sf5009: okay this is namex and she's talking about if viable but non-culturable 
bacteria really exist 
sf5010: hi i'd like to welcome you to my seminar which as namex 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 be-, 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 con-, 
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 inf-, 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 je-, 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 er 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 sh-, 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 a-, 
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 st-, 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 num-, 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 impo-, 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 dorm-, 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 clair-, 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 s-, 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 via-, 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 me-, 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 
sf5009: okay thanks very much er er 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 
sf5010: er 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 
sf5009: why 
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 
sf5009: yeah 
sf5010: okay 
sf5009: anyone else 
sf5011: er can i ask what natural environments can these viable but non-
culturable cells actually be isolated from 
sf5010: er there's been a number of studies done pseudomonas fluorescens has 
been studied that's been from soil er 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 
sm5012: er in the er a related area in the environments er can they switch from 
being viable but non-culturable to being culturable 
sf5010: er like i just said er basically it's not clear whether they are viable 
but non-culturable within the environment however as i said to namex er 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 er this would really be the only alternative to keep the the genes going 
sf5013: er can i ask would it be more costly to test water using the new 
diagnostic methods you've suggested 
sf5010: yeah er i mean just with the deve-, the dev-, development's going to be 
costly for example they are developing er gene probes for campylobacter jejuni 
at the moment but they are proving quite costly i believe so i think probably 
tra-, 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 
sf5013: mm 
sf5010: it's not really to their advantage 
sf5009: go on you go 
sf5014: i was just going to ask er do you think that there are actually being 
severe problems 
caused by these organisms that we can't culture 
sf5010: er certainly campylobacteriosis is a very serious and common disease er 
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 
sf5015: er how do the cells actually maintain viability during starvation 
sf5010: er it's not entirely clear it's thought that there may be some sort of 
store a pool of er 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 
sf5009: anyone else 
sm5016: yeah can i just ask er what's the difference between the will be viable 
but non-culturable cells and a dormant cell is it just the metabolism 
sf5010: er 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 
sm5016: how do they how do they show they've got activity 
sf5010: er 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 
sf5009: 
sf5013: 
this is namex he's going to be discussing why bacteria produce antibiotics 
sm5012: okay thank you er basically er the easy answer is we don't know there's 
there's quite a few theories on the subject er and i'm planning to take you 
through some of these er 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 er there were quite a few so it's not a 
comprehensive er study of the theories er 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 er the problems with theories er and then draw 
some conclusions at the end okay firstly defini-, definition of antibiotics er 
Waksman 
who worked on penicillin in the forties er defined them as any microbial 
product which in low concentrations er can inhibit or kill susceptible 
microorganisms and this has since been updated to include er sethynts-, 
synthetic and wholly synthetic antimicrobial compounds er but for our purposes 
we're going to stick to the earlier definition er all antibiotics are secondary 
metabolites but not nes-, er it doesn't doesn't necessarily follow that 
secondary metabolites are all antibiotics er secondary metabolism is the 
synthesism of metabolites that play no further apparent function in er 
metabolism er in nineteen-ninety five per cent of secondary metabolites 
discovered had no antimicrobial activity at all er and a point which er 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 er 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 er production of antibiotics is a 
phylogenetically diverse ability by which er for example in phenazine 
biosynthesis it reproduces by er pseudomonas aeruginosa er a gram-negative and 
also by actinomycetes er which are high-G-plus-C gram-positives er but not by 
the some of the intervening organisms er so in this case it could be for 
example of introduction of er the ability in er a comp ancestor and has been 
lost in the intervening organisms or er 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 er 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 er they have er generally complex structures er a quick 
example of that that is if i can find it er vancomycin on the left er quite a 
large antibiotic the group here attaches to the top there just wouldn't fit on 
the er diagram and then there's er the core structure of phenazine which is 
obviously a lot more simple er but it is quite a job to for it to be 
synthesized owing to the er the three benzene ring structure er s-, the forms 
from the products of primary metabolism er such as amino acids shikimic acid 
and acetate er 
synthetic and resistant genes are always in cassettes in bacteria they're 
always located in the same locatio-, er together on the genome or in er a 
limited number of examples together on a plasmid er but that is er er this i 
think is two examples of that er production is very tightly regulated er and 
this applies to the whole of secondary metabolism er examples include in the 
streptomycetes er regulation by cell density er in quorum sensing er controlled 
by gamma-butyrolactone er also by growth rate er often it's been associated 
with secondary er secondary stationary phase er the ability to produce 
antibiotics er although there is some evidence that it they can be produced at 
other growth stages er but usually the growth rate is slowing and the P-P-G-P-P 
er is a factor that can stimulate this and slowing growth er other examples 
we've got er sigma factors er which come in at this stage er and there's 
there's a 
stationary space sigma factor signa sigma S that controls antibiotic synthesis 
and sigma H a has been implicated not in streptomycetes but in the er earlier 
attempt at phenazine er and also nutritional factors which can play er can come 
in and inhibit at this point and this point and here as well er certain 
antibiotics will not be produced if you have a particular carbon source er 
right that's the mention and importantly there's been a commercial drive for 
research er 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 er so er 
there is a potential that there are a large number of secondary metabolites 
with very different purposes er that just haven't been discovered yet okay er 
now on to the theories end 
product selection is er the first one as i mentioned earlier er examples i'll 
come to later but er 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 er it's a similar it's a similar theory er right 
we'll start with end product selection this assumes er these these theories are 
all based in evolution and involve the ability to produce secondary metabolites 
and main concentrating antibiotics er 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 er 
examples of this er in erwinia carotovora er antibiotic synthesis occurs under 
quorum 
sensing control er and er their substrate er is er potatoes and that sort of er 
vegetable er and er they produce extracellular enzymes and to protect the 
investment of the extracellular enzymes and er their nutrient source they also 
produce er the antibiotic to wipe out any competition er streptomycetes er 
regulation of antibiotic production has been linked to er product-, p-, er the 
production of aerial mycelium er 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 er a 
prebiotic role for antibiotics er this goes back to the primordial soup where 
there were apparently er amino acids er and small peptides er closely related 
to present day antibiotic type molecules er and i'm going to introduce you to 
the ribozyme which you've never come across before which is er 
catalytic R-N-A which is believed to have produced the first peptide bonds er 
and once you get the ribozyme er the antibiotics were thought to interact with 
it and stabilize the peptide bonds or catalyse their performance of the 
ribozyme er and once the ribozyme evolves into the ribozome er the binding 
sites of the antibiotics were maintained and er later it became evident that 
the antibiotic er instead of now affecting the action of the ribozyme instead 
inhibited it er which er evidence for which er mainly comes from studies 
involving the the observation that er the antibiotics often act on the ribozome 
er mechanisms okay problems with these theories in general er the pathways are 
very complicated for er antibiotic production the the they're often branched 
and produce more than one antibiotic from a from the same er beginning pathway 
er and the they've postulated that retroevolution which has been er 
implicated in er the evolution of most biosynthetic pathways cannot work for er 
antibiotic synthesis er you get a large diversity of antibiotics produced 
sometimes in a single organism and er 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 er this also goes for pleiotropic effects er 
why would a bacteria need to produce an immune modulator er of a mammal when 
it's perhaps had no contact with them there must be some other purpose for it 
er immediate biosynthetic precursors of the final project er product are often 
inactive so why have you got these large pathways in between of inactive things 
er and then it just happens that the last er stage is the active er molecule 
and again er the indus-, the industry has er been a problem in research see if 
that's working okay so we move on to overflow metabolism that's probably it er 
it's 
important to remember that er antibiotic producers are often er in found in 
oligotrophic environments where there's low nutrients such as soil and water 
but with low nutrient availability er and therefore their primary biosynthetic 
pathways er tend to be not very tightly regulated they tend to be 
constituitively er switched on er and er okay i'll come to come to the next bit 
in a minute okay the er the production process itself of antibiotics is 
metabolically and genetically expensive and it's very tightly regulated er 
which suggests that in itself it has worth to the ord-, er to the organism 
otherwise you'd use a a a a more efficient means of er producing them and so 
the suggestion is that this is a safety mechanism er if you're in an 
environment where you're constantly producing all your intermediates and 
suddenly externally something becomes available er you have an internal build-
up of amino acids or something that you can't get rid of and er it's suggested 
that these can 
be shunted into the secondary metabolic pathways and ejected from the cell er 
problems with this er 
sf5013: namex two minutes 
sm5012: okay quorum sensing er has been shown to occur er why would you want 
all the organisms in the area to suddenly er get rid of all their intermediates 
er for the primary synthesis er resistance genes are required er what would be 
the point in producing a toxic er 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 er we 
it's very hard to design experiments that reflect what goes on in the 
environment because er because secondary metasm-, metabolism is so subtly 
regulated er it's 
difficult er to get the same conditions in a lab as you would in the 
environment er 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 er most worked on strains produce er 
the theories i came across were all looking they they were all looking for a 
universal theory to cover all secondary metabolites er which i don't think can 
be the case because you have things like erwinia which produce er an antibiotic 
given at a very specific time er er for an obvious purpose but then the 
streptomycetes which er seem to have very different control mechanisms er but 
it is possible to combine the appro-, the approaches and both the products and 
the process is necessary er so perhaps they had an evolutionary role as 
prebiotic effectors and then selection favoured the metabolic shunt and then 
you realize that the key s-, well didn't realize they didn't evolve to that the 
end products er could be turned into something useful and provide a selective 
advantage in terms of being er a biosite er just one further point if you 
compare another er killing mechanism used by bacteria this time maybe 
enterobacteriaceae and bacteriocins they have er 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 
sf5013: thank you very much namex can i just start the questions by asking er 
you said that antibiotics have many other functions 
sm5012: mm 
sf5013: such as being immune modulators have you got any other examples 
sm5012: yes er 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 er sex pheremones and er signalling 
molecules between in within symbioses and things like that 
sf5013: okay thank you anyone else got a question 
sm5016: yes sorry can you just explain what you mean by er retroevolution 
sm5012: right er if you have okay we're going back to an early evolutionary 
cell er and it hasn't got any biosynthetic pathways at the moment sorry i need 
a scribbling thing er er it produces all its er needs to take in all amino 
acids from its environment er and so it has er let's use histidine as an 
example it has er a decorative enzyme for histidine and then it modifies that 
enzyme and produces a second enzyme er and see what they er enzyme two which 
allows it to also take in er a precursor of histidine er and then that provides 
a selective advantage over its other relations er its competitors and so we 
start to build up a pathway backwards you don't start with th-, 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 
sf5013: any other questions anyone no can i ask then er how would work to 
regulate antibiotic production 
sm5012: okay er in phenazine the example i've come across is on phenazine 
biosynthesis by pseudomonas er and it linked er a a t-, a two-component 
signalling system with er production of the antibiotic er and it seemed this is 
under er control er produced by all the other bacteria so when a certain level 
er is attained in the environment er 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 er individually 
sf5013: mm okay thank you 
sf5010: er are there either of the two hypotheses er that you personally favour 
which do you think is the most likely 
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 er one or the other but there there there are possible 
things wi-, 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 er they all have the same purpose 
sf5013: thank you 
sm5012: thank you 
sf5013: anyone else no 
sf5017: 
this is namex and he's going to be talking about intracellular bacterial 
parasites of amoebae 
sm5018: thanks for the introduction er 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 er legionella 
pneumophila which has turned out to be the er first discovered er 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 er i'll then cover briefly a second example which is that of 
burkholderia cepacia which is er the most recently discovered intracellular 
bacterial parasite er 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 er er er ubiquitous protozoa that are predators of 
bacteria in soil and aquatic environments and they uptake bacteria by the 
process of phagocytosis er degradation of the bacteria contributes to soil 
fertility and in a-, aquatic environments the amoebae play an integral role in 
cycle of nutrients in the aquatic food chains er and then about twenty years 
ago or so it was er discovered that not all bacteria are suitable food sources 
and er 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 er the first set here which includes er 
legionella pneumophila legionella-like amoebal pathogens which are unculturable 
legionell-, legionella-like er 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 er and then there's 
er a second group here which one of them consists of vibrio er and acanthamoeba 
undergoes multiplication and then there's a final set which there's a er where 
it's just more a survival stage and here you have er just a hartmannella and 
acanthamoeba and you've got things like 
er 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 er er and so to summarize then the 
fate of the internalized bacteria can f-, ooh er can fall into three categories 
so there's er multiplication and lysis just er plain old multiplication in 
order to survive and so let's go on to legionella pneumophila so er this was 
the first er intracellular bacterial parasite that was discovered er and in the 
human lung legionnaire-, legioni-, e-, legionella pneumophila is an 
intracellular parasite of alveolar macrophage and it's er the the causative 
agent of legionnaires' disease er and Pontiac fever now er for treatment and 
prevention of these the current situation is for er treatment you've got 
antibiotic treatment with 
erythromycin and other er antibiotics that can penetrate eukaryotic cells er 
and prevention which has also been mentioned before is er practised at the 
institutional level with with er dechlorination and U-V irradiation and 
superheating and other continuous processes er to to go on to the role of 
protozoa in legionnaires' disease er there are many protozoan hosts which have 
been identified in the environment er in outbreaks of legionnaires' disease 
both the amoebae and the bacteria have been isolated from the same source of 
the infection er the following replication within protozoa the bacteria show a 
rise to resistance to harsh conditions such as er the chemical disinfectants 
and biosite and which i introduced there er also in the on lysis of the 
protozoa the bacteria released in respirable size er compartments as such which 
can be er 
inhaled and also bear resistance to harsh conditions and and that points also 
er fits with er like i say that er it can infect mammalian cells a lot better 
and also the final point there is er there are no documented cases of 
transmission between individuals of just it's the bacterium alone so therefore 
the f-, 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 er 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 er attachment of the bacteria into 
the amoebae is by a host receptor on the amoebae which is er galactose acetyl 
galactose in English is a er a lectin type molecule er it's er also er a 
tyrosine kinase receptor so er under tyrosine dephosphorylation it leads to 
recruitment and rearrangement of the cytoskeleton er and that's 
shown on this figure here just talk through this er er so here you've got the 
er lectin receptors on the host with the er phospate on the tyrosines and also 
these are other proteins on the base which also phosphorylate it and er upon 
signal transduction mechanisms from the bacterium here this leads to er 
dephosphorylation of these proteins and uptake of the bacteria by a process of 
receptor mediated endocytosis er there's also there's er an unknown mechanism 
whereby the colony infection er er affects gene expression of the host here er 
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 er you can almost see er 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 er the next stage is growth of the 
bacterium in a phagosome which is an endoplasmic reticulum surrounded er 
portion of the cell and also there's an addition of phagosome acidification and 
lysosome fusion so the bacteria can persist in this er these phagosomes are 
just shown here er bottom down there where you've got er 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 er this is by the process here of er a stringent 
response model which is in the diagram there so er 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 er leads to activation of 
regulators su-, such as er stationary phase sigma factors R-P-O-S and you get 
these switch on of genes er which leads to secretion of enzymes and lysis of 
the host so er to summarize this is covered in the top diagram so here you have 
er the motile bacterium and then it attaches to the amoebae here forming the 
phagosome and then you get this er replicate replicative state and then the 
switch on of of virulence genes by the decrease in amino acids concentrations 
and er this leads to lysis of the host here also er the infection of the human 
is by the er in the er mov-, remov-, er movement of the er amoebae into the al-,
alveolar macrophage down the lungs and you get the legionnaires' disease 
right so second example is that of er burkholderia burkholderia cepacia which 
is er an opportunistic pathogen for about forty per cent of cystic fibrosis 
patients er and it causes a necrotizing pneumonia which is er an enhanced 
immune response which lead us leads to lung tissue damage er the pr-, main 
problem here is that er there's the treatment with antibiotics is er 
problematic due to an intrinsic antibiotic resistance in the bacterium er and 
research had proved that amoebae turned out to be the reservoir and possibly 
the vehicle of transmission of this bacterium er it's more a survival stage 
because er 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 extrasta-, r-, 
extracellularly on secreted amoebal products er then the bacteria are released 
in these membrane bound vesicles and transported 
to the lower respiratory tract and er 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 ne-, nasal 
mucosa where the temperature there is thirty to thirty-two degrees er the 
evolutionary role of amoebae and macrophage er it's turned out that infection 
of the two hosts has had a a common molecular basis and er 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 er potentiates infection in both hosts er they've also done a fair 
few genetic studies on legionella and they've found er there's two gene loci I-
C-M which is intracellular multiplication cluster and P-M-I which is protozoa 
macrophage infectivity which er 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 er so there's been a a theory 
proposed that there's been coevolution of protozoa which has allowed er the 
legionella pneumophila to develop multiple redundant mechanisms and parasitize 
the protozoa of these er 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 er the er bacterial amoebal interaction provides a 
means of transport and transport survival and replication the er intracellular 
niche provides protection against adverse environmental conditions in the 
treatment of parasites and other other components there's er 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 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 
sf5017: right has anyone got any questions 
sm5018: 
sf5010: er in the two cases that you discussed 
sm5018: yes 
sf5010: do you know if er if the amoebae themselves may contribute towards the 
disease caused by the bacteria within them whether or not 
sm5018: er 
sf5010: they may influence the disease itself 
sm5018: er the er i don't think that's the case i think more it's er 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 infections go 
sf5017: anyone else 
sf5013: how great is the diversity of amoebae in capacity to less to bacteria 
is it just like a 
sm5018: er 
sf5013: a single dense amoebae 
sm5018: er the i think they've worked out there's thirteen hoped-, thirteen 
different of each strains of amoebae that can be infected by er 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 
sf5013: yes thank you 
sf5017: anyone else got any questions 
sm5018: oh 
sf5013: is it quite a problem do they do they facilitate severe cases 
sm5018: er i think it's the only means of transmission really so i i presume it 
occurs in all cases of legionnaires' disease anyway so 
sf5013: mm-hmm 
sm5018: i presume presume it's a fairly severe problem 
sf5010: 
do you think it would be useful to er 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 
sm5018: er well yeah i mean that's how they're doing this prevention mechanism 
where they're doing er continued chlorination and er tre-, 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 
sf5010: are there any chemical compounds 
sm5018: not that i'm aware of no i mean apart from chlorination but 
sf5010: okay 
sm5018: there aren't any that i'm aware of 
sf5017: anybody else thank you