through today, basically, a set of examples of specific infective positions which show particular virulence factors... er manifest themselves in terms of clinical disease. We start with er er... a fine and exciting topic for Monday morning... the topic of boils and in their finalist form... they're shown here as starike. But, the sa the very same organisms responsible for that relatively moderate appearance can also produce blocked er... region of Prince Charles, the carbuncle with which in it's most er... florid form can break down and produce the series, but really effectively interconnecting. If we... sample one of these cavities in which the process is er... which produce boils er... are going on, almost inevitably we find this organism staphylococcusorius... and offspotulates have been clearly fulfilled with organism and this disease... most notably by a bunch of of medical students who are subjected to all sorts of tortures by their professor of microbiology back in the nineteen fifties.... And over the years it's been possible... to identify a number of factors produced by these bacteria that seem to make sense in terms of the pathology and the conditions. Now the pathology is by... a chief information... and i in fact, the chief information could be provoked merely by the presence of organisms. You will, you will recognise... from your immunology... that the presence is certainly foreign... antigenic materials... can lead... immediately to, to the acute alternatives. But, notable inside... er... the material... that characterises the boils and abscesses, there is a cell death, both of bacteria and of the... both the defending cells and the structural cells. And, to a very large extent tri cell death can be related to the ability... of staph orius to produce elicit toxins, toxins which actually leave the cell death and. But the characteristic of this puss forming organism is that the legions it produces are wooled off, that is they're relatively localized. And... although the er, the causal relationship is not completely established, it is a very helpful way of remembering that one of the... principle distinguishing features of this organism which separates it from other members of the genus staphylococcus... it pr it produces this enzyme to coagulate things on... and the effects of this enzyme are illustrated here as against the control preparation, you see... a clot form due to the action of this enzyme on clotting practice which has been put into this... test tube serum. So this is almost certain a virulence factor but is also very useful in differentiating... pathogenic species of staphylococci from non-pathogenic species. And, as as you'll be hearing in later... er stages of the course... there are many non-pathogenic species... of staphylococci. It's worth also mentioning, since you saw it in the practical class, that staphylococci in general produce this enzyme catalase... all of these er lo thi this is the enzyme which breaks down hydrogen peroxide and detoxifies it. All of these factors together... lead us an to recognise as it a puss forming organism... which... is often recognised by the term hyogenic organism, ability to produce puss in er the form of reactions... it initiates. Now I want... at least, to intro introduce the possibility in the series of diagrams I'm going to show you in this lecture... that we can relate it to the four stages... of pathogenesis that we, we outline briefly... er, in the last lecture. The first stage being the establishment, the next stage being penetration, the third stage being damage, what actually produces the damage in the disease, and the fourth stage is, if the disease is going to last for any period of time... the ability of the organism to persist. I'm going to introduce this... abbreviated momentictiture here in the hope that during the course of the lecture I'll be able to add... comments about which of these factors lead to which of these results. Er, but if we run out of time and I find we haven't we we we can't fit it all in I'd like you all to attempt... to do this for yourselves after er, the lecture is over. Okay? So, establishment for staphylococcusorius is often not an important feature because... in the majority of infections we're talking about an endogenous infection, the organism was there in the first place. Penetration... in many instances is not related to a property of the organism itself, in most instances it's re related to... some form of injury in which the organism is moved from it's normal skin location to a deep tissue location. So we won't mention these factors here, although... in... in some instances... er, these... properties are important to talk about for staph orius. The damage, is fairly obvious to see is produced by... these two... and it's worth pointing out that persistence... can be related to this catalase enzyme. Staphylococci have some degree of resistance to killing by phagocytes and yo you should recall that phagocytes... er, one of the mechanisms by which phagocytes kill intercellular organisms is the production of reactive oxygen intermediates... and catalase is one of the ways that these can... detoxified... and th there's... fairly good evidence that the catalase enzyme enables... er,staph orius to persist. Okay. So, that's, that's the first example of a very common condition.... Let's move on now to consider a very important... type of infection... such as meningitis. You recognise by... particularly common in children not recognised by the particular posture that they adopt and extended neck, rigid neck due to the inflammation of le, lymphees... which I will resist being a reflection of the neck, completely rigidly. It's a disease which, if it a, they're fortunate enough to re, recover from the disease, if it's been severe enough the inflammation may have... involved... er, some of the cranial nerves, and here we can see a third nerve palsy er as a result of... er, the process. If you take a sample... er, of ce the cera cereblis fluid... during the course of acute bacteria meningitis you will find it's drowning, drowning, not due the fact that it's an organism, but present, being due to the presence of... puss cells. This is another biogenical fluid... and, in about a third of the cases you will find... this, very faintly with them
[speaker001:] Mm mm.
[speaker002:] which you can just about see here... a in conjunction with the puss cells with which it is... busily
[speaker001:] [cough]
[speaker002:] the life and death, life and death... tackle. This organism... is the fastidious grand negative... rod you saw in the erm... practical class... innocuous influenzi.... And it is a... a very important pathogen responsible for about a third of the cases of meningitis in in this country... almost exclusively in children. Now, the process we recognise... in the pa pathogenesis... of meningitis due to this organism... is as follows, first of all...... and then it is able, by, mechanisms not completely described but... we could probably involve this transpschycosis er phenomenon that er, I I briefly mentioned last time. First of all, penetrates from the into the bloodstream and then... into the cerebral spinal fluid. Now... the basis for the pathogenesis is not well enough established for us to expect you to understand... the details of how it achieves these processes, but one thing which is... remarkably clear... is that the organism... is a capsule producing organism... and that once it gets into the er... cerebral spinal fluid that predominately is an acute inflammatory.
[speaker001:] [cough]
[speaker002:] We do recognise establishment factors, that is... adhesions... in this part of the process... and it is very clear that the capsule... is one of the factors which allows the organism to pe, persist because it helps the organism to resist phagocytosis. You'll notice that the damage in this instance is actually the result of the acute inflammatory response, not particularly some form of direct noxious effect... of the organism, so... the contribution of the host is absolutely critical. Now, the reason we... we focus on the capsule as illustrated on the right here... is... that... hymophylus... have many different capsular types, but but only one type appears to be associated with meningitis.... This type B capsule refers to a particular pulse
[speaker001:] [cough]
[speaker002:] and... has... it's so important in the pathogenesis of the capsule that antibodies to it... can be used er,ca, er can actually completely prevent the disease... and we end up with a capsule vaccine based on the type B pulse. Plus, referencing with the other two organisms which produce... meningitis in, in humans... it is very prominent that both of these, licea meninigitipitus... and staphylococcus are also very prominent capsule mechanisms. Hello!
[speaker001:] That's like the type.
[speaker002:] H I for hymopholus... influenzi. And that's, that H I is actually the... th th H, H I B is the acronym for the vaccine. There's a new vaccine introduced last October... which is being er, used for the first time in this country to prevent meningitis in children.
[speaker001:] Is that just the name of the vaccine?
[speaker002:] H, well H I B is a, an abbreviation for the strain of hymopholus that produces the infection, but the name of the vaccine the H I B vaccine, yeah. Okay? I should say that a, a lot of this information will be covered again... during this course, what I'm trying to do now is to bring together... the... the themes in a pathogenesis of infection that I introduced last week into the practical context of clinical infections. And to to, to try and illustrate to you why we focus on particular virulence factors for particular organisms.... Move on now to... to look at... an area that's er... not frequently... er... doesn't impinge a great deal on life in this country but it certainly does for travellers... and for many people in the developing world. This is the topic of secretory diarrhoea... produced by infective agents. This is a dramatic disorder of the small intestine which produces a massive outflow of water electrolytes that can be fatal within a matter of hours after the onset of disease! We recognise... two agents... which are er, responsible in bacterial causes of this process,th there are many others but these are certainly the most important. choleri, the cholera vacilus... and... er, an acronym here, ETEC, I wouldn't worry about... taking down the full er, er..., it actually stands for e entrotoxigenic ecolytes, it's a strain or a group of ecolars... that are able to produce this effect. These diseases are common in areas er where an out pouring of water from the individual is squared up with a ou, out pouring of people into the water, then you can appreciate that that transmission... is very er, readily promoted under those circumstances. The effects on the, the individual are very dramatic! Dehydration, er can... produce death within a matter of fe, er a few hours and... and an understanding of the mechanism by which this dehydration... leads us into... one of the most effective forms of heat treatment er, ever produced. A prominent feature of secretory diarrhoea is that... effective and normal bowel in,yo you ca, you can ignore this... sign... are almost indistinguishable, so despite this dramatic change in the physiology of the small bowel, borthalogically it actually looks quite normal. And you have a normal meconium. Now, the organisms that produce... this effect haven't, have... this target which is around zero for the choleri, the villi of a small intestine... and... the... both of these organisms have... pro... prominent adhesion mechanisms. Let's just show you something with that. In an affected small bowel... you will see... the grey background with these tiny little curved... rods present... the whole area of the er, the villi is covered by the organisms which are er... stuck down effectively by the processes which you ca, you can't really see them in the transmission... micrograph, but they are attached to specific receptors on the surface of the entrocite membrane.... Once they've got there, the organism... really goes no further. Both... the cholera organism... and the ETEC organism... secrete toxins... referred to here as C T for cholera toxin and LT for the le Lethane toxin. It is... very striking that these toxins are actually almost exactly the same molecule! So that although these are... er... fairly different relatives in terms of bacteria... it's striking that the ETEC organism has been able to acquire in some way almost exactly the same gene that's present in choleri. And, this toxin when released from these tightly organisms... disorders the er... the physiological activity of the lining cells, the empha cells. The molecular... oh we've lost a slide! The molecular basis for this is very well understood and... forms one of the important themes in... er... the effects of e, exotoxin in human cells. This toxin is composed of two components er, of binding components and... active component. Binding component binds to specific receptors on the emphacyte and the active component penetrates into the cell and... inactivates the controlling mechanism on the production of. And this is the cell that er, is possessed by several other toxins that affect, not just the gut but other parts of the body as well.... I should point out that in... one of the features of adhesions is that it takes two to tango, so to speak! Where specific adhesions are involved... the... the, the structures that are, are most commonly associated with a, require a specific receptor... for the adhesion reaction to take place... and that if... the potential host doesn't have specific receptor... they're not susceptible to that particular strain of organisms.... Okay. Let's move on to look at another... fairly dramatic infection... which is a case of diphtheria.... That, which is er... a infection ca characterized by this... profuse athema of th the... base of the neck, referred to as bull neck... if you look inside this child's er pharynx you see this very messy... inflammatory process at the back and the whole of the respiratory mucosa is beginning to detach in what's referred to as a pseudo-membrane. In, fatal cases this pseudo- membrane can extend right from the top of the respiratory track here down into bronchi, and you can get these enormous casts of mucrotic material... er, starting in the pharynx and working their way right down in the bronchi.... It's been known for a very long time that from these cases you can isolate this organism C diphtheria bacterium...... which you saw in the practical classes and has this distinctive stayed property where er certain granules can be stayed up and also the arrangement of the cells is rather reminiscent of what called Chinese lettering.... The picture, the acute picture which you've just seen is... virtually exclusively due to the production of a toxin... and that toxin produces really... a necrotic pharyngitis... inflammation of the pharynx... associated with necrosis and detachment... of what we call the pseudo-membrane. In... very severe cases... the toxin gets into the blood stream and their paralytic effects... toxin i i is to some extent a neuro-toxin, and also there are effects of the heart. So... although... for the majority of cases... that a fatal... as a result of diphtheria... the fact that, due actually to the pharyngitis and the pseudo- membrane obstructing the respiratory passage. In later stages, the minority of cases may go on to produce these results.... Well this is a disease due to another exotoxin... which, in practical terms is of importance to you because it's the basis for a test for identifying the. The toxin... may or may not be present in strains of... er... C diphtheria... and this is... just an immunological test so I wo won't explain it in detail but what you can see is a... a strip of er, filter paper which has been bathed in anti-toxin and... growth of various different strains of the organism... erm... which, some of which do produce the toxin... you can see lines of precipitation here... and some of which don't produce the toxin. And that test... has to be applied to es each eyslate... because... this toxin is not part of the genome of... Cherani bacteria diphtheria, it's actually a farged mediated toxin, it's actually encoded for by a bacteria farged. So when
[speaker001:] What's paresis then?
[speaker002:] Paresis, paralysis.
[speaker001:] Oh!
[speaker002:] I thought you were doing neuro-sciences course? The... as I said this, this is required for identification for the organism, but... the to the toxin has been... recognised for a very, very long time and... right from er, the early days... the possibility of inactivating the biological properties of a toxin... while retaining their immunological properties has been attractive to people seeking to prevent these diseases. And, if you treat... this toxin with something like... formaldehyde or ethanol, you end up by inactivating it's... toxic properties and... you end up with what, what we call a... toxoid, something that retains immunological properties, it's able to stimulate specific immunity to the toxin... but it doesn't have... any of the toxic effects... and we end up with this toxoid vaccine... against diphtheria.... And all of you, I would expect, have received the toxoid vaccine against diphtheria. Curiously it has led effectively to the irradification of Cherani bacterium diphtheria from the circulating respiratory flora in this country... and the reasons for that are are quite obscure! Another im prominent toxoid vaccine which you should all have received and be... regularly receiving boosters for is the tetanus... vaccine, and really, a very similar situation pertains here, that the toxin is so important in the pathogenesis of disease, tetanus, that simply immunity to that one factor can lead to immunity... er... to the disease as a whole.... So, I've gone through... a series of examples... of exotoxins... I want to just... er, as far as is possible summarize... the general properties of exotoxins. First thing to be said about them is that they're all proteins... covering a very wide range of molecular weights... between three and three hundred kilobocals... three thousand to three hundred thousand bocals. And they have very specific impacts because they all result... their effective result on an interaction with a specific receptor... and... er... an activity I would express on that receptor or as a result of the er... erm... the uptake of the toxins. Consequently,. As I've indicated before... you can divide them into patha and physiological effects... and... membrane damage more specifically than what I said before... membrane damage is the... i i is a particularly important thing.... Your patha and physiological toxins are often sub-unital structure... and... they make the most, the momegictiture most widely used... is an A plus B sub-unit structure.... The B is a receptor... and A is often, although by no means always, an enzyme... and... in the case the cholera toxin... rotusis toxin... and... several other erm pseudo-membrane toxins and several others, diphtheria toxin... they have the specific A viral... I think they put the... group onto a target molecule and disorder itself. The membrane doesn't use toxins... like the lytic toxins that staphylococcusorius expressed er... they're often detected by their immunelytic properties, see the in the er, in the bacterial passage.... I'm sure you've experienced in many instances here, the effect of lytic toxins... in er a boil or some... some other unpleasant minor lesion!... Gonna go on now... to talk about a severe... condition... which may start out... that with a minor... skin rash like this, this is called a petechial... skin rash... they later progress to rather more generalised rash... and in it's most severe form... it's mucrotic effects on the skin... and... er, obviously a lethal outcome! This type of picture is almost exclusively... related to the isolation of grand negative organisms from the blood, these may be phacili or... cocci, doesn't really make any... er, any difference. It's the grand nega negative... cell wall structure... that seems to matter. And we now know... that these... ghastly effects are the results of... what we referred to in the last lecture endotoxins.... You will hear... in... considerable detail in a later lecture about how... septic shock and endotoxin... work. At this stage it's worth remarking that wa... there are perhaps three... prominent features of endotoxin effects... first of all, they promote physcocine release and th the the two pro, prominent phsycocines affected are I O one, Interlooping one... and TMF, tumourmacrosis factor which I... hope you heard about in the... immunology course. I O one is... the molecule most closely associated with the mi, the effects of lodose endotoxin... and that is... fever... I O one... er, when released into the blood sys bloodstream... affects the hypothalamus and... th i one of the principle reasons why... infective conditions lead to fever. In more severe situations there's... uncontrolled continent acto activation... and uncontrolled clotting factor, activation and it's really that last... feature that's particularly responsible for the er... er... ghastly skin disease you saw... i in the last sla, in the previous slide!... Now the eff... the endotoxin as I've... is almost exclusively associated... with the grand negative cell wall.... And again in... abbreviated form... that, I just want to take you through the molecular basis for endotoxin action. Here is the positive wall with it's... large... er... expansive petroglycan... grand negative with an inner... and an outer membrane, and the petroglycan sandwiched in between. Here we have a diagram of a grand negative organism with it's inner membrane and outer membrane... illustrated here. Very easy to reproduce endotoxic shock in in experimental animals and... er, it was found fairly early on that... the effects, er the endotoxic effects were almost exclusively associated with the outer membrane. And detailed chemical strains of the outer membrane... show that it has a slightly different structure from normal unit membranes. Expand that up, you'll end up with... a normal inner leaflet to the membrane, a phospholipids... but... the outer the, er the outer layer is almost exclusively... composed of this stuff, lycropolysacrulite... I mentioned this in an earlier lecture but we need to go into... in a little bit more detail.... This lycropolysacrulite is... can then be broken up into three different sections... the internal membrane section... is referred as lipid A. Lipids in general are not very antigenic erm... er... it's interesting... to note that lipid A, when it's purified from the other components is... almost totally responsible for the toxic amount... of any property. The polysacurised component of composed of a four region... to which the lipids are attached, and that outer region is... er, it extends out into the environment....... Now, the importance here is that... both of these polysacrulite components are based in general the outer region is highly variable, that's to say that... very, er that most of organisms... possess a unique outer region to that polysacrulite. And by contrast, the poor region, certainly amongst the grand negative bacilli... is very similar across a wide range of different species... and this provides a very important opportunity to produce immunity to endotoxin directed against the core... and... this... possibility has recently been exploited by a production of the molecule antibody directed against the core... polysacride of the erm... er er, of... ecoli. So, this has been shown to be effective in... moderating... the toxic... events which occur in grand negative shock. Building on this approach... and something which I think you'll see... certainly during the course of your... your medical studies... antibodies directed against... tumourler proce, tumourler process factor are now being used to try and interrupt the chain of events... at a slightly later stage.... So that's... that's the endotoxin story. I want now just to summarise and er... some of these... the features I'm going to point out I've... I've already stated so you may... may just want to sit back and just take this in.... If we compare endo and exotoxins... endotoxins are only associated with grand negative organisms... whereas exotoxins maybe produced by either grand negative... or grand positive.... Endotoxins are exclusively cell associated in their production.... Exotoxins may also be cell associated, they've, they may not be a sa, they're not a structural component, but, particularly in grand negatives... exotoxins er, are released first into the peraplasmic space, the space between the two membranes, and... the the actual release... into the medium... er, may require cell. So,tha they are both cell associated and cell free.... Endotoxin... from different organisms... although it may be of... of different potency... has essentially the same biological effects. So, a wide variety of different organisms... can produce endotoxic shock. But exotoxins from different organisms... produce... really very different... biological effects. Even, even though they may be er,the they may work as a result of different o of the same enzyme activity in some cases... the target for that enzyme activity... and the receptors that have put it into the specific cells that are targeted... lead to really very different biological effects. In general terms... endotoxins are a very low potency... about point... O one of a gram is lethal to one person... some exotoxins are incredibly potent, and they're being the, the subject of er... a certain amount of er... weapons research. The same goes for O one of a gram... for example, toxin and it gives you a hundred thousand... er as as I've... indicated here, there's a, a wide range for the potency.... Endotoxins are heat stable, and this is very important when we... try to produce materials that are safe... for intravenous injection. Whereas, for the most part exotoxins are... destroyed by heat into a hundred degrees centigrade. And finally... treatment with er... formaldehyde or ethanol has no effects on endotoxins... but... in a number of instances... formaldehyde or ethanol will lead to... er, the production... of toxoid. So, I think you should now have... a very clear basis for discriminating between the two sorts of... two sorts of toxin.... And... we can move safely on to another... another, and the final really, final topic about the patterns... of pathogenesis.... Let's move on now, briefly, to consider this disease, tuberculosis.... This is a, a normal test x- ray... the commonest manifestation of tuberculosis is... the disease of des destructive regions... in the upper poles... of the lungs... and pathologically... the very nasty destructive lesions er, are appearing... in... in the area. And in a mi, in a minority of cases... you may get a sort of forminant region, this is... er small scale, it's perhaps a centimetre apart, across... a series of lung lesions containing these little necrotic due to the er... the presence of tubercle... cila.... Although this was... this organism, microbacterium tuberculosis, named tuberculosis... was discovered back in eighteen eighty... we... we really understand the pathogenesis of the disease very poorly!... What we know for sure is... is really outlined here.... The organism is in... i is... inspired into the alveolar spaces... and it's taken up by alveolar, the standard... er abbreviation for macrophage... and, the organism... i in contrast to many others which we've sort of er... die fairly rapidly after being taken up by these phagocytic cells, says Goodee! That was exactly what I wanted to happen! And it grows... very rapidly, within macrophages... and... after er... a period of time is actually disseminated into the circulation. Now, this happens in nearly all... er... cases o of tuberculosis... but, interesting, the vast majority... of infections microbacterium tuberculosis are asymptomatic... and the reason for that is that after a period of growth and dissemination does not lead to clinical symptoms, cell mediated immunity, C M I, comes into... comes into play... and you end up with a balance between the macrophages, and fi... and M T B, where there's a constant turnover of... macrophages... dying, and M T B... winning out.
[speaker001:] Excuse me!
[speaker002:] Hello! Yep?
[speaker001:] What's the M T B?
[speaker002:] Microbacterium tuberculosis. Okay. Thank you.... You have a balance between the effects of macrophages which have been activated by... cell mediator immunity... and... the lethal effects of microbacterium tuberculosis. In individuals... in whom some mediated immunity is not well developed... the growth and defemination,de defemination phase may take over altogether. If the C M I doesn't lead to this kind of balance... then... it can have this sort of effect... and... get rapidly fatal tuberculosis occurring at the time of the primary... infection. But it's believed that the majority of cases of tuberculosis, that we actually see, are so called post-primary tuberculosis where all of this process is taking place asymptomatically... and... the host is in this state of balance but something else comes along and stresses cell mediated immunity... and the control breaks down so that th... the organism M T B... wins out very dramatically over the, over the macrophages and you end up with this post-primary pattern... of tuberculosis. An example of th, of the stress might be... the administration of steroids... or the occurrence of another disease, most notably, in this day and age of course, aids, would lead to... T B coming out in this way.
[speaker001:] Is this a type of miliary?
[speaker002:] Miliary er i... it... it refers, well you've heard of millet seed and it's... it refers to a seeding of many different lesions... er, around the body... it means the disseminated form of... of tuberculosis where the lesions occur all over the place.
[speaker001:] Mm.
[speaker002:] You'll hear, you'll hear these things repeated many times again, both in the pathology, and in the microbiology course.... So, that's er, really a very different pattern of infections... where the virulence factors are not clearly identified. Let me just finish off now... by... illustrating what leads... to the outcome and how a a of a particular infectious condition... and the combination of the factors that we think lead to these outcomes. I think, the best analogy I can produce for you is that a... a balance... in which... various factors act on the side of the host... and various factors act on the side of the organism, and the outcome... is disease and death if the organism wins out... and outcome is resistance or recovery... if the... the host wins out. And we could... really, add in for each organism in different weights... the significance of each of the factors on each side. So, for example, if we took diphtheria... we could say that the toxin... was... by far the most important and weighty factor, and that if we knock the toxin out... then the disease can't occur. And, similarly on the... the host side, we could say that antibody was the most important feature. In... another example in tuberculosis... we could say that the ability of the organism to persist inside macrophages... was the most important feature... and that effective cell mediated immunity... was the most important feature... on the host side. So, I hope I've introduced to you the very different styles by which microbes produce disease... and indicated the balance... of factors involved.