INFD3012 Lecture Notes (2) PDF

Lecture 2 - Hepatitis Virus and Disease Hepatitis A and E are more acute and endemic areas compared to B, C and D. Generally transmitted via blood transfusion or sexual. Carrier state - ability to transmit. Fecal-oral transmission - e.g. ingestion of contaminated water/food....

Lecture 2 - Hepatitis Virus and Disease Hepatitis A and E are more acute and endemic areas compared to B, C and D. Generally transmitted via blood transfusion or sexual. Carrier state - ability to transmit. Fecal-oral transmission - e.g. ingestion of contaminated water/food. Hep A and E tend to resolve much quicker than other types. Acute - can spontaneously resolve with own immune sys. Hep B + C viral particles able to live in hepatocytes without damaging cells unlike some viruses which damage the cell, usually host immune response is the cause of liver disease. Perinatal - womb/uterus, early age from mother to foetus. Serology - testing blood + what kinds of antigens (anything eliciting immune resp, Ag) present. - Detection of surface protein antigen (enveloped protein, coated in diff virus/proteins). - Surface antigen - diff types of proteins on surface of virus. - Elicits imm resp against surface protein. - HBe - envelope protein. - How much surface antigen present vs amt of antibody being produced. Active virus - prod its coatings, envelopes, surface proteins (chronic or acute stage of virus). Envelope antigen last thing prod by virus to enable viral spread. Presence of surface antibody in large quantities can enable spontaneous clearing of virus in cases of reinfection - i.e. amount of antibody must equate or exceed the amount of antigen present. Takes on average 14 days to prod antibody in response to antigen. In case of HBV look for DNA to detect, RNA for other types of hep viruses. LHS is acute infec (weeks), RHS is chronic infec (years). IgM is first AB to respond in case of infec. Able to prod responses (antibodies) to diff parts of virus itself - e.g. core protein (centre, wrapped around by surface envelope proteins). IgM should eventually go down due to seroconversion into IgG, not the case in chronic infec where high levels of IgM are maintained. In chronic infec core antibodies prod not as good as surface/envelope being prod (stops entry into other cells). Diff clinical conditions reflect diff amts of proteins and antibodies within blood. Based on presence/absence of diff serological markers able to determine chronicity or time frame of partic infec. Tolerance - on edge from going acute to chronic. Control - have ability to suppress virus. ALT usually not present in acute, usually a sign of chronic conversion of Hep B (leaks out from hepatocytes into blood). Infec control - e.g. report to authorities to ensure treatment. Unable to get vaxx (recomb HBV surface antigen based) if immunocompromised. Higher levels of viral DNA copies present = increased risk of chronic infec + downstream HCC. Risk of HCC varies based on age of onset, immune state + subset of HBV. Integration of HPV DNA into cells increase infectivity + imm state. Hep D is always coinfection with Hep B (req HBV to survive). Hep D unable to prod its own envelopes/surface proteins.  Hep D does not change between isotypes and variants of it. Hep D envelope derived by surface antigen of HBV. HDV can be acute like HBV if you have right treatments. Interferons - particles our imm cells prod that interfere with viral replic. IFN induction does not work in treating HDV well, works for HBV. Coinfection of HDV + HBV leads to chronic infec quite quickly. Injection drug use - e.g. contact with contaminated needles. Other sources of transmission of HCV (esp in healthcare facilities) - inappropriate disposal of blood products. Vertical transmission in HCV20 cells/mL indic some form of infec within neurospace. - Non-treponemal tests to test CSF. - FTA is a type of treponemal test, many false positives when looking @ CSF. Better + easier to be treated early on (can get tested early), treatment varies dep on state of condition (e.g. stage). Alternative drugs are antibacterial if penicillin not working or allergic - prim, secondary + early latent stages mainly, more systemic in tertiary stage. For neurosyphilis need IV as need to get penicillin in rapidly + dissem thru entire body. - Alternatives are stronger antibiotics. Want to do follow ups as antibiotics may have not worked straight away. Reassess for bacterial presence @ given month time points w diff titers (assess for increase in titer = bac load). Neurosyphilis - treated pretty much rest of life. Treatment of syphilis varies on the basis of stage. Lecture 5 - Parasites in the Clinic Not everyone presents to emergency department. Not every doctor’s office is good at travel medicine - i.e. may not know of diff types of diseases around the world. STIs on the rise after COVID lockdown. Always need to take into account patient’s travel histories. Case study 1. - Asplenic - without spleen (i.e. removed). - Hematuria - blood in urine. - Collected in EDTA tube to stop coagulation with EDTA. - Thin blood smear taken from EDTA tube + stained (giemsa stain - stains internal components of different parasites, RBCs not stained in this case). - Blue string like feature observed in RBCs should not be present in healthy patient - could be the cause of disease. - Check if intraerythrocytic (inside RBCs) or in circulation - indication of what parasite may be. - Caused by Babesia sp. Genus - ring like structures are typical of this parasite. - Tetrad (4 quadrants) observable. - Can be either intracellular or extracellular - important to distinguish between parasites (some have to reside in cell whilst others don’t have to). - Followed up by PCR diagnosis to confirm condition. - Human’s not part of the life cycle - unfortunate interaction with particular lifestyle of parasite. - Usually from infected tick eating off mouse - replication usually occurs within mouse. - Tick sucks blood from infected mouse → fertilisation within gut of tick (forming of sporozoites, more active part of parasite that can infect human cells). - If tick bites human is ingesting blood + pouring out sporozoite into blood and infecting cells. - Replication occurs in human cells but not as great as in mice. - Lack of proper screening + giving blood donations - can be infecting other people downstream. - RBCs get recycled through spleen - lack of spleen = infection not getting cleared out properly. - Treatment length/medication dependent on circumstances - e.g. how infected you are/parasite load. - Clindamycin - antibiotic. - Most treated with single drug, can use combination therapy in some cases (e.g. no spleen). Case study 2. - Parasite under microscope had teeth like structures (spines) - wiring into person’s eye (inside, hard to remove). - Identification of parasite under microscope - can see diff kinds of spiracles on mouth part, whether it has circular spines, parasite has ridges (similar to what is observed in maggots).  - Ocular myiasis - larvae/maggots attaching to surface of skin (eye in this case). - Sheep botfly - usually infects sheep, somewhere person has come into contact with a particular mosquito or fly (injected larvae onto it). - Determined solely on morphology of parasite. - Location in patient important - i.e. could be a diff parasite if diff part of body which is covered up, eye is an easy access area for fly to come into contact with. - Often see lots of flies around sheep's nose - larvae deposited around eyes + larynx area as well. - Have to attach onto mucosal membranes - hence usually not seen on skin, eyes have many mucosal layers. - Likely attached at point where it was little egg + hatched over period of couple of days - when larvae attached. - Mandibles - graft onto eye/mucosal area. - May get from sheep botfly itself (e.g. area of high risk, farmland) or mosquito (vector), when sucking blood drops off eggs of larvae + infect host. - Larvae quite big - does not secrete any toxins, does not damage host (just feeds off blood + mucosal areas). - Treatments provided are for humans (just surgical removal), different if for sheep (can get treated). - If travelling to high risk areas - having mosquito repellant, etc to prevent this form of transmission (another part of prophylactic treatment for sheep botfly). Case study 3. - H&E stain - nucleic acids. - Giemsa stain - extracellular matrix of tissue. - Helped distinguish between genetic material and foreign bodies in this case. - Caused by Leishmania sp of parasites. - Were types of spherical ovoid structures in nucleus that were not foreign - would not be in host cell if foreign, helps us determine what kind of parasite it was. - Bacteria have flagella/cilia - help it move around. - Sand flies (vector for parasite - i.e. transmission) endemic to Peru - travelled to by patient. - Lesions can be highly ulcerated - pus, fluids, scratchy, itchy. - 30 species of Leishmania. - Integrated into pathway of parasite, humans not usual host for it to replicate in (usually in hind gut of sand fly or mosquito). - Sand fly takes blood meal + simultaneously injects phagocytic sporozoites. - In this case human stage starts with macrophage (phagocytic cells that take up parts of particular Leishmania) → starts to propagate within macrophages, helps it replicate so it can disseminate out once it kills off the macrophage (hence distributed across body quite easily, thru lymphatic system). - Following release of sporozoites/small parts of parasites, if bitten by uninfected sand fly, infects sand fly and cycle continues on. - In terms of treatment have to think about individual - where it is being contained and contracted, depending on what part of world it is from determines what species + how it is treated. - History of infection - i.e. has individual had previous infection or travelled to other parts of world as well.  - Where is the infection showing - i.e. just on skin, mucosal regions (harder to treat than if just on topical surface of skin). - Ointment can be used instead of oral agent if lesion easily located on skin + if being treated early on. Case study 4. - CT scan of chest - presence of air bubbles where they shouldn’t be present. - Biopsy stained with H&E - abnormal structure. - First may appear to be lung cancer, however non-smoker, hence more skewed towards parasite or infectious agent. - Circular observations that shouldn’t be present in normal tissue. - If dog not treated for heartworm, individual can also be infected with it. - Need to assess morphological features, number of reproductive tubules, how it actually eats + presence of lateral ridges on surface of worm. - Dog infected by vector such as mosquito. - Humans can either get directly from mosquito which has bitten infected dog, or from faecal areas or in close contact with saliva from infected dog. - Not routine in some parts of world to treat dog for heartworm. - Different larval forms of worm - starts off quite small in mosquito, but once it gets into host (i.e. dog) able to replicate and grow quite large. - Humans are suboptimal host for this partic heartworm, hence usually don’t get infection with heartworm in heart (i.e. anything that goes into human heart dies off), however in dog happily lives. - Surgical removal of worm. - Lung is primary place where heartworm resides in human (but can travel to/reside in other places) - once worm in lung, immune system has surrounded it (macrophages, B cells form granuloma, similar to TB) → removal of entire lesion. - Effective treatment for dogs - monthly tablet or yearly injection. Case study 5. - No medication prevention. - Schuffner’s dots, Maurer’s clefts - diff kinds of Plasmodium falciparum (causes malaria). - Falciparum is the major one - most commonly found in Africa + sub saharan Africa. - Thick + thin smears - depends on how much of volume you’re using (i.e. taking blood - drop of blood or more liquids, determines how much you can visualise). - Schizont - infect hepatocytes (liver) and generally found within liver, merozoites found floating around in blood itself. - Infected w RBCs - main part infected by Plasmodium falciparum initially (mode of dissemin in body). - Visual microscopic test + PCR - some malaria looks exactly the same (some Plasmodium look like other Plasmodium), but treatment varies hence need to determine which one is infecting. - Different parts of world more endemic than others (malaria) - not common in Aus. - Have effective treatments - if caught early prior to mosquitos getting around to bleeding people, not much of a concern. - Only certain subtypes of total species infect humans, but are able to infect other animals. - Treatment depends on where in world you are.  - Partic parasite has become drug resistant in certain areas - affects how it is treated. - Antimalarial drugs. - Some things in terms of treatment not to be given to pregnant women - may cause spontaneous abortion. - Chloroquine + doxycycline - major drug treatments for partic parasite (inhibits life cycle of malaria) → using diff kinds of drugs helps to clear off malaria. International travel - consider what drugs/prophylactics you may need to take (malaria is biggest one, but other like TB + STIs can be prevented as well). Hep A + E - contam food + water. Vector borne - thru mosquitos, have prevention but hard to stop mosquitoes. Lecture 6 - Malaria and the Host Different subspecies of Plasmodium. Mortality underreported - many deaths in third world countries not represented in statistics. Plasmodium falciparum - mortality not just directly from initial infec, but from complicated malaria. Plasmodium vivax - different complications due to increased parasite and immune distress in area. Life cycle does not change much amongst diff types of Plasmodium. Anopheles mosquito → bitten by mosquito, injects sporozoites (form of Plasmodium itself) → preferentially infect hepatocytes (don’t replicate in blood, replic in hepatocytes to start life cycle of protozoan Plasmodium). Liver stage (first stage) → sporozoites go into hepatocytes + replic → form merozoites (life cycle part that infects RBCs) → once RBC infected, causes for it to explode + can start replic again (asexual cycle of protozoan) → prod schizont (whole bunch of merozoites formed in RBC, usually ends up in lysis of RBC). Sometimes (not always) can get sexual cycle for Plasmodium → producing gametocytes, taken up by mosquitos, given back as sporozoites (gametes transformed into this in back and hind gut of mosquito) in humans. Two key stages of life cycle in humans → liver + blood (preference for hepatocytes + RBCs). After 5-7 days all sporozoites in hepatocytes, get replication in liver. Merozoite morphology - if stained with certain stains look like small rings (trophozoites). Schizont ruptures infected RBCs (48h). Parasitised RBC - becomes sticky (don’t want this, want them to flow thru → parasite does not want this flow thru to spleen and consequent eat up by macrophage), sticks to endothel → major problem with complicated malaria (cerebral - blood clots in brain, pulmonary edema - lung area). Immune resp 12-13 days after - cytokines, interleukins prod due to parasitised RBCs. When we want something to be killed by immune sys, always sequestered in membranes → in this case have many exploding RBC prod sticky contents which triggers imm sys to go overdrive + prod imm resp. Non-imm resp - in certain ppl, dictates whether it goes from uncomplicated to complicated malaria. P. vivax - has long incubation time (i.e. can persist in liver for v long time). In some cases some may think it is resolved + have not kept up w treatment (i.e. usually take malaria tablets one week once back, good for some but for this one if not taken constantly get relapse), but due to long incub time may get relapse. P. falciparum - infects normal healthy RBCs. P. vivax invades RBC within 42-48h of its formation + fewer merozoites formed. Duffy RBC antigen - chemokine recep on RBCs (found on some people), stops HIV (in many cases gets uptaken as opposed to infecting macrophages) → good to have in HIV cases, but in malaria preferential to be uptaken into RBCs. P. ovale - 48h to prod merozoites. P. malariae - 73h to prod merozoites. Subtype of Plasmodium infected with - different ways of identifying which malaria + treating it. Under microscope after staining can see differences in subtypes of Plasmodium (i.e. different RBC morphologies). Trophozoites + schizonts quite similar in P. falciparum - small + punctate, many stages within liver itself rather than peripheral blood.  P.vivax - trophozoites + schizonts look diff, amoeboid + large (hence RBCs expanding, bigger components within them) + young RBCs bigger in size than older ones. P. malariae - look like bands more than punctate/amoeboid shaped structures. Tufted ends - membrane ruffled. P. ovale - looks like macrophage under microscope more than RBC. Not all mosquitoes can carry malaria, only if within this species family. Sporozoite production occurs in mid or hind gut of mosquito - hotter weather favours life cycle of Plasmodium + mosquito (shorten time taken for sporozoite to replic within mosquito). 16-33 deg Celc (and under 2000m altitude) - avg temp req for anopheles mosquitoes to survive within (likely to be more safe in cold climates from being infected + getting to around 40 deg Plasmodium + mosquitoes don’t do as well comparatively). Longevity - if mosquito has one big meal and doesn’t have to eat for a few days, helps Plasmodium replic (not losing sporozoites/other parts of it when mosquito bites another person). Behaviour - most mosquitos in dusk + dawn, most cases occur inside + evening (not outside). Breeding - presence of stagnant water around, can increase amt of mosquitoes there vs if dry + arid (won’t have as much + thus lower transmission). Anopheles gambia - highest efficiency in prod Plasmodium and transmitting as vector. Epidemiology varies quite widely depending on where it is in world, human state + type of Plasmodium. Climate change - increase in global temp help facilitate spread to cooler regions of the globe (become more temperate). Seasonal rainfall - has role in how mosquitoes transmitting + replicating, increased amounts will result in increased cases + spread. Depending if it is fresh/salt water, marshes or swamps increase spread compared to other parts. Genetic factors - human host, i.e. factors which don’t favour RBC entry, will have less Plasmodium in those sorts of pop’ns. Increased prevalence in third world countries as comp to second + first world countries - impact malaria on being able to access preventative measures. Cases from international travellers returning who had not taken correct prophylactics. Anaemia + fever occurring - imm resp to damaged RBCs + hepatocytes from infection. Anaemia - directly as a result of parasitised RBCs, reduced amount due them being exploded (signals imm sys) hence lowered iron which leads to anaemia. Normally everything in imm resp sequestered into memb, when you have bacteria/bug that is exploding does not care abt that hence getting contents which are highly immunogenic (immunogenicity factors which help increase imm resp). Fever - biggest imm resp → fatigued, increased body temp, myalgie), mostly due to TNF alpha (prod by imm cells affects cardiac output + cerebral imm resp/thinking patterns + how glycolytic components in muscle work, decreases ATP produc hence get fatigue → all due to macrophage or DC resp (part of innate imm sys). Adaptive imm sys - B cells prod antibodies, T cells help kill infected cells (hepatocytes/RBCs). Direct component of Plasmodium itself is elucidating an imm resp in this partic species of Plasmodium. Increases diff receps that help sequester RBCs. Can have diff types of receps thru body depending on types of tissues we have. PfEMP1 - anchorage point of infected RBCs to endothelium by Plasmodium itself, binds to CD36 → adhesion can start after 12h of being infected (i.e. once merozoites released + gone into RBCs, can increase anchorage point) → quite quickly can get sequestering amount of RBCs in vessels → can get diff types of blood clotting (particular in brain, but can get other areas such as  lung endothelial tissue, placental if malaria contraction occurs while pregnant, any type of tissue where RBCs flow thru can get these complications → complic vs uncomplic malaria). Receps which RBCs are anchoring to via PfEMP1 are endothel receps. CSA only a concern when pregnant during malaria contraction, main ones are CD36 + ICAM-1. Diff genes have high selection preference/pressure on diff types of Plasmodium in diff regions of the world. Duffy antigen (P. vivax uptake facilitated) expression absent in most people from Central Africa - hence P. vivax not very prevalent in region. Most people have normal healthy RBCs (great for Plasmodium uptake) - in case of anaemia or sickle cell disease, thalassaemia + melanesia, these change RBC morphology/how it works, which does not help Plasmodium (i.e. more protected against malaria). Diff structural abnormalities → HbS (sickle cell shape as opposed to more open and ovoid part of RBC normally in there - merozoites cannot get in and replic, hence don’t get infected), thalassaemia (small RBCs = not enough space for merozoites to get in and replic), melanesia (morphology change of RBC causing inability of Plasmodium to infect areas). Spread of diff types of Plasmodiums, selection pressures tell which ones are going to be more prevalent, hence should take into account travel history to help treat partic malaria. DCs + macrophages have PRRs (detect diff pathogen molecules on cell surface + elicit imm resp) → GPI (on falciparum @ surface from merozoites), have TLR2 (can see partic antigen from parasite) → imm resp (DC will phagocytose, uptakes antigen + presents it to CD4 + CD8 T cells, can also have dsDNA uptaken by DCs to activate imm resp). Depending on type of antigen cell is producing → cytokine produc → fever resp + recruit + activate other immune cells. If activating Th1 cells or subset of CD4 helper cells → large amts of interferon prod from DC or adaptive imm sys (macrophages love interferon - type of cytokine activates it and upregulates phagocytosis to rid of infected RBCs if they happen to get to areas they need to for imm resp such as spleen → though parasite sneaky, has anchorage pts + makes RBCs sticky to prevent from getting where it has to for macrophage response). Cycling effect w imm sys - macrophages start prod cytokines that cause fever + other recruitment of imm cells. If hepatocytes infected cannot easily engulf then as they’re sitting inside lover and not free floating in blood → where CD8 T cells + NK cells to kill intracellular parasite → kills off sporozoites found in liver cells. Adaptive imm sys plays important part generally after 14 days post infec or reinfection with malaria → if getting sporozoites still transmitted through blood, mosquito not injecting straight thru to liver → if have antibodies floating around in blood, can tag it, opsonise sporozoite → can be uptaken + destroyed, stop entry into hepatocytes, good at stopping init infec w sporozoites, but doesn’t always happen v quickly (i.e. in case of first exposure where time taken to develop imm resp) → if it enters hepatocytes, have activated CD8 T cells, can kill off infected cells, but if this not gotten in time or it all happens at once (i.e. do not have enough CD8 T cells @ time) → release of merozoites (may have antibodies against this as opposed to sporozoites → protection). If all evaded and gone into RBCs, merozoites still prod antigens that may become on cell surface of RBCs or RBC may change in shape - imm resp (i.e. opsonization by ABs or upregulation of macrophages in spleen). Sexual reproduc - gametocytes also prod imm resp against it. B lymphocytes prod antibodies. Clinical features from imm resp. Many children affected by Plasmodium due to lack of great imm resp at their age. Asymptomatic does not help with life cycle - unsure if we are spreading or not.  Decreased amt of moderate transmission + low transmission + travellers (i.e. if going to Africa + coming back have lower risk + transmission, but can have severe + symptomatic disease if prophylactics not taken/lack of protection against mosquitos taken and get bitten by a lot of diff ones). Pregnant women have higher risk + higher risk of prod symptoms. Uncomplicated malaria: - Doesn’t usually start straight away for liver. - Merozoites cause of RBC destruc vs hepatocytes. - Falciparum is major one. - Symptoms range depending on individ, parasitic load + how imm resp works. - Symptoms cycle again due to amt of RBCs lost vs amt of merozoites forming. - If infec cont and no medical treatment seeked - enlarged liver (hepatocytes exploding + infected by sporozoite cycle), anaemia from RBCs, enlarged spleen (increased amt of macrophage activity req for infec clear up + dead RBCs + bone marrow compensating, cyclic effect). - Easy to treat if treated in time. Complicated malaria: - Don’t know if infected, don’t get treated + constant imm resp + change in parasitic load over time - multisystem effect (in Asia more travellers vs residents of country). - Cerebral malaria - adhesion of blood vessels in brain → blood clots + brain damage (depends on location of brain/what part of it vs symptoms) → most ppl get seizures or comas quite quickly due to widespread, but some people can have localised locations in brain + can have speech, auditory or ophthalmic issues. - Getting rid of blood clots + reducing stickiness - can recover well if treated in time, but if lack of blood and oxygen to brain over time brain dies off. - Severe anaemia + renal impairment/failure (due to destruc of other imm resp in body) due to RBCs being killed off for some time. - Not getting good glycolytic change, not getting nutrients spread around body - hence severe anaemia. - Infec in lung - respir distress → liver failure. Uncomplicated more common, get infec + treated + survive (complicated is end life cycle after no treatment). Malaria diagnosis can vary in diff areas - can use macroscopic differentiation to observe diff life cycles of diff types of Plasmodiums (can look quite diff) + RBC morphology changes. Microscopic analysis good for quick check, but needs to be confirmed via other kinds of tests → RAT, nucleic acid PCR (clinical tests) to find out which subspecies of Plasmodium as treatment dependent on where it is found + subspecies + age + complications. Types of treatment change depending on things you are trying to treat - e.g. allev symptoms can have diff antibiotic combinations if req (treating active life cycle of parasite itself). As P.vivax + P.ovale have long life cycle in liver important to do preventative treatment long term for these types of Plasmodium. Preventing spread - prophylaxis if travelling around, diff types of primaquine or chloroquine if looking for other types of Plasmodium. Most of the time suppressive treatment - not bad if getting early on treatment, can take some time to treat it.  Radical treatment depends on type of infection - P. vivax + P. ovale due to long incub period. Generally all single doses over a period of time used to treat these types of infecs. Like most parasites become drug resistant - hence important to know travel history/residency, many regions in world (mostly Africa + parts of South America) have become chloroquine resistant (major treatment used many yrs ago, now need to use combination therapies or other types of antimalarial drugs to combat those type of resistant species) → genetic testing to ensure getting correct treatments. Minimise contact with vector - i.e. not getting bitten by mosquito → e.g. repellant usage esp in early morning and evening (when mosquito most active). Areas that have lots of swamp land - routinely go out monthly, esp in warmer seasons + spray certain water areas (vector control, indoor for residential but not as efficient as cannot have person sitting inside whilst spraying dangerous chemicals + not as easy to do every time). Controlling @ larval stage - spraying groundwater, removing stagnant water (great breeding ground for anopheles mosquito) → stopping mosquitoes from replicating + spreading. See GP for prescrip for malarial drugs - taken two days before travel usually for most cases in world + continued for abt a week after return → most likely protects against most kinds of Plasmodium you come across. Depends on whether pregnant or infant on how you get treated, majority of ppl just prophylactics. Recently - malarial vaxx, not greatest efficacy but good for endemic countries (used mostly for children in Africa that have lots of high transmissions of malaria) → has to be used quite soon from infec, ideally before infection. If alr have complicated malaria vaxx won’t rlly do anything - req drug combinations working. Being vaxxed means imm sys helps stop replic in body - hence if bitten by mosquito, unlikely for them to uptake sporozoites + further spread. Lecture 7 - Central Nervous System Infections Neurons in PNS housed in collections of ganglia (collections of neurons) that sit outside of spinal cord (up its length) - responsible for receiving sensory input (sending info into ur brain) or relaying motor neuron messages out from brain. Injury in brain hard to repair - i.e. wound in periphery typically heals, but loss of neurons which make up brain (post mitotic cells - no longer actively dividing, hence cannot divide more to make more to replace them, only to a very small extent, but have to reroute info pathways in brain but of high difficulty to do). Vertebral column protects spinal cord. 3 layers of meninges underneath bones of our skull, between underlayer of skull + brain - wrap around brain + go all the way down spinal cord. - Dura mater - outermost layer → thick, fibrous layer made up of many fibroblasts (v tough, protective layer). - Pia mater - innermost layer → adheres v tightly to brain + goes all the way down spinal cord, v thin + delicate (one fibroblast cell layer thick). - Arachnoid mater - in between dura + pia layers → sits under dura mater + adheres more tightly to it, get projections of arachnoid mater which project down into pia mater (spider web appearance in cross section), gap between arachnoid mater + pia mater (subarachnoid space where cerebral spinal fluid found). CSF is a liquid which provides cushioning to brain + nutrients - where major arteries + blood vessels which supply brain found. Blood brain barrier - additional protection from infec of brain. - Selective gating system - control as to what is trafficked into brain (i.e. keeping out toxins, pathogens + keep imm surveillance low as don’t want lot of inflamm in brain - cytokines, chemokines, signalling in brain not good for neurons + supporting cells present). - In periphery endothelial cells have gaps/junctions that cells can traffick in between → in brain supplying vessels, endothelial cells lining interlock with one another (tight - controlling/limiting movement of imm cells from periphery into brain, only enter if req). - Low imm surveillance in brain - ideal for pathogen if it manages to enter, protected from imm sys to some extent. - Additional layer of control + to check those endothel cells + BBB intact - interacts with astrocytes (glial cell in brain) + pericytes (cells which control vascular tone). Pathogen reaches critical threshold/certain amt circulating in bloodstream (e.g. during viremia/bacteremia) → come up and flow thru endothelial cells or blood vessels that line brain + still able to cross BBB. Direct infection of endothel cells which form BBB - e.g. poliovirus → facilitate entry into brain. Some able to transit between tight junctions endothel cells form thru signalling to endothel cells + tricking them into loosening up junctions to allow them to squeeze in between. Some cause complete break down of BBB - lose endothel cells, all falls apart. Hard for us to understand what is happening in scenarios - see a snapshot of what has happ in moment and not the events leading up to it (can have animal models, but don’t always fully recapitulate what happens in a human → some understanding comes from animal model, other from studying autopsy samples - piece tgt what we think has happened).  In some cases virus/bacteria use trojan horse method by infecting imm cells that circulate in periphery - imm cells may traffick into brain occasionally, taking pathogen with them → e.g. HIV, patients would often have HIV assoc dementia due to virus entering + infecting astrocytes + lead to long progressive dementia → have antiretroviral therapy which suppresses viral loads, not observed as much now (in case where used). PNS interacts directly with CNS - taken advantage of by viruses (e.g. HSV, VZV infecting sensory neurons - travel between skin + sensory neurons, sometimes go the other way + jump into CNS, get up via spinal cord + into brain to establish infec, rabies virus gets into brain thru trafficking via motor neurons + getting up into spinal cord + brain, measles can do this too). Olfactory neurons - unique subset of neurons, help with smell (sense what is around person) → creates weakness in armour of protecting CNS → have cribriform plate (base of skull, has little gaps in it - allows axons from olfactory neurons to project down into top of nose + help smell) at top of nose where olfactory bulb is. Pathogens which can infect olfactory neurons can use that as a highway straight up into brain - can be used by several diff pathogens → Naegleria fowleri is an example, but rare infec (brain eating amoeba, enters via olfactory neurons). - See infection assoc w activities where ppl dealing with fresh water - where pathogen found. - Trophozoite, protist. - Activities where fresh water can get sprayed up into nose at high velocity - pathogen takes advantage + able to grab onto olfactory neurons straight up into brain, gets into brain + amoeba starts eating away @ brain cells → infec, high mortality rate. - Often observed in children. Direct spread - e.g. can be assoc w infec of sinus or ear (not controlled, pathogen keeps eating away + gets into brain), can be via surgery (i.e. any neurosurgery), trauma (e.g. no helmet + fall of motorcycle - skull broken + pathogens able to get in), use of cotton/earbuds (case where person who used them perforated their ear drum, pathogen got straight thru into brain + caused abscess). Once pathogen in brain can set up diff types of infec. - Meningitis - inflamm of meningeal layer, infec restricted to outer layers that wrap around brain + spinal cord (inflamm is conseq). - Encephalitis - where infec is in brain itself (neurons or supporting glial cells directly infected and killed/worn down - bacteria in there, usually viral cause). - Abscess - collection of pus from infec. - Can have combinations of infecs - e.g. meningoencephalitis (both in meningeal layer and progresses to encephalitis), encephalomyelitis (infec of brain + spinal cord). Pyogenic - assoc w lot of inflamm. Aseptic - nothing grows on bacterial culture. Meningitis more distinctive symptoms - neck stiffness (both meningeal layers run down spinal cord, become inflamed due to infec → moving neck = stretching meningeal layers, if inflamed info relayed is stiffness + soreness → indication it may be myelitis if seen in combination w headache + fever). Photophobia - sensitivity to light due to involvement with optic nerve. Neurological deficits more assoc w encephalitis.  Meningitis - talking abt absence of involvement of infec in brain + spinal cord itself, infec strictly restricted to meningeal layer → important in viral context. Group B strep from vaginal tract during delivery - pregnant women often screened to see if they carry this, risk for baby, can often be treated to eradicate bacteria + ensure safer delivery. Listeria - seen in older age group as well, bacterial infec assoc w weakened/naive imm resp/sys. Neisseria meningitidis - big one, meningococcal, partic for ppl in age grp of uni students + a bit younger. Haemophilus influenzae - bacterial infec, now vaxx against type B, observe nontypeable forms of it. Neisseria gonorrhoeae is a related bacteria to neisseria meningitis - similar causes, diplococci bacteria that have little pili on outside. Nasopharynx - nasal passages. Humans only natural host for Neisseria meningitis bacteria - 12 diff serogroups (5 cause disease, abt 1 in 10 people asymptomatically carry it) → typically carried for a while, infec cleared + may pick up another one and asymptomatically carry it → what determines if bacteria then able to go on and cause meningitis not sure of (sometimes assoc w virulence mechanisms of bacteria picked up, tend to see clusters, not always the case). Typically by age of adolescent, quarter of pop’n carrying it, though not all end up w disease (meningitis). In case where strain is highly pathogenic - way in which it causes meningitis is to first cause septicemia (infec of blood + spread around body) → results in typical rash (causing blood vessels/capillaries in skin to burst open - bleeding). Bacteria have to reach threshold/certain amt in bloodstream to be able to cross BBB + get into meningeal layer to cause infection → high morbidity + mortality, often sufferers left with long term sequelae bc of it. Way in which it causes disease/leads to problems with functioning of CNS - once bacteria in CSF (nutrient rich + kept @ great temp, not a lot of imm surveillance) perfect niche for bacteria to replicate + survive in → bacteria need virulence mechanisms such as capsule which allow for it to survive in bloodstream + make it that far. Imm sys as a result senses this and mounts strong imm resp to protect brain → inflamm (lot of imm cells rush to area + increase in BBB permeability) → thickening of CSF due to presence of bacteria + imm cells → damage to brain itself caused by cytotoxicity of bacteria (may be releasing toxins or other things which trigger imm resp) + imm resp. Vasculitis as a result of too many imm cells trying to come out of those blood vessels - affects flow of blood → patients can have stroke as brain not getting properly oxygenated + supplied by blood. Or could end up w hydrocephalus, high intracranial pressure - bones of skull are fixed + brain is a certain size, putting too much fluid into space can cause brain to herniate + be squeezed out of confined space of skull → can be lethal. Viral meningitis → typically most people recover, may feel unwell for a bit → caveat for when only talking about meningitis (i.e. infec of fibroblasts usually in arachnoid matter, viral infec limited to that) → if viral infec spreads beyond this point is a diff story. Enteroviruses - things related to polio but not usually polio - spread seasonally, peaks in viral meningitis often seen around spring when viruses circulating. Herpes viruses can do this usually during primary infec phase. Mumps was major cause of viral meningitis prior to vaxx. Immunisation status - e.g. vaxx against mumps, visit to Japan (no vaxx against mumps as issue with initial vaxx rolled out, hence regular cases still observed), visited area where arbovirus circulating).  Lumbar puncture to sample CSF - culture to look for bacteria, bacteria changes protein and glucose levels, molec studies to identify organism (e.g. PCR). Stool culture + throat swap for traces of viruses like coxsackie + echo - generally spread via faecal oral route. If meningococcal suspected - medical emergency + treatment needs to be started ASAP → if child comes into ER irritable, have headache and showing signs of meningitis are looking for rash and then start treatment ASAP → infection can become lethal within 24h of infec, needs to be dealt w v quickly. Ideally try get lumbar puncture to confirm, but may not be time and may be better to treat straight away → lose info as to what sensitivities bacteria may be, what antibiotic resistance it may have + what best treatment is → need to select antibodies able to pass BBB + be effective. Viral cause → not much can be done (less options) - manage patient, usually full recovery if it stays at meningitis. Diff viruses tend to like to set up house or infect diff regions of brain - not understood, may be base on diff types of neurons found → e.g. HSV likes temporal lobe (sets up there and directly kills neurons - focal necrosis), JEV sets up in thalamus → sometimes affects brain, diff regions of brain responsible for diff things. Neurological symptoms from patient may hint as to what region of brain impacted + what pathogen it may be. Classic triad of symptoms patient w encephalitis presents w, abnormalities of brain func signal infec is encephalitis (e.g. slurred speech). HSV most common cause of sporadic encephalitis partic in western countries. Normally HSV gives cold sore, HSV encephalitis killed off part of brain (is missing from image). Murray valley encephalitis - circulates around kimberleys, in Aus → JEV related → tends to set up residency in thalamus, mainly imm resp causes neuronal damage → typically fall into coma w this virus. Arbovirus - only spread by mosquitos, no person to person transmission, but can still have big outbreaks within mosquito pop’ns. Rabies still big problem (i.e. widespread), esp in Africa (rabid dogs + other animals), lyssavirus (bats natural hosts) → preventable (have vaxx), if overseas and get bitten by rabid dog can receive post exposure prophylaxis in form of antibody therapy which stops infec, 100% fatal if not treated. - Virus in saliva of rabid animal (transmission) - virus enters + replicates in muscle tissue → slow course (can take two months) → aims to get to motor neuron endings, once reached used as highway straight up to CNS → neurological symptoms start → one week til patient death (become v aggressive in this time, afraid of water - virus buys time to get to salivary glands → gets them to lash out and try bite someone else for spread). - Brain of person w rabies - not a lot of obvious signs that infec was there, not known fully how it causes damage to neurons (assumed it exhausts them/system and person dies - v little imm resp + cell death, unless stained for not lot of indic of virus presence). Nipah + Hendra - has been seen in horses in Aus, people who have cared for these horses have contracted infec → 12 fatalities out of 12 cases, high morbid and mortal → in asian countries like Malaysia + Bangladesh is where Nipah observed (fruit bats roost in trees, people collect date palm sap contaminated by bats carrying virus or spread to domestic animals like pigs which live under date palms, then humans can get infected after caring for pigs). - Virus spreads everywhere thru body, desyroys BBB.  Treatment of encephalitis - physical exam, travel histories, clinician looking for classic triad of symptoms, CSF for molec studies of viruses (herpes virus infec have RBC presence bc of destruc occurring in brain, capillary leakage = blood in CSF appears) → not many options for treatment, antivirals need to be able to get past BBB (don’t have a lot of useful ones, hence not lot we can do). Brain abscess - bacteria (usually, can be fungal) has gotten in + set up residency, begun to replicate → infiltration of neutrophils surrounding it (pus), focal inflamm of pus + bacteria, necrosis in brain as a result. Can have abscess of periphery and bits of it can break off and travel up into brain or vice versa (i.e. from brain to e.g. heart, daughter abscess). Pathogens responsible dependent on source of infec - e.g. direct trauma (pathogen living on skin that has gotten in + caused infec). HIV patients - low CD4 T cell counts, Toxoplasma gondii (risk for pregnant women) carried by cats + excreted in their faeces (if pregnant do not handle kitty litter). M. Tb not always located in lungs - can spread. Haematogenous - spread from bloodstream. Can see abscess on MRI. Can flush pus with antibiotics/antifungal (for fungal cause) after drainage - keep patient on antibiotics/antifungal, get culture of what’s there to determine sensitivities + pick best one for treatment. Prion - protein. Protein itself doesn’t reproduce (i.e. doesn’t make more of the same protein, but it causes healthy protein which folds in a partic way to misfold into prion form → converting folding of protein into a diff form → creates more from existing healthy form of protein). Prion protein cellular - PrP^C. When protein misfolds becomes PrP^SC (scrapie form). All have/encode PrP protein, typically in C form but if exposed to or start misfolding PrP^C to get PrP^SC, can becoming ongoing cycle + get more converted over time. Normal cellular protein expressed on outside of neurons + other cells (PrP). Lack nucleic acid - sets it apart from other types of infectious agents which have it as their means of transmitting genetic info + establishing infec. Misfolded form is a highly stable structure (globular protein which folds, composed of alpha helices, when it misfolds takes on beta sheet conformation, cross fibular/amyloid protein fold → characteristic is that it is highly resistant to degradation → need to expose to 10 M NaOH/urea to get it to misfold and breakdown - hence diffic to get rid of protein). Not typical latency period as assoc w HSV - have exposure to protein then have 10-15 yr gap b4 disease, what happens within that time in body not well known (likely slow build up of protein converting normal to scrapie form until threshold reached). Once neurological signs shown, rapid disease course (maybe not compared to bacterial/viral infec) - months to live @ this pt. No one has recovered, no treatment. Brain begins to look like sponge (TSA). Brain from sheep w scrapie looked like sponge - recognised sheep were suffering from this disease, showed neurological signs and unable to stand up and walk properly, typically began to rub themselves against fence posts (lose wool). Treated agent with everything known to destroy nucleic acid and still able to transmit disease - decided it was protein responsible.  First recog human form of disease - Kuru (100% mortality rate + neurological symptoms, 12 months following showing sign of disease death occurred). Infected person who has died - brain shared amongst tribe as part of ritual → transmitted infection. Only recognised when brain sample of someone w Kuru sent to vet in London + compared to brains of sheep w scrapie → able to show it was transmissible to chimpanzees. Practice of cannibalism stopped due to education, eradicated from PNG. Deposition of abnormal PrP leads to more neurons being killed off → spongy appearance of brain. As self protein - typically do not get imm resp, hence no form of infiltrate observed to try control, generally rare disease + no treatment. Unusual - disease can be acquired, genetic or sporadically occur. One of the proteins accidentally misfolds occasionally → scrapie form → slowly catalyses/converts other normal proteins to scrapie form → disease. Can be genetic/inheritable as encoding genome, may acquire mutation in gene which favours misfolding of protein into beta sheet formation. GSS + FFI assoc w diff mutations in proteins, determine how they misfold or timing + how fast it is to cause disease. Medical procedures like neurosurgery. Exposure to any forms of prion disease (even if genetic, i.e. taking genetic misfolded protein from someone with GSS and giving to someone who doesn’t have genetic mutation) can transmit disease → all forms of prion disease + CJD are transmissible. Species barrier - cannot get scrapie (cannot be transmitted to humans, but can be to cows → mad cow disease) → cows started to show neurological signs + became sick (looked like they had scrapie to some extent) after being fed animal products of those who had scrapie. Cows who were infected were in our food chain - new form of vCJD (variant CJD) observed in young people following infected cow consumption (transmission of misfolded form of protein + led to CJD outbreak in young ppl). Usually sporadic CJD + genetic forms occur later in age (i.e. peak around 60s, bit earlier for genetic). Epidemiological evidence, biochem analysis of protein + misfolding, slight seq variations + what happens w misfolding supports link between outbreak of mad cow disease + vCJD occurring in children + humans. Prion transmission - neurological surgery (dura mata grafts, growth hormones derived from human pituitary glands - good way to spread if picked up from someone who had prion disease surgery, instruments get contamin, hard to clean, can transmit). Lecture 8 - Pathogenic E. coli and the Urinary Tract E.coli can be pathogenic in other parts of body as well → e.g. brain for neonates, bloodstream (bacteremia), kidneys (go thru tubules + into bloodstream). Diff kinds of E.coli infect/invade diff parts of intestinal sys (small vs large). Nephritis - inflamm of kidneys. Increasing age increases risk for contracting UTIs. Are other kinds of viruses/bacteria that can cause these infections but mainly is uropathogenic E.coli (UPEC). Complicated cystitis - ascending infection up into kidneys usually. Depending on where source is coming from could be E.coli or staph. Dysuria - pain during urination. Clinically may have pain during sex, amt you usually discharge can change, may/may not get pelvic tenderness (some may get v acute pain to touch), only in bladder + imm sys not alerted as of yet (not much communic between imm sys and fluid contained in bladder - takes a while to get imm resp). Complicated UTI - gone up thru bladder → ureters → kidneys. Klebsiella usually in gut, staph aureus colonises in skin areas. Kidneys not immunoprivileged site - has lot of immune surveillance (immune resp - cytokines + chemokine rel increase in body). Abdominal pain as rising up to where kidneys located - not so much bladder infec, more kidneys but does not mean bladder is not infected (usually have both at same time, not all bacteria has travelled up necessarily if ascending infec). Bacteremia (bacteria in bloodstream) more common when infec travels to kidneys due to abil to filter blood to rid of diff components into urine → bacteria takes advantage of this + goes back other way. Bacteriuria - bacteria in urine. CFU = colony forming units. Could be asymptomatic for months/years and unknown if infected unless tested. Symptomatic UTI - pain, tenderness, pus in urine + imm resp. When talking abt UTIs usually acute - is in small phase, otherwise if getting for months or yrs (i.e. constant bouts of recurrence) → chronic UTI. Pathogenesis diff bc of host, imm state, haemostatic states, urine (big part of why we have bladder). Changes in osmolality - drinking lots of water one day will have weak urine, compared to if dehydrated. Changes in osmolality, pH and organic acids of urine have ability to change way UPEC is there or whether it survives in environ. Urine generally considered good culture media, however if you have normal homeostatic way (synthetic urine prod in lab to grow bacteria bc of ability to have high amts of salts, sugars + acids) sometimes also have antibacterial agents that are secreted into bladder as well - affect abil of pathogenesis if UPEC infec occurs. Body trying to make good environment for foetus to grow (during preg), all nutrients expelled into urine @ same time, bacteria happier that way. Getting rid of bacteria - voiding bladder over period of time, flush out bacteria. Adhesins prod on surface of bladder cells - stop bacteria from attaching to it + help clear off using urinary flow. Bladder has mucosal layer + other types of cells.  Uromodulin - little proteins that can attach to surface of bacteria (usually type 1 pili), blocking it so bacteria cannot bind + can get taken out as urine passed thru → innate/rudimentary means of protecting bladder + kidneys from infec w UPEC. Kidneys have more immunogenicity than bladder. Epithel cells can also prod cytokines + chemokines (as well as imm cells), can sense foreign invader → cytokines that cause fever, recruit macrophages and neutrophils req to clear off extracellular bacteria. IL-8 - biggest for UTIs, helps in neutrophil recruitment → pus (mainly dead neutrophils) in urine. PMNs - polymorphonuclear cells (neutrophils - half life approx 6h, come in clear infec and die - pus formation). If observe pus in urine means u have UTI and body trying to clear it. 10% undiagnosed, 1% pop’n contrib lot to it + other factors such as klebsiella or staph aureus. Have distinct virulence mechanisms to evade host imm sys + invade us - ascending infec up to kidneys. Each type of E.coli has a slightly diff way it attaches + has its abil to secrete diff things. Main thing - attachment to bladder cells itself. Squamous epithel cells, has good abil to stop things squeezing in thru it - where bacteria attaches + enters cells directly. Umbrella cells of bladder - thick layer for protection us + against pressure → diff amts of urine over time, stretching/shrinking of bladder, need to make sure it is quite tensile strength as well. Facet cells sitting on external surface of bladder, facing into where urine is. Have proteins coating outside of cells (like biofilm/mucosal layer) that is helping protect those cells → where UPEC comes into it, comes binds to these partic layers of uroplakin → done using type 1 pili. P pili + type 1 pili (attaches to uroplakin w adhesin molec) - attaches slightly differently. Have quite a diff number of diff structural proteins - FimA, F and G (structural parts - rod, @ top is v flexible end), FimH (adhesion molec of type 1 pili sys, recognising uroplakin on surface of epithelial cells, binds to it, beginning of UPEC invasion into epithelial cells). Electron micrograph - blue uroplakin + epithel cells of bladder, yellow rod shaped bacteria → anchorage pts, anchored into bacteria to epithelial cells (stuck there + not being flushed off in urine, can start invasion of epithel cells). Type 1 pili is anchorage point for UPEC infec. Ascending infec - P pili. Pyelonephritis - kidney infecs. Not all UPECs have abil to go up into kidneys, majority are able to due to expressing P pili (rod shaped). Schematic - diff adhesion molecules @ top, structural proteins embedded into cell wall. Pap protein = FimH (equivalent) - flexible + has hinge region, where it looks for glycosphingolipids on kidney epithel (where it attaches + anchors into it). PapG - distal protein akin to FimH. Asymptomatic don’t express P pili as not actually binding to kidneys → no imm resp → no symptoms. P pili triggers host imm resp → innate imm cells have TLRs looking for PAMPs (TLR4 - recruitment of neutrophils). Schematic - diff attachment proteins, toxins/adhesins it can rel, flagella (major for ascending kidney infec), P-type pilus (kidney attachment), LPS (toxin gram -ve bacteria prod), type 1 pilus (FimH attaching to bladder cells) → how it becomes good pathogen to cause UTIs. Extracellular space - where urine would be, have bacteria coming into this partic space → FimH binding to uroplakin on cell surface → changing actin molecs in cells + how polymerisation  between actin and rest of cell works to come in and create quiescent intracellular reservoir (once inside cell + bac sits there for some time + starts to replic - symptomatic responses due to PRR expression inside epithel cells + cytokines prod), allowing for bacteria to open up + go inside partic epithel cell → have diff types of expressions of proteins being turned on in this partic case, get efflux of diff types of integrins at cell surface (help bacteria bind later on). Test done to see how bacteria integrates inside cells - can cultivate + keep FimH, attach to some inert beads (FimH now on surface as it would be on bacteria) → inert beads come into contact with actual epithelial layer (uroplakin) + bacteria get uptaken due to attachment w FimH (changes + signals actin polymerisation, cytoskeletal rearrangements + inert beads get uptaken as E.coli would). Worked out that FimH both adhesion and effector molec of type 1 pili sys. Diff types of molecs have to work to help invasion → CNF-1 (cytoskeletal rearrangements within epithel cells), apoptosis (bigger thing in terms of helping defence, usually have lots of exfoliation of our bladder cells over time, can get rid of as you expel urine + helps to detach bacteria, but once bacteria has invaded bladder cells, apoptosis occurs, needs to get out of cells + go up and change how it’s infecting → tissue damage, not done in a partic way that is sequestered from our imm sys → becomes immunogenic/help start inflamm process (symptomatic UTIs)). Diff types of adhesion molecs (apart from type 1 pili + p pili) that bind to integrins that are also on host cells, collagen (big structure in many of our cells or extracellular space) - how bacteria can move and ascend up to kidneys. Bacteria commonly assoc w toxins - prod alpha haemolysin, type 1 sys (increase in complications + clinical severance). High conc - much better at being able to travel up to kidneys, getting out into bloodstream + circulating across body, increased risk of complicated UTIs + clinical severity. Lower conc - doesn't help it move away, helps imm sys to be alerted + modulates way imm sys works. Diff types of toxins - Sat/Vat causes tissue damage, allowing it to move thru, explode/open cells, HIyA causes apoptosis of cells, turning caspases on, changes way calcium fluxes in bladder + kidney cells as well (becomes detrimental over time to host), changing the way we get actin membrane ruffling (aid bacterial attachment, opposed to what we want, getting rid) → all happens within urine + kidney space. Big antigen that has to be expressed on UPEC to go from uncomplicated to complicated = H antigen. Diff tests done to show how bacterial load can disseminate thru body if it does/doesn’t have presence of H antigen. Graph - WT vs KO in flagella, those w high amts of H antigen ascended well into both kidneys, in KO didn’t ascend. Bacteria goes thru → destroys host cells → imm resp (extracellular bacteria in this case, even tho hiding inside bladder cells → need neutrophil recruitment, critical to phagocytose, engulf + get rid of bacteria). P pili → stim TLR4 → IL8 prod → neutrophil recruitment (recog bacteria as foreign + causes imm resp). Low expression of IL8 recep correl w high UTI incid (don’t get increased amt of neutrophils, decreased amt aiding UPEC to invade + slow down imm resp/clearance in this case). TLR on cell surface - recog antigen (e.g. P pili, part of P pili, PapG) → turning on + getting phosphorylation of diff adapter proteins (e.g. TRIF → turn on endosome increases → prod MHC expression to present antigen to other imm cells OR MYD88 → directly activate NF-kB → turns on range of transcription factors, helps recruit and cause inflamm such as IL1 + IL6 → fever + pain in assoc areas).  KO in animal model vs low amt of recep in human → e.g. PapG, type 1 pili not being recog + stim TLR4 (KO), asymptomatic bacteria (low levels human) as not alerting imm sys, doesn’t mean bacteria not there (imm sys v slow at recog or cannot recog). KO in other recep types → IL8 recep, if can still recog + prod via TLR4 but cannot recog IL8 → increased recurrence in kidneys, comes more acute + symptomatic. Exfoliation - easiest way of clearing bacteria (any part of body w epithel cells) → first thing done within bladder area → good @ helping most of the time, but large amts of bacteria increases infec risk. Downside - once mature cells rid of, immature cells more vulnerable at this state (i.e. if have high bacterial load in bladder and not all cleared off, easy entrance for UPEC infec). Introd bacterial load into bladder of mouse, can start seeing dissemination of bacteria over few hours (C), bacteria shed off but some still present (D), after period of time would replic quite quickly (much quicker than we can mature cells) → increase of colonisation → infec of kidney cells. Filamentous regions of UPEC infec can pop in and out of cells, flagella important to attach + ascend up into kidneys (mouse bladder cell). Initial have attachment (type 1 pili), bacteria floating around in urine (have Tamm-Horsfall protein binding to surface of bacteria, rendering it unable to bind, doesn’t always work and get attachment to cell surface) → uroplakin on cell surface of epithelial cells where bacteria has attached → start invasion → diff types of cytoskeletal arrangements to come into it → epithel cells sense some sort of change/danger/damage + start to prod IL6 + IL8 (imm resp turned on) → macrophage + neutrophil recruitment → sometimes, but unknown if of help during UPEC infec + UTIs, damage in epithelial cells can reduce nitric oxide (may interfere attachment or replic process) + epithel cells prod defensins (small peptides that interfere w attachments + other parts bacteria/cell memb → may have some sort of defence against it, not known whether it helps or not at this stage) → replic inside epithel cell if don’t get imm resp in time → explosion/rupture, can lose some of this during exfoliation but allows larger amounts of CFUs to be in bladder + immature imm cells following exfoliation (persistent bacterial presence) → ascension up into kidneys. Not entirely sure whether B + T cells have big role in this case - done in extracellular space (unable to get there), hard to get thru T + B cells thru epithel tight junctions (macrophages + neutrophils much more easily migrate thru there) → not many antibodies prod that can help, no vaxx in this case. These types of bacteria can also create biofilm on surface of epithelial cells, can become quite dams or concentration areas for this as well (aids bacteria in this case + has big role in complicated UTIs). Looking at how biofilms can be broken down using drugs to help treat UTIs. Entrance (take into account diff factors and influences that start off infec) - going up urethra to bladder, type of pili sys attaching to epithel in bladder vs kidneys is import, TLRs + ILs we have that recruit imm cells (partic neutrophils), type of important factor E.coli must express to get up into kidneys (H antigen, flagella → nephrons, nephritis caused). Complicated pt of UTI - if not treated (i.e. asymptomatic early on) in time → bloodstream → bacteremia thru body or septicemia if body reacting in that case (not good systemically) → earlier known symptoms, better to get treated. UPEC doesn’t travel too far elsewhere @ that stage (doesn’t have ability to really affect other places in this case compared to other types of E.coli → have had instances where it has come into lungs/brain in immunocompromised ppl). Lecture 11 - Fungi and Clinical Disease Until 1966 fungi classed in plant kingdom - share more in common to animals than they do plants. Spores generated at end of mycelium - dissemination of spores effective way for fungi to reproduce in environment. Fungi break down organic matter by secreting cohort of enzymes - e.g. cellulases → return to soil. Mycorrhizal fungi is non-pathogenic. Microscopic part of fungus - hair like extensions observable in soil, get very entangled with roots unable to distinguish where plant root begins and where fungus ends → symbiotic relo (fungus provides plant with phosphate + get sugars in return). Fruiting body of mushroom is macroscopic component responsible for spore produc, microscopic myecelia below soil. Antibodies for penicillin isolated from fungus/mould growing on rotting fruit. Yeast are biological factories used to make vaccines. As yeast are eukaryotic, used as model organisms to understand basic cellular function. Saccharomyces cerevisiae - brewing yeast → better understanding of how cell grows, divides → what goes wrong when cancer develops. Candida (gut resident commensal) derives food from us, no benefit for us → when environ in gut changes (e.g. had antibiotics/become sick), Candida can become more aggressive + can become pathogen (host harmed and organism derives benefit). Sepsis - bloodstream infection. If balance shifts, organism can shift from commensal to becoming pathogen. HIV - target CD4 T cells, have low levels (host immunocompromised), more susceptible to infections. COPD - chronic obstructive pulmonary disease (lung complications). Organ transplant recipients - altho can tissue match individual, body still has tendency to want to rid of organ → need to have lifelong low level of immunosuppression → prone to fungal infection. If have B cells proliferating out of control, need chemother to dampen down/kill B cells but at same time compromising ur imm system + becoming prone to getting infection. Cases where imm sys over reactive - rheumatoid arthritis, severe asthma. Candida - dimorphic fungus, has yeast form → when it becomes more pathogenic starts to make hyphae, are tissue invasive. Aspergillus - in hair like extensions has fruiting body struc that makes spores (how it disseminates). Cryptococcus - polysaccharide capsule is one of its most distinguishing features, most common cause of fungal meningitis worldwide. Inhalation of small desiccated yeast cells - how we get infected. Lung infection if immunocompromised → in case of cryptococcus infection can get into blood + dissem into brain to cause meningitis. Spore dispersal is effective means for its spread + reproduction of organisms. Taking lot of antibiotics - dampens number of bacteria, Candida may multiply more rampantly + can give host conditions like thrush (topical infection). Breach in gut wall, critically ill - much more dangerous bloodstream infec could occur (fungal sepsis). Scratching fungus into eye - fungal keratitis, infecs mainly occur in South America. Catheter - facilitate administration of drugs, represents open portal to environment → need to be careful to practice strong aseptic technique (can introd infecs thru catheter).  Scratching insect bite adding additional trauma - scratching mucor spore into wound when immunocompromised → tissue necrosis. Eukaryote - has membrane bound organelles. Some diff between fungal + human cells → exploited for new drug development. Fungal membrane has diff sterol to humans (ergosterol vs cholesterol). Bacteria much more primitive than fungi, considered primordial bacteria - many millions of years of evolution separating bacteria from fungi, have good drugs to treat them but have problems w resistance. Some antifungals toxic. Intravenous expensive - have to go to hospital for administration by trained staff. C. auris is newly emerging. C. gattii is sister species to C. neoformans. Most fungi from environment and are opportunistic pathogens. Candida - most commonly acquired hospital infec (breach in gut wall, catheter). Yeast form less pathogenic than hyphal (more tissue invasive). Trying to make more drugs for systemic infections (getting under control). Lot of bacteria also form biofilms (difficult media to infiltrate/penetrate by imm cells) → fungi protected in biofilm, challenges for drug treatments. Of all species calling bloodstream infec w Candida - 5 species cause the most common ones → need to be concerned about other species of Candida even if not as prevalent as they’ve become resistant (been exposed to drug + devel resistance or may be intrinsically resistant to common drug fluconazole) → may have somehow been exposed to chemical that looks like azole, hence when confronted with drug alr has all machinery it needs to kick it out of cell. Differentiate between diff strains of Candida - chrome agar test (preliminary diagnosis, not final) → diagnosis confirmed w molec tests. C. auris first discovered in patient w ear infec → resistant to lot of hospital grade disinfectants. Most fungi cannot be transmitted person to person (such as thru sneezing coughing). C. auris v hard to decontam → hospital outbreaks. Are fungi in environment becoming more predisposed to high temps - will be good at infecting humans (37 degree body temp). Preliminary diagnosis using chroma agar - get partic colour and colony appearance → need more definitive tests to confirm C. auris. Look under microscope - C. auris grows as cell aggregates + quite thermotolerant. Mass spec - FDA approved method → TOF = time of flight → used in combination with molec tests like PCR/seq of ITS seq of 23SrDNA. New method - combines culture of fungus in blood bottle with multiplex PCR test → take sample from patient, put in blood bottle, goes in machine (incubator), once fungus multiplied robotic arm take sample, isolates DNA + puts onto multiplex PCR test → diagnosis within 10 mins. Mucor - causes necrotic condition → termed black fungus bc necrotic tissue is dark. Severe covid - blood oxygen levels drops, had oxygen masks + tank on → believed that bc equipment not sterilised very well + humid climate of India → fungi thrive, got infection when breathing in oxygen → mainly affects nasal regions + spreads to eyes, but can go to brain (lot of patients had untreated diabetes, high levels of acidity in blood conducive to mucor infec + being treated w steroids due to severe covid) → epidemic. BAL = bronchoalveolar lavage sample (e.g. sputum sample or tissue biopsy) → need to train people what fruiting body of mucor looks like → throw any antifungal at it until something works (empirical therapy). Cryptococcosis - polysaccharide capsule is a diagnostic marker for having infec, can easily spot cryptococcus in patient sample (negative stain - India ink, can see capsule light up), in phylum  basidiomycota, related genetically to certain species of mushrooms + another plant fungal pathogen. Of patients with no immunosuppression, likely patients had some sort of underlying immune condition yet to discover (e.g. defective macrophages in innate imm resp). High rates of HIV in Sub-Saharan Africa. Climate change may have impact on distribution of fungi around world - outbreak observed in non-tropical region (tropical regions ideal for fungi to thrive). C. neoformans + C. gattii both encapsulated yeast and hard to distinguish under microscope → can still find C. gattii infecting immunocompromised patients, but has preference for immunocompetent patients. India ink - cheap and effective. Lateral flow assay - similar to covid/preg test → only req small amt of patient sample, antibodies in test strip recognise capsular polysaccharide → effective test. Cell wall and membrane sterol exploited for drug development. 5 major classes of drugs to treat infections - three of these target sterol ergosterol or its synthesis. Polyenes which bind to ergosterol, get toxicity due to subtle differences in structures (to cholesterol). Fungicidal - kills fungi. Drug synergy - when two drugs added tgt, better effect received than if used each drug by itself. Not metabolised in liver - able to take other drugs + no risk that those drugs will be metabolised differently → no toxic effects bc other drugs taken not broken down as quickly as they should be + excreted from body. Taking amphotericin B for long time → renal failure, req kidney transplant, clinicians don’t like to keep patients on this for long time. Azole - blocking step in ergosterol synthetic pathway (conversion) → get lanosterol intermediates in membrane instead of ergosterol, unable to do job of ergosterol → membrane perturbations + disruption in function (similar to what you get with amphotericin B. Extensive resistance as azoles don’t kill fungi, just prevent growth → potential to get cross resistance. Plants also get fungal infections + animals (e.g. chickens) → azole like drugs being used in environ → resistance building up in environment → if immunocompromised and get infected w fungus alr seen (e.g. azole in environ), chance that azole treatment in clinic alr seen it + knows how to deal with it. Cytochrome P450 - enzymes in our liver that detoxify compounds, if inhib by fluconazole, unable to metabolise statins + antibiotics (become toxic). Azoles most developed antifungal class - toxicity not as bad as amphotericin B, but issues with resistance. Beta-1,3 glucan synthase - enzyme sits between membrane and cell wall + links glucans together (form struc of cell wall). Cryptococcus - most critically ranked. 5-FC - inhibits DNA + protein synthesis → exploits enzyme that converts cytidine to uridine, but when competing with metabolite 5-FC + it gets incorporated into RNA + DNA → nonfunctional DNA + RNA → inhibition of protein + DNA synthesis. Fungal infections more of a problem in developing countries than in western countries. Cryptococcus infection - administered AmB in first stages of treatment (i.e. first two weeks) in combination with 5-FC → AmB is toxic, but combining two as treatment get additive/synergistic effect (need less of each drug drug req when used together vs amt req when used alone) + prevent issues w resistance developing due to having combination therapy.  Mechanism - AmB binds to ergosterol (disrupts membrane, makes it leaky), increases penetration capacity of 5-FC. In bacteria transposons + plasmids can flip around from one cell to the next very quickly - resistance big problem w bac. Resistance unlikely to spread as rapidly in fungi as seen in bac. As using drugs similar to what is used in clinic and environment, resistance should still be monitored. More drug resistance in environment, esp of Asian countries, being observed. Ways in which resistance develops - mutation in drug target (e.g in azole which target conversion of lanosterol to ergosterol, enzyme which does conversion has mutations in it + azoles no longer able to recognise enzyme). Fungi good at mounting stress responses + upregulating drug efflux pumps (don’t just pump out drugs, things like nutrients and glucose also pumped out - i.e. drugs pumped out of cell along w other things cell doesn’t want). New antifungals - have made advancements in enhancing existing drugs that target cell wall + new first class drugs (i.e. novel drug targets). Caspofungin - old style echinocandin. Ibrexafungerp - can be taken as a tablet (i.e. no hospital visits req) + broad spectrum activity. Thrush acquired - diflucan (fluconazole) provided @ chemists, now have option of non-azole drug. Orphan drug - used to treat rare fungal infections (targets diff enzyme than 5-FC - same enzyme in human cells, but made 1000x more specific for fungal enzyme in pyrimidine biosynthetic pathway than human enzyme). Fosmanogepix - first in class drug, get converted into its active form (manogepix) + blocks GPI anchor biosynthesis (cell wall enzyme but diff MOA to echinocandins). Gw1 enzyme - responsible for synthesis of GPI anchor proteins (important for strong cell wall - lack of these means cell wall is weak). Making existing drugs more effective - being done for AmB (is toxic + not v bioavailable - i.e. not readily available to the places it needs to go) → to increase bioavailability + ability to penetrate biofilms (i.e. able to kill fungi in biofilm) put into liposome. AmB - highly lipophilic fat like molec → explains why not v bioavailable, not soluble in water. Combination therapy - reduce toxicity. Statin/blood pressure drugs - pharmaceutical companies would not mind as much to go thru whole drug discovery process, drug used everyday (i.e. long term usage) = higher revenue. Drugs used to treat diff conditions (e.g. cancer) can be repurposed for treatment of diff conditions (e.g. antifungals). E.g. discovered fungal pathway v important in fungal virulence (i.e. enzymes which convert A → B → C - blocking any of these genetically = disruption of many functions important for pathogenesis) → developing drugs to block pathway + kill fungi. Looking @ analogous pathway in humans, lot of redundancy (i.e. have 3 enzymes converting A → B) + enzymes only 20% similar → gives opportunity to make drug more specific for human enzyme comp to fungal (e.g. like olorofim) → in human pathway dysregulation can occur to cause things like diabetes, obesity + uncontrolled cell division → have access to many compounds being used to treat human conditions which can be repurposed/dual purposed to use as antifungal. Fungal infections only increasing in prevalence as we have more immunocompromised individuals in society → can cause outbreaks (e.g. cryptococcus gattii + candida aureus). Earlier (good) diagnosis + earlier start to treatment = better outcome/prognosis. Lecture 12 - Antibiotic Resistance and the Need for New Drugs Multidrug resistant + pan drug resistant gram negative bacteria prevalent in hospital settings. MRSA - pathogen of high concern, large burden on healthcare. VRSA - not observed before, recent emergence of strains (classes of gram positive bacteria that are pan resistant rather than gram negative, the ones we are usually concerned abt). What distinguishes drug resistant TB from other pathogens = length of time needed to deal w M.Tb (chronic course of infec). Some of the antibiotics used for treatment can be quite toxic. Infection with XDR - very small number of options for treatment. Kind of coming back to pre-antibiotic era in some scenarios - for some strains have no means of treatment. Concerned about drug resistance primarily bc morbidity + mortality, increasing rate of infection in hospital + cost of complications it causes → also major burden to healthcare system. First line drugs - standard treatment. Resistance to drug - move up drug line → increased costs associated with moving up drug lines. Treatment costs (XDR) - involving hospitalisation, drug treatment → can range up to $1 million, with undefined outcome (i.e. may not survive). Once bacteria exposed to new antibiotic, tends to develop resistance quite quickly → similarly observed for even fourth, fifth line drugs. HAIs in US (in general even) are typically bacterial - e.g. bloodstream infections from catheters, aerosol infection via respirator, surgical site infecs such as worms or pseudomonas infec. Increased antibiotic use = increased resistance (shown by diff regions of globe having diff rates of resistance based on antibiotic use). Aus has low resistance but relatively high use - likely due to how we are more strict regarding dispersal of drugs, though there are some cases where antibiotics are provided either as a preventative measure or in cases where it is unknown whether it will work or not (need to limit this as we give chance for resistance to arise with more widespread usage). Many antibiotics developed early on (50-60s), explosion → innovation gap, reduction in amt of antibiotics generated → new classes come in 2000 onwards. Was a phase where antibiotic resistance was increasing but number of new drugs being created was decreasing. Many early drugs identified were natural products - tend to come from larger circulating organisms such as actinomyces (many drugs commonly derived from this). Earlier on was relatively easy to discover a broadly active natural product from actinomyces → increasing difficulty as time has gone on, have to screen more naturally occurring organisms → become more restrictive (i.e. most drugs discovered now have narrow spectrum, only against gram +ves and -ves). Broadly active antimicrobials, give advantage to these organisms in nature - many come from soil, water from ocean (used by organisms as their defence against other bac/organisms). Broadly active antimicrobials have been dispersed v widely thru actinomyces - 1/10 chance if taken 100 actinomyces species of finding broadly active molec (good chance) → once found may need to look at 1 in 1000, then 1 in 10000, then 1 in 100000 to find new antibiotic that has a v narrow range. Actinomyces were initial pool of antibiotics used in 40-60s - exhausted, need more creativity in terms of finding broadly active drugs. ESKAPE pathogens - escape drugs, acronym for diff pathogen classes.  Enterococci - something distinct from other drug resistant bac is that it is pretty much one clonal type of enterococcus faecium responsible for most infections → these strains are typically naturally resistant to many antibiotics, in partic aminoglycosides + ampicillin + beta-lactams, very good at acquiring vancomycin resistance (bugs considered serious threat, not as many deaths as staph aureus but still high number of deaths per yr). Vancomycin resistance varies per region in Europe - relative change between 2012/13 to 2018, have some regions with major increases in resistance (even though rates are low in some regions), other regions have gone from higher rates to lower rates and have achieved stabilisation → each healthcare sys per region has diff approach to dealing w bac (reflected by resistance rates). How vancomycin resistance occurs - transposable elements (things that jump around genomes, called vancomycin cassettes, many diff types of these) → Van-A first discovered and most relevant. Cassette inserted into bacteria - i.e. vancomycin sensitive enterococcus which acquires this cassette from another bacteria, jumps into bac + inserts into genome → expression of diff enzymes. Vancomycin resistance - typically in bacteria susceptible to vancomycin have cell wall precursors that are made (important for development of peptidoglycan/cell wall synthesis), within precursors being build up to make cell wall have amino acid repeats (D-Ala-D-Ala repeats) → vancomycin (drug) can see these precursors attached to it, stop cell wall from being made + kill bacteria. Vancomycin worked effectively for long time until resistance detected in 1980s. Bacteria modifies cell wall precursors → all genes present on makeup of genes doing multiple things → either building up diff precursors (instead of D-Ala-D-Ala have D-Ala-D-X or D-Ala) → when vancomycin comes along, unable to bind → cell wall synthesis (vancomycin no longer active against precursors). Vancomycin genes - have enzymes on there that degrade D-Ala-D-Ala (degradation of vancomycin binding precursors + synthesis of new ones) → vancomycin resistance development. Penicillin one of the first antibiotics discovered, clinically used in 1940s, 50% of isolates resistant to penicillin mid 1950s to early 1960s, now have strains of staph aureus resistant to both penicillin + methicillin + vancomycin. MRSA - methicillin resistant staph aureus, though resistance much broader than methicillin now (highly drug resistant strains) → 2 types. MSSA - methicillin sensitive strains → MRSA strains more virulent. HA-MRSA - associated with hospital acquired infections (e.g. open wounds, catheters, ppl who are more immunocompromised). CA-MRSA - in community, skin and tissue infecs (e.g. flesh eating bacteria commonly assoc w this). CA-MRSA usually more virulent than HA-MRSA. All strains that have developed methicillin resistance - due to incorporation of mecA cassette (defines/encodes methicillin + broader resistance → CA-MRSA have smaller mecA cassette (i.e. when things inserted into bacteria, often a fitness cost as bacteria prod energy for survival, but if they bring on things such as plasmids or insertions, need to generate energy towards that → inserting large chunks of DNA could be an issue for growth of bacteria → likely part of the reason why CA strains have less necessity to prod/use lot of energy to keep cassette likely more virulent, don't have to deal w incorporating this additional cost). CA strains can be more lethal + express PVL gene (extra toxin). HA-MRSA - low drug resistance + tend not to have PVL toxin + have diff inserted mec types (larger).  CA-MRSA - can be in many diff settings, tend to be persistent w beta lactams, have PVL + diff mec types. First identified/most studied resistance phenotype in bac - staph aureus → penicillin + methicillin resistant → many drugs interfere w capacity of bac to build cell wall, methicillin resistant bacteria (mecA gene/cassette → encodes for altered penicillin binding protein → methicillin/penicillins would normally bind to protein made in cell wall, get killing of bac → in case of altered PBP antibiotic cannot bind + don’t get destruction of bacteria (resis mech). Way toxin works - PVL phage (start w methicillin sensitive bac), phage carries gene for toxin + inserts gene cassette into chromosome of staph aureus → PVL toxin produc → damage → have PVL positive strains that acquire methicillin resistance. Not always that u have methicillin strains that acquire resistance, often tends to be other way around (can also have methicillin sensitive strains positive for toxin). Implications of toxin - can potentially be directly toxic on host cells (i.e. toxin can be released by bacteria → tissue destruction) → more common toxin is rel + binds to surface of cells (e.g. polymorphonuclear cells) → 2 outcomes → if have lot of toxin may get apoptosis of cells (level of tissue destruction), high levels of toxic may cause lysis of immune cells, rel of toxin, inflamm + tissue destruction/damage. Naturally acquired vancomycin resistance (VISA) - v low/intermediate levels, scenarios observed in laboratory, cell wall thickening of some bac, reduced ability to uptake vancomycin, no major clinical impact observed, not as much of a concern. High level resistance to vancomycin (VRSA) - end up w bac that have same lvl of resistance (i.e. staph aureus that has same level of resistance as enterococcus → bc staph aureus strain acquired vancomycin resistance from enterococcus/VRE). Vancomycin staph aureus resistance identified in 2004 - found strain of staph aureus that had all common resistance mechs, within had acquired vancomycin cassette (from VRE) → found due to isolating this partic strain from catheter of individual who was in hospital, able to isolate vancomycin resistant staph aureus from tip of it + VRE from same location → genetically typed them, vancomycin cassette was same in both bacterial strains (i.e. caught in act of VRE transferring its vancomycin cassette to staph aureus) → first example, highly concerning as end up w highly resistant superbugs. ESBL - as opposed to methicillin resistance, are new enzymes that give greater resistance to more drugs (cephalosporins). Beta lactamases induce resistance for beta lactams. Klebsiella can have lot of these mutations. Common mechanisms of resistance encoded by plasmids, relatively easy for bac to move around (esp within themselves + then other related bac). For two classes how it works - have normal beta lactamase resp for coming in + lysing beta lactams (those antibiotics no longer useless) → mutated to extend spectrum (broad specificity), around active site (req for their mode of action, hydrolysing antibiotics). SHV - similar mainly in Klebsiella. TEM + SHV - do similar things + have mutations in existing beta lactamase to give them broad specificity. CTX - more concerning, recently emerging, acquired from new class of bac (Kluyvera - non pathogenic, circulate in environ + contain lot of beta lactamases - diff class hence look diff to other ones) → pathogenic bac finding beta lactamases from environment + using them to hydrolyse drugs (partic classes of cephalosporins). Called CTX bc of activity against beta lactamases → have activity in hydrolysing v broad spectrum of cephalosporins (commonly used drugs). Closer tgt dots are = more similar protein seq are.  Dark grey, white + grey quite distinct but still grouped tgt → outside of that have completely distinct classes (v diff structurally). Many classes of beta lactamases circulating in bacteria → highly problematic, role of these is to hydrolyse/break down diff classes of antibiotics as encountered by bac. Carbapenems - another important drug class, esp w Klebsiella → antibiotics work against bac that contain these extended spectrum beta lactamases → can be good, showed there are many ESBLs circulating + would be good to have smth that can deal w it, though bac like Klebsiella have carbapenem enzymes (can hydrolyse diff drug class + are transferred on plasmids) → those infected with Klebsiella bac expressing carbopenemases, relatively high mortality rate → evolution of bac to deal w new drugs as they’re being developed. Drug resistant superbugs → in India identified strain w many diff classes of drug resistance against many drugs (inc carbopenemases) → Klebsiella NDM-1 strains (strain resistant to pretty much anything + was able to transfer this around) → highly problematic. Acinetobacter baumannii - strain less prevalent + concerning 20-30 years ago, has started to take off in prevalence (partic in hospital setting), have broad level of carbapenem resistance. Early 2000s prevalence relatively low (0-20/30%), within 5 years some provinces up to 70% resistance - very quick, strain of concern as u have pan drug resistance strains (can’t deal w). Pseudomonas aeruginosa - great imitator, has picked up on all resistance mechanisms, can find it in many settings (e.g. bloodstream infecs), v prevalent in burn + surgical wounds → v problematic + diffic organism to treat → has many chromosomal mechanisms for drug resistance. Efflux pumps - as soon as drug comes in can pump it out. Can modify outer membrane permeability - reduce influx of drugs. Inactivate antibiotics using drugs. Have diff OXO classes of carbapenemases or beta lactamases that are present + circulating → pseudomonas v good @ picking these up. Clavulanic acid - molec used to combine w beta lactamases to improve activity of enzymes → OXO types diffic to treat w next gen treatments. Alteration of drug targets - such as in penicillin resistance. V diffic to treat, lot of research into dealing with this. Lecture 13 - New Approaches to Drug Design 3 classes of antibiotics - natural (derived from naturally occurring sources, tend to be bacteria and other organisms), semi-synthetics (largest class as of now, break down to create alternative product that will be active against bacteria that may have had resistance to initial drug, e.g. penicillin derivative), synthetic (smaller class, in some cases is targeted or in other cases is by chance). Scaffolds/major drug classes identified occurred quite some time ago (semi synthetic). Recreation of natural product or new derivative by complete synthesis. New classes of antibiotics discovered, but was innovation gap where not much advance in drug discovery. Last 30-40 years - major increase in identification/development of antibiotics/bacteria resistant against. Resources + funding to be able to screen, identify, produce/manufacture, distribute drugs @ scale that is req - pharmaceutical companies not attracted to this in terms of new abx produc. Identified some of the v useful drugs relatively quickly, now have less capacity to identify some of these newer compounds. Many of the drug classes that were easiest to find/were most useful had alr been discovered - ongoing issue in getting pharmaceutical companies enthused to look @ identification of new drug products. Antibiotic usage vs other drugs (i.e. such as statins) - usage for antibiotics is only when person needs it and is short term, whereas for drugs like statins they are used everyday + antibiotics more cost effective (not expensive - drugs for general good). Academia + small biotechs do initial discovery - once smth useful discovered larger company takes that on. Genomics has not been overly useful. Tetracyclines - try to get around fact that bac are good at pumping antibiotics out of cell. Cephalosporins - typically bacteria contain beta lactamases or extended spectrum beta lactamases which are able to lyse or hydrolyse drugs and stop them from working. Tigecycline inhibits protein synthesis - kills bac. Tetracycline is drug we commonly use to do this w partic mode of action - binds to ribosomes (smaller subunit of it) → problem is many bac have efflux pumps which r quite good at pumping tetracycline out of cell (i.e. as drug comes in it is also removed) → develop modified drugs to overcome this (i.e. tigecycline, derivative of tetracycline, has additional moiety on drug, means it still binds to same target site but not removed by efflux pump → identified resistance mechanism, tried to counter it but also kept activity of drug). Can also try to identify drugs that work in a similar way but look v diff (e.g. retapamulin is a diff drug that we use which also inhibits protein synthesis, but binds to diff part of ribosome, is not targeted by efflux pumps, hence drug is not removed by tetracycline resistance mechanisms).  Have orig antibiotic class → diff generations (bc develop resistance to one partic class, diff generations are modifications of prev gen which have developed resistance) → e.g. tigecycline is tetracycline w modified side chains. Cephalosporins - 5 generations, bac good at prod beta lactamases (en