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<u who="sf5009"> okay this is <gap reason="name" extent="2 words"/> and she's talking about if viable but non-culturable bacteria really exist </u><u who="sf5010"> hi i'd like to welcome you to my seminar which as <gap reason="name" extent="1 word"/> said is on the topic of do viable but non-culturable bacteria really exist this is a controversial topic in microbial sphere at the moment and i hope to give you an introduction to viable but non-culturable cells and also some of the evidence that has been stated in studies recently i'd also like to tell you about the importance of viable but non-culturable cells to public health as water and the food sources cannot have been contaminated right the first thing that i'd like to do is give you a definition of a viable but non-culturable cell they can be defined as cells which could not be cultured by methods which would normally be used with

that organism however the cells do still <trunc>be</trunc> appear to be viable because they possess apparent cell integrity they have measurable cell activity and in some cases have apparent capacity to regain culturability such viable cells can be identified using a number of techniques these include measurement of dehydrogenates activity within the cell microautoradiography and direct viable counts direct viable counts are not actually suitable for gram-positive organisms as they use nalidixic acid okay you may ask yourself why it's important that we determine if a viable but non-culturable state exists it is believed that the viable but non-culturable state makes it very difficult to determine accurate numbers of bacteria in public food and water supplies the viable but non-culturable state has also been implicated in many diseases most notably campylobacteriosis and cholera the viable but non-culturable state can also make it difficult to

diagnose sources of infection a number of studies have shown that clearly implicated sources of infection often can't produce any culturable bacteria as stated a number of species have been implicated in the viable but non-culturable state these include vibrio for example vibrio cholerae the causive agent of cholera and vibrio fischeri a symbiotic organism of squid also escherichia for example E-coli salmonella for example salmonella typhimurium aeromonas for example aeromonas hydrophila and salmonicida these are common bacterial contaminants of fresh water supplies also legionella for example legionella pneumophila the causive agent of legionnaires' disease and probably most notably as most studies have been done on this organism campylobacter for example campylobacter jejuni as stated earlier the viable but <trunc>con</trunc>

non-culturable state is controversial at the moment and many studies have been carried out however no consensus has yet been reached i'd like to present you with the <trunc>inf</trunc> with the studies that show for and against the viable but non-culturable state many studies have been carried out that do claim to have isolated bacteria within the viable but non-culturable state these include studies in campylobacter pseudomonas fluorescens streptococcus faecalis and enterobacter aerogenes these studies have used a variety of methods including microscopy acridine orange direct counts and direct viable counts theoretically the viable but non-culturable state has also been implicated in the epidemiology of some infectious diseases for example cholera and campylobacteriosis it is thought that the viable but non-culturable state may explain the absence of culturable infected bacteria from clearly implicated sources of infection this may be seen for

example in an infected water well that has caused a cholera outbreak which would not give any culturable vibrio cholerae a couple of studies have also suggested that viable but non-culturable cells may explain latent infections however little information is available on this i cannot tell you if this is true or not most recently a lot of research has been done into the viable but non-culturable state that has stated against the viable but non-culturable state existing the main part of this evidence is that the culture medium for some bacteria is inadequate and that some bacteria may be culturable under different conditions to those which may normally be used cells may also be ultimately culturable this means that they may be culturable after a certain period of time just not within the time span that they are studied also many studies may have actually disregarded the presence of a limited number of cells that never lost culturability and these may

account for the the seen increase in colony forming units the consensus for the microbial sphere also assumes that only cells which are viable can multiply however this does not concur with the idea of viable but non-culturable cells obviously if the viable but non-culturable state exists it is important to realize what causes it many suggestions have been made most notably the suggestion that it may be a starvation or stress response a number of studies have been carried out using campylo <trunc>je</trunc> jejuni and legionella pneumophila where they are starved in drinking water for over three weeks this such a starvation or stress response may be induced to enable bacteria to survive adverse environmental conditions other factors which may be important may be desiccation or metabolite accumulation these factors may lead to cellular factors such as damage to or lack of an essential component or

D-N-A damage meaning that the cell cannot multiply minimum concentrations of cellular components may also be important for example ribosomes within the cells a factor which is seen in vitro is something called substrate accelerated death this is where if a substrate is included in a recovery medium which was originally limited this may actually lead to lower numbers of colonies being seen the last two the activation of lysogenic phage or the activation of suicide genes within the cell may push the cell from a state of culturability to viable but non-culturability however it should be noted that these factors do not account for why a cell may grow a media at one point in time but not another it may be that critical genes cannot be expressed or key resources have fallen below the threshold value however it has also been suggested that it may be a deliberate process which may be genetically determined and regulated such as a developmental

process like sporulation okay what happens to cells in the viable but non-culturable state most notably it's unknown from many bacteria what actually happens as little research has been done however # a study was done recently with campylobacter jejuni where it was starved of water for over three weeks the resulting microscopy showed this here we have a rod-shaped organism and the cocci these are both actually campylobacter jejuni however unfortunately this picture hasn't turned out too well but in the original it was clear that there was cytoplasmic shrinkage and flagella fragmentation of the rod however the cocci <trunc>sh</trunc> showed neither of these factors and still appeared to be viable as mentioned earlier viable but non-culturable cells may regain culturability this is a complex process and the question that must be asked is are increases in colony forming units from <trunc>a</trunc>

below to above threshold levels due to cells changing their phenotypes or simply from the regrowth of a small population of previously undetected cells a number of processes or states have been suggested to be involved in such a regain of culturability this may involve regrowing of cells that were originally not detected but were culturable the recovery or repair of injured cells a move from a <trunc>st</trunc> a dormant state to a viable but non-culturable state resuscitation of cells and limited cell division which is where cells can only multiply for a <trunc>num</trunc> a limited number of times as this is a controversial topic i'd like to give you my opinion i believe that the viable but non-culturable state is very complex and much more research needs to be done however i do believe that viable but non-culturable bacteria exist within the environment opposed <trunc>impo</trunc> posed upon them within the artificial culture mediums

which we use i believe that possibly the viable but non-culturable state is a consequence of the limitations of culture mediums used for particular organisms the definition of non-culturability clearly depends upon the efficiency of such media of recovering damaged or <trunc>dorm</trunc> dormant organisms our culture media may not be as good as was previously thought and some organisms such as campylobacter jejuni which are notoriously difficult to culture may need new culture mediums developing for them more research is obviously needed but i believe if we can understand the viable but non-culturable state more we may be able to use this information to help us to improve our culture media there are a number of things that we can learn from the viable but non-culturable state i believe that present methods of assessing bacteria in public water and food supplies may underestimate the total numbers of

bacteria present this may be especially be a problem in untreated or low free chlorine residual water this may produce an underestimate of coliforms and total heterotraits the research has shown that if only plain counts were used for detection within water a number of organisms would not be detected these include klebsiella pneumoniae streptococcus faecalis enterobacter aerogenes and micrococcus flavus possibly the preferred approach lies in eliminating the need for culture by using a direct detection method such alternatives are being studied at the moment including genetic probes and fluorescent antibodies i believe that <trunc>clair</trunc> a clear definition must be made between cells which are not immediately culturable and those which really are non-culturable not immediately culturable cells may become culturable after a <trunc>s</trunc> a certain period of time and could therefore cause problems with human health i believe that the most important thing that we can

take away is that we do need to improve our methods of detection especially in public water and food supplies as this can be a human health hazard in summary i believe that viable but non-culturable bacteria do exist under certain conditions they may play an important role in infectious diseases and may also be detrimental to human health when found within water supplies and food sources <trunc>via</trunc> the viable but non-culturable state may be an adaptive response to differing environments or it may indeed be a quirk caused by in vitro culture methods alternative culture methods <trunc>me</trunc> may be needed and new diagnostic methods such as gene probes and fluorescent antibodies should definitely be investigated more research is needed within this area and hopefully when more research is conducted a definite answer will be able to be given to the question do viable but non-culturable

cells really exist </u><u who="sf5009"> okay thanks very much # # i'd just like to ask once these cells are in a viable but non-culturable state can they exist in it indefinitely or do they eventually die </u><u who="sf5010"> # they can exist for a certain period of time it hasn't really been studied because the research has only been started to be done but they can actually revert to a culturable state as well if you put them into animals go to animal passage or maybe in a different culture medium change the constituents then they can actually become viable and culturable so it's quite possible that within an environment they may not indefinitely stay in that viable but non-culturable state </u><u who="sf5009"> why </u><u who="sf5010"> because as a viable but non-culturable cell they need to maintain their viability so i personally would say that there's got to be a stop point they can only go on being viable for so

long </u><u who="sf5009"> yeah </u><u who="sf5010"> okay </u><u who="sf5009"> anyone else </u><u who="sf5011"> # can i ask what natural environments can these viable but non-culturable cells actually be isolated from </u><u who="sf5010"> # there's been a number of studies done pseudomonas fluorescens has been studied that's been from soil # fresh water environments sea water ground water food samples so basically anywhere but whether or not they actually exist within the natural environment or are just of our own making is not yet clear </u><u who="sm5012"> # in the # a related area in the environments # can they switch from being viable but non-culturable to being culturable </u><u who="sf5010"> # like i just said # basically it's not clear whether they are viable but non-culturable within the environment however as i said to <gap reason="name" extent="1 word"/> # animal passage so if they were taken up by

a host within the environment it's quite possible that they may switch to being viable # this would really be the only alternative to keep the the genes going </u><u who="sf5013"> # can i ask would it be more costly to test water using the new diagnostic methods you've suggested </u><u who="sf5010"> yeah # i mean just with the <trunc>deve</trunc> the <trunc>dev</trunc> development's going to be costly for example they are developing # gene probes for campylobacter jejuni at the moment but they are proving quite costly i believe so i think probably <trunc>tra</trunc> traditional methods are cheaper but of course the water companies have only got to keep those bacteria below a certain number so it's not really in their </u><u who="sf5013"> mm </u><u who="sf5010"> it's not really to their advantage </u><u who="sf5009"> go on you go </u><u who="sf5014"> i was just going to ask # do you think that there are actually being severe problems

caused by these organisms that we can't culture </u><u who="sf5010"> # certainly campylobacteriosis is a very serious and common disease # whether or not they're actually caused by viable but non-culturable cells is another thing i think it's just that in our detection methods they don't show up as being culturable so we don't recognize them for example you may study a food source and there may not actually be any culturable bacteria but the campylobacter may be there so yes i think it is a serious problem </u><u who="sf5015"> # how do the cells actually maintain viability during starvation </u><u who="sf5010"> # it's not entirely clear it's thought that there may be some sort of store a pool of # sort of proteins and nutrients within the cell but obviously that can only be kept going for a certain limit of time but it's thought that that's sort of done at a very low rate so you wouldn't

really notice it so it's like a stable process </u><u who="sf5009"> anyone else </u><u who="sm5016"> yeah can i just ask # what's the difference between the will be viable but non-culturable cells and a dormant cell is it just the metabolism </u><u who="sf5010"> # a dormant cell if you look at a dormant cell it actually doesn't seem to have any activity whereas a viable but non-culturable cell is shown to have definite cellular activity whereas dormant cells as far as i know don't show any cellular activity </u><u who="sm5016"> how do they how do they show they've got activity <gap reason="inaudible" extent="1 sec"/> </u><u who="sf5010"> # you can look by direct viable counts and other methods it's i mean dehydrogenase activity within a cell as well is a good indicator whether it's viable </u><u who="sf5009"> <gap reason="inaudible" extent="1 sec"/> </u><gap reason="break in recording" extent="uncertain"/> <u who="sf5013">

this is <gap reason="name" extent="2 words"/> he's going to be discussing why bacteria produce antibiotics </u><u who="sm5012"> okay thank you # basically # the easy answer is we don't know there's there's quite a few theories on the subject # and i'm planning to take you through some of these # but to start with i'll just give a bit of a a definition and some background on antibiotics and then i've divided the theories into two main areas # there were quite a few so it's not a comprehensive # study of the theories # the first one is end product selection and the second is detoxification which i'll explain later i'll give you some examples of these and # the problems with theories # and then draw some conclusions at the end okay firstly <trunc>defini</trunc> definition of antibiotics # Waksman

who worked on penicillin in the forties # defined them as any microbial product which in low concentrations # can inhibit or kill susceptible microorganisms and this has since been updated to include # <trunc>sethynts</trunc> synthetic and wholly synthetic antimicrobial compounds # but for our purposes we're going to stick to the earlier definition # all antibiotics are secondary metabolites but not <trunc>nes</trunc> # it doesn't doesn't necessarily follow that secondary metabolites are all antibiotics # secondary metabolism is the synthesism of metabolites that play no further apparent function in # metabolism # in nineteen-ninety five per cent of secondary metabolites discovered had no antimicrobial activity at all # and a point which # i'll come back to it's quite important in some

of the theories and one of the problems with them is that antibiotics have pleiotropic effects # in that they don't necessarily just act as antibiotics they can be immune modulators and they have all sorts of other functions right this is just a bit of revision on antibiotics # production of antibiotics is a phylogenetically diverse ability by which # for example in phenazine biosynthesis it reproduces by # pseudomonas aeruginosa # a gram-negative and also by actinomycetes # which are high-G-plus-C gram-positives # but not by the some of the intervening organisms # so in this case it could be for example of introduction of # the ability in # a comp ancestor and has been lost in the intervening organisms or # converting you could have a a gene transfer later on between the two species or simultaneous evolution of the

same ability and the pathway for phenazine biosynthesis # shares some similarities with tryptophan synthesis so they could perhaps have evolved them separately okay sorry i just lost my overhead it isn't on the floor is it right sorry about that okay # they have # generally complex structures # a quick example of that that is if i can find it <gap reason="inaudible" extent="1 sec"/> # vancomycin on the left # quite a large antibiotic the group here attaches to the top there just wouldn't fit on the # diagram and then there's # the core structure of phenazine which is obviously a lot more simple # but it is quite a job to for it to be synthesized owing to the # the three benzene ring structure # <trunc>s</trunc> the forms from the products of primary metabolism # such as amino acids shikimic acid and acetate #

synthetic and resistant genes are always in cassettes in bacteria they're always located in the same <trunc>locatio</trunc> # together on the genome or in # a limited number of examples together on a plasmid # but that is # # this i think is two examples of that # production is very tightly regulated # and this applies to the whole of secondary metabolism # examples include in the streptomycetes # regulation by cell density # in quorum sensing # controlled by gamma-butyrolactone # also by growth rate # often it's been associated with secondary # secondary stationary phase # the ability to produce antibiotics # although there is some evidence that it they can be produced at other growth stages # but usually the growth rate is slowing and the P-P-G-P-P # is a factor that can stimulate this and slowing growth # other examples we've got # sigma factors # which come in at this stage # and there's there's a

stationary space sigma factor signa sigma S that controls antibiotic synthesis and sigma H a <gap reason="inaudible" extent="1 sec"/> has been implicated not in streptomycetes but in the # earlier attempt at phenazine # and also nutritional factors which can play # can come in and inhibit at this point and this point and here as well # certain antibiotics will not be produced if you have a particular carbon source # right that's the <gap reason="inaudible" extent="2 secs"/> mention and importantly there's been a commercial drive for research # people looking for antibiotics have been driven by industry and so if it doesn't have an antibiotic function they're not going to find it # so # there is a potential that there are a large number of secondary metabolites with very different purposes # that just haven't been discovered yet okay # now on to the theories end

product selection is # the first one as i mentioned earlier # examples i'll come to later but # this is just illustrating antibiotics and that sort of thing and then there's detoxification and overflow metabolism which has also been called metabolic vomit # it's a similar it's a similar theory # right we'll start with end product selection this assumes # these these theories are all based in evolution and involve the ability to produce secondary metabolites and main concentrating antibiotics # it assumes that the product is useful to the organism and specifically tailored to its particular purpose and therefore it provides a selective advantage to that organism to have that ability # examples of this # in erwinia carotovora # antibiotic synthesis occurs under quorum

sensing control # and # their substrate # is # potatoes and that sort of # vegetable # and # they produce extracellular enzymes and to protect the investment of the extracellular enzymes and # their nutrient source they also produce # the antibiotic to wipe out any competition # streptomycetes # regulation of antibiotic production has been linked to # <trunc>product</trunc> <trunc>p</trunc> # the production of aerial mycelium # the two are synthesized at the same time and so perhaps this is a a method of protecting the aerial mycelium and wiping out any competitors for it and then a slightly more interesting theory # a prebiotic role for antibiotics # this goes back to the primordial soup where there were apparently # amino acids # and small peptides # closely related to present day antibiotic type molecules # and i'm going to introduce you to the ribozyme which you've never come across before which is #

catalytic R-N-A which is believed to have produced the first peptide bonds # and once you get the ribozyme # the antibiotics were thought to interact with it and stabilize the peptide bonds or catalyse their performance of the ribozyme # and once the ribozyme evolves into the ribozome # the binding sites of the antibiotics were maintained and # later it became evident that the antibiotic # instead of now affecting the action of the ribozyme instead inhibited it # which # evidence for which # mainly comes from studies involving the the observation that # the antibiotics often act on the ribozome # mechanisms okay problems with these theories in general # the pathways are very complicated for # antibiotic production the the they're often branched and produce more than one antibiotic from a from the same # beginning pathway # and the they've postulated that retroevolution which has been #

implicated in # the evolution of most biosynthetic pathways cannot work for # antibiotic synthesis # you get a large diversity of antibiotics produced sometimes in a single organism and # for this for end product selection to work you would expect a single broad spectrum antibiotic to be produced and to wipe out all the competition rather than favouring lots and lots of different ones that are perhaps ineffective # this also goes for pleiotropic effects # why would a bacteria need to produce an immune modulator # of a mammal when it's perhaps had no contact with them there must be some other purpose for it # immediate biosynthetic precursors of the final project # product are often inactive so why have you got these large pathways in between of inactive things # and then it just happens that the last # stage is the active # molecule and again # the <trunc>indus</trunc> the industry has # been a problem in research see if that's working okay so we move on to overflow metabolism that's probably <gap reason="inaudible" extent="1 sec"/> it # it's

important to remember that # antibiotic producers are often # in found in oligotrophic environments where there's low nutrients such as soil and water but with low nutrient availability # and therefore their primary biosynthetic pathways # tend to be not very tightly regulated they tend to be constituitively # switched on # and # okay i'll come to come to the next bit in a minute okay the # the production process itself of antibiotics is metabolically and genetically expensive and it's very tightly regulated # which suggests that in itself it has worth to the <trunc>ord</trunc> # to the organism otherwise you'd use a a a a more efficient means of # producing them and so the suggestion is that this is a safety mechanism # if you're in an environment where you're constantly producing all your intermediates and suddenly externally something becomes available # you have an internal build-up of amino acids or something that you can't get rid of and # it's suggested that these can

be shunted into the secondary metabolic pathways and ejected from the cell # problems with this # </u><u who="sf5013"> <gap reason="name" extent="1 word"/> two minutes </u><u who="sm5012"> okay quorum sensing # has been shown to occur # why would you want all the organisms in the area to suddenly # get rid of all their intermediates # for the primary synthesis # resistance genes are required # what would be the point in producing a toxic # substance when you're trying to get rid of an influx of potentially toxic substances and you produce very large molecules which is wasteful in effect of biosynthesis okay so my conclusions as # we it's very hard to design experiments that reflect what goes on in the environment because # because secondary <trunc>metasm</trunc> metabolism is so subtly regulated # it's

difficult # to get the same conditions in a lab as you would in the environment # research is biased to overproducing antibiotic strains in the environment that there is evidence that they produce it in very small quantities as opposed to the masses that # most worked on strains produce # the theories i came across were all looking they they were all looking for a universal theory to cover all secondary metabolites # which i don't think can be the case because you have things like erwinia which produce # an antibiotic given at a very specific time # # for an obvious purpose but then the streptomycetes which # seem to have very different control mechanisms # but it is possible to combine the <trunc>appro</trunc> the approaches and both the products and the process is necessary # so perhaps they had an evolutionary role as prebiotic effectors and then selection favoured the metabolic shunt and then you realize that the key <trunc>s</trunc> well didn't realize they didn't evolve to that the

end products # could be turned into something useful and provide a selective advantage in terms of being # a biosite # just one further point if you compare another # killing mechanism used by bacteria this time maybe enterobacteriaceae and bacteriocins they have # shorter unbranched pathways so you would expect evolution to favour shorter unbranched pathways if you were merely using antibiotics to kill other things thank you </u><u who="sf5013"> thank you very much <gap reason="name" extent="1 word"/> can i just start the questions by asking # you said that antibiotics have many other functions </u><u who="sm5012"> mm </u><u who="sf5013"> such as being immune modulators have you got any other examples </u><u who="sm5012"> yes # i've sort of used the terms antibiotic and secondary metabolite interchangeably because there are so few non-antibiotic ones but you also find there's # sex pheremones and # signalling

molecules between in within symbioses and things like that </u><u who="sf5013"> okay thank you anyone else got a question </u><u who="sm5016"> yes sorry can you just explain what you mean by # retroevolution </u><u who="sm5012"> right # if you have okay we're going back to an early evolutionary cell # and it hasn't got any biosynthetic pathways at the moment sorry i need a scribbling thing # # it produces all its # needs to take in all amino acids from its environment # and so it has # let's use histidine as an example it has # a decorative enzyme for histidine and then it modifies that enzyme and produces a second enzyme # and see what they <gap reason="inaudible" extent="1 sec"/> # enzyme two which allows it to also take in # a precursor of histidine # and then that provides a selective advantage over its other relations # its competitors and so we

start to build up a pathway backwards you don't start with <trunc>th</trunc> the most extreme thing that you take in and convert it all the way down to histidine you build the pathway backwards hence retroevolution i think </u><u who="sf5013"> any other questions anyone no can i ask then # how would <gap reason="inaudible" extent="1 sec"/> work to regulate antibiotic production </u><u who="sm5012"> okay # in phenazine the example i've come across is on phenazine biosynthesis by pseudomonas # and it linked # a a <trunc>t</trunc> a two-component signalling system with # production of the antibiotic # and it seemed this is under # <gap reason="inaudible" extent="1 sec"/><gap reason="inaudible" extent="1 sec"/> control # produced by all the other bacteria so when a certain level # is attained in the environment # a certain number of other bacteria is attained in the environment they start producing the antibiotic because it's then advantageous to them to start protecting

their nutrient supply as opposed to attempting to take on a large nutrient source # individually </u><u who="sf5013"> mm okay thank you </u><u who="sf5010"> # are there either of the two hypotheses # that you personally favour which do you think is the most likely </u><u who="sm5012"> i i there's there's lots of holes in both arguments really i don't think you can sort of the because there's so little research done i don't think you can definitely say # one or the other but there there there are possible things <trunc>wi</trunc> combining the approach and it's not necessarily true that you have to have the same purpose for a molecule throughout its evolutionary history it can change purposes and still retains bits and pieces and i i don't think it's true that you can you can say that # they all have the same purpose </u><u who="sf5013"> thank you </u><u who="sm5012"> thank you </u><u who="sf5013"> anyone else no </u><gap reason="break in recording" extent="uncertain"/> <u who="sf5017">

this is <gap reason="name" extent="2 words"/> and he's going to be talking about intracellular bacterial parasites of amoebae </u><u who="sm5018"> thanks for the introduction # well first of all i'd like to start with a introduction to how i'm going to present the talk and i'm going to start with a brief introduction to the role of amoebae environments and this will be followed by an overview of bacterial pathogens that survive in an amoebae and the outcome of that protozoa uptakei'll then concentrate on # legionella pneumophila which has turned out to be the # first discovered # intracellular bacterial parasite and the role of the amoebae in legionnaires' disease which has been introduced before and also the mechanism of the bacterial and amoebal interaction # i'll then cover briefly a second example which is that of burkholderia cepacia which is # the most recently discovered intracellular bacterial parasite # i'll then go on to cover the evolutionary role of protozoa and

macrophage which are the human reservoir of the intracellular bacterial parasites of amoebae and then i'll do some conclusions right then so first of all i'd like to do an introduction to amoebae they've been termed the Trojan horse of the microbial world # # # ubiquitous protozoa that are predators of bacteria in soil and aquatic environments and they uptake bacteria by the process of phagocytosis # degradation of the bacteria contributes to soil fertility and in <trunc>a</trunc> aquatic environments the amoebae play an integral role in cycle of nutrients in the aquatic food chains # and then about twenty years ago or so it was # discovered that not all bacteria are suitable food sources and # this is for two possible reasons there's one is an inability to take up or to kill and digest the bacteria or also the bacteria may be resistant to uptake by production of things

such as toxins and toxic pigments now i'd just like to go over some examples of some bacterial pathogens of amoebae # the first set here which includes # legionella pneumophila legionella-like amoebal pathogens which are unculturable <trunc>legionell</trunc> legionella-like # microorganisms which have been shown by genetics to be similar to sarcobium lyticum and there's finally listeria monocytogenes and in the middle here you've got the protozoal hosts and there's either acanthamoeba naegleria or hartmannella and also here there's the outcome of the protozoal uptake and there's multiplication and cell lysis # and then there's # a second group here which one of them consists of vibrio # and acanthamoeba <gap reason="inaudible" extent="1 sec"/> undergoes multiplication and then there's a final set which there's a <gap reason="inaudible" extent="1 sec"/> # where it's just more a survival stage and here you have # just a hartmannella and acanthamoeba and you've got things like

# mycobacterium leprae opportunitive opportunistic mycobacterium pseudomonas alcaligenes faecalis coliforms burkholderia cepacia which is the other organism i'm going to cover i'm going to give you my # # and so to summarize then the fate of the internalized bacteria can <trunc>f</trunc> ooh # can fall into three categories so there's # multiplication and lysis just # plain old multiplication in order to survive and so let's go on to legionella pneumophila so # this was the first # intracellular bacterial parasite that was discovered # and in the human lung <trunc>legionnaire</trunc> <trunc>legioni</trunc> <trunc>e</trunc> legionella pneumophila is an intracellular parasite of alveolar macrophage and it's # the the causative agent of legionnaires' disease # and Pontiac fever now # for treatment and prevention of these the current situation is for # treatment you've got antibiotic treatment with

erythromycin and other # antibiotics that can penetrate eukaryotic cells # and prevention which has also been mentioned before is # practised at the institutional level with with # dechlorination and U-V irradiation and superheating and other continuous processes # to to go on to the role of protozoa in legionnaires' disease # there are many protozoan hosts which have been identified in the environment # in outbreaks of legionnaires' disease both the amoebae and the bacteria have been isolated from the same source of the infection # the following replication within protozoa the bacteria show a rise to resistance to harsh conditions such as # the chemical disinfectants and biosite and which i introduced there # also in the on lysis of the protozoa the bacteria released in respirable size # compartments as such which can be #

inhaled and also bear resistance to harsh conditions and and that points also # fits with # like i say that # it can infect mammalian cells a lot better and also the final point there is # there are no documented cases of transmission between individuals of just <gap reason="inaudible" extent="1 sec"/> it's the bacterium alone so therefore the <trunc>f</trunc> the final point there it it's it's clear that there must be an association of the legionella with the protozoa for the main # the the that being the major factor in the continual presence of bacteria in the environment so to go on to the mechanisms of interaction # attachment of the bacteria into the amoebae is by a host receptor on the amoebae which is # galactose acetyl galactose in English is a # a lectin type molecule # it's # also # a tyrosine kinase receptor so # under tyrosine dephosphorylation it leads to recruitment and rearrangement of the cytoskeleton # and that's

shown on this figure here just talk through this # # so here you've got the # lectin receptors on the host with the # phospate on the tyrosines and also these are other proteins on the base which also phosphorylate it and # upon signal transduction mechanisms from the bacterium here this leads to # dephosphorylation of these proteins and uptake of the bacteria by a process of receptor mediated endocytosis # there's also there's # an unknown mechanism whereby the colony infection # # affects gene expression of the host here # also there's a second mechanism of uptake by coiling phagocytosis where the mechanism's not known but it has been shown to be occurring energy as if it had been done electron microscopy so i've just

quick slide here i've # you can almost see # this is the membrane of the protozoa with these bacterium in the centre here and it's uptaken dissident coiling phagocytosis so this point that was just raised today where invasion of the host is by this process of a cytoskeleton-independent receptor mediated phagocytosis or coiling phasgocytosis # the next stage is growth of the bacterium in a phagosome which is an endoplasmic reticulum surrounded # portion of the cell and also there's an addition of phagosome acidification and lysosome fusion so the bacteria can persist in this # these phagosomes are just shown here # bottom down there where you've got # this is the endoplasmic reticulum surrounded phagosome here and there's free bacterium in there so with the final stage is a lysis at about ten-to-the-four motile bacteria and # this is by the process here of # a stringent

response model which is in the diagram there so # when amino acids decrease in concentration you get a switch on a P-P-P-G-P by a rel-A which is the by the gene rel-A which is a P-P-G-P-P synthetase and this # leads to activation of regulators <trunc>su</trunc> such as # stationary phase sigma factors R-P-O-S and you get these switch on of genes # which leads to secretion of enzymes and lysis of the host so # to summarize this is covered in the top diagram so here you have # the motile bacterium and then it attaches to the amoebae here forming the phagosome and then you get this # replicate replicative state and then the switch on of of virulence genes by the decrease in amino acids concentrations and # this leads to lysis of the host here also # the infection of the human is by the # in the # <trunc>mov</trunc> <trunc>remov</trunc> # movement of the # amoebae into the <trunc>al</trunc> alveolar macrophage down the lungs and you get the legionnaires' disease

right so second example is that of # burkholderia burkholderia cepacia which is # an opportunistic pathogen for about forty per cent of cystic fibrosis patients # and it causes a necrotizing pneumonia which is # an enhanced immune response which lead us leads to lung tissue damage # the <trunc>pr</trunc> main problem here is that # there's the treatment with antibiotics is # problematic due to an intrinsic antibiotic resistance in the bacterium # and research had proved that amoebae turned out to be the reservoir and possibly the vehicle of transmission of this bacterium # it's more a survival stage because # they've shown from research that there's only a one roughly a one log increase in bacterial numbers so it's seventy-two hours so it's not replicating in the amoebae whereas replication is <trunc>extrasta</trunc> <trunc>r</trunc> extracellularly on secreted amoebal products # then the bacteria are released in these membrane bound vesicles and transported

to the lower respiratory tract and # causing infection which appears to be more effective at thirty degrees than any other temperature and this is also this coincidence that in humans the amoebae colonize teh anterior <trunc>ne</trunc> nasal mucosa where the temperature there is thirty to thirty-two degrees # the evolutionary role of amoebae and macrophage # it's turned out that infection of the two hosts has had a a common molecular basis and # the mechanism of the interaction appears to be the same there's this growth in the endoplasmic reticulum surrounded phagosome and also this expression of a surface protein R-I-pim which # potentiates infection in both hosts # they've also done a fair few genetic studies on legionella and they've found # there's two gene loci I-C-M which is intracellular multiplication cluster and P-M-I which is protozoa macrophage infectivity which # have a definite requirement in both hosts and also

there's a mil loci which is only required for infection in the macrophage so there's a possibility that this loci is required at a later stage enabling the bacteria to adapt to survival in the macrophage # so there's been a a theory proposed that there's been coevolution of protozoa which has allowed # the legionella pneumophila to develop multiple redundant mechanisms and parasitize the protozoa of these # intracellular growth and survival mechanisms and if it's turned out that some of these would be useful in the colonization of macrophage so to conclude # the # bacterial amoebal interaction provides a means of transport and transport survival and replication the # intracellular niche provides protection against adverse environmental conditions in the treatment of parasites and other other components there's # the amoebae acts as vectors of transmission and in the case of legionella and as a survival state in the case

of burkholderia and there appears to be an evolutionary relationship in intracellular growth between amoebae and the macrophage so just to finish i'd like to leave a final thought with a quote from a paper i <gap reason="inaudible" extent="1 sec"/> from Barker and Brown where they state that an intraprotozoal growth of bacteria may well optimize their potential for virulence inside the protozoal horse they may be adapting to the human Troy thank you very much </u><u who="sf5017"> right has anyone got any questions </u><u who="sm5018"> </u><u who="sf5010"> # in the two cases that you discussed </u><u who="sm5018"> yes </u><u who="sf5010"> do you know if # if the amoebae themselves may contribute towards the disease caused by the bacteria within them whether or not </u><u who="sm5018"> # </u> <u who="sf5010"> they may influence the disease itself </u><u who="sm5018"> # the # i don't think that's the case i think more it's # a transmission thing where the

amoebae are transmitted to the host and then the bacterium lies in the amoebae and then infect the macrophage so i think that's when the <gap reason="inaudible" extent="1 sec"/> infections go </u><u who="sf5017"> anyone else </u><u who="sf5013"> how great is the diversity of amoebae in capacity to less to bacteria is it just like a </u><u who="sm5018"> # </u><u who="sf5013"> a single dense amoebae </u><u who="sm5018"> # the i think they've worked out there's thirteen <trunc>hoped</trunc> thirteen different of each strains of amoebae that can be infected by # legionella i'm not sure about the others but there's there's nowhere near as many amoebae as there are types of strains of bacteria </u><u who="sf5013"> yes thank you </u><u who="sf5017"> anyone else got any questions </u><u who="sm5018"> oh </u><u who="sf5013"> is it quite a <gap reason="inaudible" extent="1 sec"/> problem do they do they facilitate severe cases </u><u who="sm5018"> # i think it's the only means of transmission really so i i presume it <kinesic desc="shrugs shoulders" iterated="n" n="sf5013"/> occurs in all cases of legionnaires' disease anyway so </u><u who="sf5013"> mm-hmm </u><u who="sm5018"> i presume presume it's a fairly severe problem </u> <u who="sf5010">

do you think it would be useful to # perhaps control amoebic populations for example and do you know of any ways of actually controlling the population say for in a for example in a air conditioning or <gap reason="inaudible due to overlap" extent="1 sec"/> </u><u who="sm5018"> # well yeah i mean that's how they're doing this prevention mechanism where they're doing # continued chlorination and # <trunc>tre</trunc> treatment with high temperatures and things just to kill the amoebae so you can take them out attacking it at the source of the infection </u><u who="sf5010"> are there any chemical compounds </u><u who="sm5018"> not that i'm aware of no i mean apart from chlorination but </u><u who="sf5010"> okay </u><u who="sm5018"> there aren't any that i'm aware of </u><u who="sf5017"> anybody else thank you

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