Advanced Microbiology Flashcards
What is a fever?
• Sign of inflamm & can be a symptom/ sign of infec
• Temp over 38 degrees
• Symptoms;
o Fever ‘burning up’
o Chills, sweats, night sweats (patient’s don’t always tell you about this)
o Rigors (uncontrollable shaking- can’t hold tea with spilling, symmetrical on body, usually associated with feeling cold followed by feeling hot)- usually caused by infec
What tests can you use when you suspect an infection?
• FBC
o Hb; not much help in infec- but anaemia of chronic disease (normocytic, normochromic) can be caused by infec
o White blood cell count (WCC)- caution- just because it’s normal doesn’t mean patient doesn’t have the diagnosis
- Can be raised in infec but other conditions too (poor specificity)- severe sepsis can lower WCC
- Neutrophils- raised in bacterial infec
- Lymphocytes- raised in viral infec
• Blood C-reactive protein
o Inflamm markers;
- C- rective protein <5mg/L= diagnosis of bacterial much less likely (–ve predicted value may help differentitae between a viral or bacterial infec)
- Procalcitonin <0.5µg/L
- (TRAIL, IL-6, IP-10)
- Raised inflamm markers support diagnosis, -ve markers make infec less likely
• Blood prolactionin (in above bullet point)
• Radiological test; chest x-ray (ring around consolidation);
o Clinical infecs- respiratory
o NB x-rays, computed tomography (CT) scanning & CT combined with PET used to support a diagnosis of infec
- How severe is infec?
o Blood lactate & blood gases can help to i.d. severe sepsis & resp failure
o CURB-65- see how severely ill patient is to see if they need to be hospitalised - What is the pathogen?
o Clues from history
o Most common bacterial cause- Streptococcus pneumoniae
o Penicillin resistance more common in Spain
o Legionella pneumonia can be acquired during hotel stays
- Only 2 reasons to carry out a diagnostic test; improve outcome, provide epidemiologicl data
- Lab plays partial role in microbiological diagnosis- results of history, examination, non-microbiological tests & lab tests combined to male diagnostic hypothesis
If CRP & procalcitonin normal- likely that it’s not bacterial pneumonia so won’t need antibiotics.
What are Methods of Microbiological Diagnosis?
• Use infec tests to confirm a clinical diagnosis
• Microbiological diagnosis relies on 3 modalities;
o Direct detection
o Culture
o Indirect- Immunological tests (antibody detection)
• Culture tests should be taken before antibiotics given
What is the use of culture?
• Isolation of viable pathogen enables;
o Identification- immediate or by further testing
o Typing- to establish organism relatedness
o Sensitvity testing- to direct antimicrobial therapy
• Not applicable to non-cultivable micro-organisms
• Needs to be done before antibiotics are started
• Certain organisms die quickly- need to do tests before start giving antibiotics
• Blood culture sampling;
o Coagulase –ve staphylococci normally on skin (can cause infec)- needs to be aspectic technique to avoid contamination
o Can improve sensitivity & specificity by way the blood sample taken
o Put some of culture on plate
o 9. Identification process
o 10. Lab will provide report
What is a Gram Stain? What colour are +ve/-ve?
• Chemical process that distinguishes cell walls that retain crystal violet & those that don’t when stained & washed with acetone
• This helps to decide which antibiotics they will respond to
• Gram +ve- purple
• Gram –ve- pink (or colour of counter- stain)
• Microbiology will give you gram satin of bacteria & shape of bacteria
• Gram +ve cocci;
o Form chains
o Form clusters
What is Sensitivity Testing?
• Needs viable micro-organisms- usually bacteria or fungi
• Agar plate spread with a suspension (organism) your interested in
• Put samples of antibiotics
• As organism grows can see which antibiotics it can’t grow around (effective) & which it can (antibiotics it’s resistant to) by measuring zone of inhibition
• Basic principle;
o Culture of micro-organism in presence of anti-microbial agent
o Work out if conc of antimicrobial that will be available in the body is high enough to kill the micro-organism
o Solid or liquid media
What are the Uses &limitations of Sensitivity Testing?
Uses & limitations
• To inform decisions on targeted antimicrobial therapy;
o Initial treatment with ‘empirical’ therapy
o Subsequent treatment ‘targeted’- need;
Isolation of micro-organism
Antimicrobial susceptibility testing
• The correlation between antimicrobial sensitivity & clinical response is not absolute
• Establishes micro-organism presence at a particular site- cultivable micro-organisms only
• Allows the use of empirical & targeted antimicrobial therapy
• Provides epidemiology & typing info
To inform antibiotic therapy;
• 1st line antibiotic choice if infec doesn’t need immediate treatment e.g. infectious endocarditis, osteomyelitis (unless patient is septic)
• 2nd line choice after empiric therapy (use very broad spectrum, then give specific)- ‘smart smart then focus’
• 2nd line choice after failure of initial therapy- uncomplicated UTI in general practice
To provide epidemiological data- ‘surveillance’;
• Sensitivity results collated locally, nationally & internationally;
o To inform local guidelines &antibiotic choices
o To provide epidemiological data (can work out trends & future resistance)
o To provide early warning of threats to public health
What is Direct Detection?
• Detection of whole organism- microscopy e.g. CSF from lumbar puncture
• Detection of component of organism; antigen, nucleic acid (DNA or RNA)
• Detection of antigen
o E.g. Legionella antigen detection test
o Adv of doing this at bedside- quick result
o Disadv- needs training, quality control, need to ensure no false +ves
o Target Ags include; polysaccharide capsule (Cr. neoformans), cell wall polysaccharides (Aspergillus galactomannan, Candida mannan).
o Solubility & distrib of Ag differs with infecting species, & is poorly understood for Aspergillus & Candida.
o Serological tests include latex agglutination (Cr. neoformans) & ELISA (Aspergillus galactomannan and Candida mannan).
o Sampling protocol i.e. use of test for screening (low pre-test probability pop’n) vs. diagnostic (high pre-test probability).
o Does result offer any adv over other methods? Does making the diagnosis influence clinical outcome?
• Detection of nucleic acid (DNA/RNA)
o Viruses- influenza
o Bacteria- streptococcus pneumoniae, 16S PCR (all bacteria have ribosomes, primers bind to conservative areas, can compare organisms- can do PCR & i.d. any bacterial pathogen, broad range PCR identifies pathogen)
o Fungi- Candida spp., Aspergillus spp.
What are the Uses &limitations of Direct Detection?
• Establishes presence of micr-organism at a particular site- cultivable & non-cultivable organisms
• Allows the use of appropriate empiric antimicrobial therapy
• Does NOT give any info on:
o Antimicrobial susceptibility
o Typing
• Is usually the fastest diagnostic method
What are Immunological Tests?
• Detection of immune response to infec
• Antibody detection
o IgM detection
o Seroconversion- change from –ve to +ve result from 1 test to a subsequent test
o Fourfold rise titre- rise in antibody conc from 1 test to subsequent test
‘Titre’ is 1/greatest dilution at which antibody is detectable
i.e. if antibody is just detectable at a serum dilution of 1/64 the titre is 64
‘Fourfold rise in titre’ would be e.g. 2= 32 or 4=64
• Serological test to look for antibodies; dilute to make serum less & less concentrated in wells in plastic trays, need to do it in pairs (acute & convalescent sample in patient)- if serum converts & no antibodies in acute but lots in convalescent- shows been exposed to pathogen
• E.g. diagnosis of disease in question can be made in Patients B &a; C- showed seroconversion & fourfold rise in titre respectively, Patient A had no signif change in titre between tests, and antibody was not detected at all in patient D
• Other immunological tests
What are the Uses & limitations of Antibody Testing?
- Confirms exposure to a specific micro-organism- cultivable & non-cultivable organisms
- Is restricted to patients with a detectable antibody response
- Is retrospective- aften too late to inform antimicrobial therapy decisions
How do you Choose the Right Antimicrobial Agent?
Know the likely organism(s)
• Body site- can be whole body or just e.g. urinary tract UTI
• Immunological status- e.g. depending on how immunosuppressed patient is
• Microbiological history- e.g. skin & soft tissue infec e.g. check they don’t have MRSA as may need to use diff antibiotics
• Microbiological history- if antibiotic they once used failed don’t use again
• Risk factors
1. Select agent with appropriate antimicrobial spectrum- to cover organisms likely causing infec
2. Match the pharmacokinetics to the patient
• Distribution e.g. which antibiotic would go to urine if UTI or CSF if meningeal infec
• Interactions
• Adverse effects
- Empirical & targeted therapy
- Route, duration (review)
- Distrib/ penetration (antibiotics get into site of infec e.g. into CSF for meningitis)
- Bacteriocidal (antibiotics kill microorganism)/ static (antibiotics stop microoganisms growing)
- Resistance
- Special situations e.g. liver/ renal impairement, obesity (some antibiotics need to be given on true/ ideal body weight), young/ elderly, HCAIs (healthcare associated infecs e.g. c. diff)
- Monitoring (e.g. gentamycin or vancomycin)- adequate dose/ levels that cause toxicity)
- Prophylaxis- surgical (e.g. high risk infec surgery) or long-term (suppression)
What do Bacteria & Fungi have in Common?
- Cell wall (physical protec)
- Cell memb(s) (bacteria may have 2 membs)
- DNA (for protein synthesis)
- Synthetic functions- protein synthesis
- Can use these to target antimicrobial agents
- Synthesis happens in the middle
What are the Differences between Bacteria & Fungi ?
DNA localisation
• Bacteria are ‘prokaryotes’- their DNA exists as a ring like struc in cytoplasm
• Fungi are ‘eukaryotes’- their DNA separated from cytoplasm by a nuclear memb (nucleus seps DNA & cytoplasm)
Size
• Bacterial cells smaller than fungal cells
Structure
• Bacterial cells uniform simple strucs (e.g. e.coli cells look the same)
• Fungal cells may have complex struc & same organism may have many diff forms (hyphae, spores etc) e.g. spores diff from mushroom
Cellular processes
• Fungal protein & DNA synthesis very similar to human processes (protein & synthesis- harder to use this as a target for antimicrobial treatment
What are Antibiotics?
• Antibiotics- chem products of microbes that inhibit/ kill other organisms
What are Antimicrobial agents ?
• Antimicrobial agents (antibacterial, antifungal, antiviral);
o Antibiotics (many made by fungi e.g. penicillin, some made by bacteria)
o Synthetic compounds (not from a microbe) with similar effect to antibiotics e.g. sulphonamide
o Semi-synthetic i.e. modified from antibiotics to make a new compound- diff antimicrobial activity/ spectrum, pharmacological properties or toxicity
• Terms antibiotic & antibacterial agent often used interchangeably
What is Bacteriostatic/ fungistatic?
• Bacteriostatic/ fungistatic- agent inhibits growth of bacteria/ fungi e.g. protein synthesis inhibitors
What are Bacteriocidal/ fungicidal?
• Bacteriocidal/ fungicidal- kills organisms e.g. cell wall-active agents
What is the Minimum inhibitory concentration (MIC?
- Minimum inhibitory concentration (MIC)- minimum antimicrobial conc at which visible growth inhibited in an artificial cuture system, if low MIC- agent active against organism it is being tested (is a good antimicrobial)
- Low MIC (e.g. 0.1 mg/L)= sensitive organism; BUT sensitivity actually depends on level available at site of infec (e.g. might be able to kill organism (have a low MIC) but can’t get into urine)
What is the Minimum bactericidal/fungicidal conc ?
• Minimum bactericidal/fungicidal conc (MBC/MFC)- minimum conc of antimicrobial agent at which most organisms killed
What is the Antimicrobial Spectrum ? When would you use the different types?
- Range of bacterial/ fungal species likely to be sensitive to a partic antibacterial/ antifungal agent
- Broad spectrum- kills most types of bacteria/ fungi encountered
- Narrow spectrum- kills only a narrow range of organisms
- Knowledge of antimicrobial spectrum vital in choosing appropriate antimicrobial agent/ combination to treat a given infec
- Aim: use narrowest spectrum agent as possible ideally only kills particular species of bacterium that is causing infec
- E.g. broad spec antibiotic in 6 yrs time might be narrow spec due to antibiotic resistance can now only kill a narrow range
Broad spectrum can decrease enterohepatic cycling= decrease effect of oral contraceptives and fit k synthesis. Can also increase INR and affect warfarin
What are features of the Bacterial Cell Wall?
• Gives bacteria structural rigidity
• Peptidoglycan;
o In gram –ve & +ve
o Polymer of glucose-derivatives, N-acetyl muramic acid (NAM) & N-acetyl glucosamine (NAG)
• Animal cells don’t have a cell wall- ideal potential for selective toxicity
What are features of the Fungal Cell Wall?
• Β-1,3-glucan
o Large polymer of UDP-glucose
o 50-60% of dry weight of fungal cell wall
o Synthesized by β-1,3-glucan synthase
• Also has some chitin in it (small proportion &; no anti-fungal agents aimed at chitin)
• Animal cells don’t have a cell wall- ideal potential for selective toxicity
What are antibacterial Cell Wall Synthesis Inhibitors?
- β-lactams (big class of antibiotics)
- Glycopeptides (smaller antibiotic class)
Other clinically useful cell wall synthesis inhibitors; cycloserine (anti-tuberculous agent) & fosfomycin (antibacterial but not available in UK)
What are Anti-fungal Cell Wall Synthesis Inhibitors?
• Echinocandins (antifungal class)
What are β-Lactam Antibiotics?
- 1st true antibiotics in clinical practise- Benzylpenicilin (penicillin G);acid labile so give parenterally
- All contain β-lactem ring; 4-membered ring struc (C-C-C-N), forms a structural analogue of D-alanyl-D-alanine
- Interfere with ‘penicillin binding proteins’ (enzymes involved in synthesis & maintenance of peptidoglycan) function; transpeptidase enzymes involved in peptidoglycan cross-linking (if stop these cell dies)
- Most widely prescribed of antibacterial antibiotics
- Phenoxymethyl penicillin (penicillin V) 1st oral penicillin
- Ampicillin 1st penicillin with activity against Enterobacteriaceae fam, can only be delivered parenterally (oral equivalent is amoxicillin)
- Meticillin- anti-staphylococcal penicillin (side chain prevents it being hydrolysed by staphylococcal b-lactamase)
- Has been superceeded by drugs such as cloxacillin & flucloxacilin
What are the different β-Lactam Antibiotics Classes?
• Penicillins;
o Benzylpenicillin, amoxicillin, flucloxacillin
o Relatively narrow spectrum
o Ends with ‘cilin’ is a beta lactam so if someone allergic to penicillin they are allergic to all drugs ending in ‘cillin’ (types of penicillins)
• Cephalosporins (all start with ‘cef’);
o Cefuroxime, ceftazidime etc
o Arranged into ‘generations’
• Carbapenems
o Meropenem, imipenem
o Extremely broad spectrum
• Monobactems
o Aztreonam (not bi-cyclic only has 1 ring, gram –ve activity only)- considered safe to give in penicillin allergy
What are Penicillins?
β-Lactam Antibiotics
o Benzylpenicillin, amoxicillin, flucloxacillin
o Relatively narrow spectrum
o Ends with ‘cilin’ is a beta lactam so if someone allergic to penicillin they are allergic to all drugs ending in ‘cillin’ (types of penicillins)
What are Cephalosporins?
β-Lactam Antibiotics
o Cefuroxime, ceftazidime etc
o Arranged into ‘generations’
What are Carbapenems?
β-Lactam Antibiotics
o Meropenem, imipenem
o Extremely broad spectrum
What are Monobactems?
β-Lactam Antibiotics o Aztreonam (not bi-cyclic only has 1 ring, gram –ve activity only)- considered safe to give in penicillin allergy
What are β-lactamase Enzymes?
• Hydrolyse (inactive) β-lactams
• Common mechanism of resistance to β-lactam antibiotics
• Diff β-lactamse enzymes confer resistance to a narrow/ wide range of β-lactams e.g.;
o Staphylococcal β-lactamase- some penicillins only
o ‘Extended spectrum β-lactamsae’ (ESBL)- penicillins & cephalosporins
o Carbapenemases (e.g. NDM1)- cebapenems
What are β-lactam/β-lactamase Inhibitor Combinations (BLBLI)?
- Give combination drug- antibiotic drug & another chemical that inhibits beta lactamase (if organism uses this to inhibit antibiotic)
- Amocixillin- clavulanate (Augmentin)- increases spectrum of amoxicillin
- Piperacilin-tazobactam (Tazocin)- increases the spectrum of pipercillin
- Alavulanate & tazobactam stop it breaking down antibiotic
- ESBL & carbapenemase BLBLIs- work in progress
- Probs with BLBLIs;
- Very broad spectrum, so predispose to C.difficile infec
- Names don’t end with –illin or start with cef- (easy to forget these are penicillins)= importance of penicillin allergy may be missed (don’t give to someone who has a penicillin allergy)
What are Glycopeptides?
Glycopeptides (Antibacetrial)
• Vancomycin, teicoplanin
• Large molecules, bind directly to terminal D-alanyl-D-alanine on NAM pentapeptides- inhibit binding of transpeptidases & so peptidoglycan cross-linking
• Gram-positive activity- can’t penetrate gram-ve outer memb porins
What are Echinocandins?
Echinocandins (antifungal)
Mode of action;
• Inhibit of β-1,3-glucan synthase (makes beta 1,3- glucan)
• Construction of severely abnormal cell wall
Examples;
• Anidulafungin
• Caspofungin
• Micafungin
Protein synthesis in bacteria
• RNA translation= protein takes place on ribosome
• Ribonucleoprotein complexes (2/3 RNA, 1/3 protein)
• 50S (large) and 30S (small) subunits combine to form 70S initiation complex (makes protein)
• S=Svedberg units; relative sedimentation rate
What are common classes of Protein Synthesis Inhibitors?
Aminoglycosides
MLS antibiotics
Tetracyclines
Oxazolidinones
What are Aminoglycosides ?
Protein Synthesis Inhibitors
• Gentimicin, amikacin
• Bind to 30S ribosomal subunit
• Mechanism of action not fully understood
What are MLS antibiotics ?
Protein Synthesis Inhibitors
• Macrolides, Licosamides, Streptogramins
o Erythromycin, clarithromycin (macrolides)
o Clindamycin (lincosamide) (pre-dispose to c.diff so used less); bind to 50S ribosomal subunit, inhibit protein elongation
What are Tetracyclines?
Protein Synthesis Inhibitors
• Tertacycline, doxycycline (type of tetracycline)
o Bind to 30S ribosomal subunit
o Inhibit translation by interfering with tRNA binding to rRNA
• Tigecycline- modern derivative of tetracyclines with a similar mechanism of action & broader spectrum
What are Oxazolidinones?
Protein Synthesis Inhibitors
• Linezolid
o Inhibits protein synthesis inititation
o Bids to 50S ribosomal subunit
o Inhibits assembly of initiation comples
o May also bind to 70S subunit
o Traets MRSA infec of chest & skin & soft tissue infecs
• Other antibacterial protein synthesis inhibitors
o Mupirocin (bacteroban is trade name)
o Fusidic acid
What are Protein Synthesis inhibitors in Fungi?
NONE
• Fungi are eukaryotes- have same protein synthesis mechanisms as humans
• So protein synthesis not a target for antifungal agents
What are common classes of DNA Synthesis Inhibitors?
Trimethoprim & sulfonamides
Quinolones & Fluoroquinolones
What are Trimethoprim & sulfonamides?
Trimethoprim &sulfonamides
• Both inhibit folate synthesis
o Trimethoprim- dihydrofolate reductase
o Sulfonamides- dihydropteroate synthetase
o Folic acid is a purine synthesis precursor
o Many bacteria make folic acid from para-aminobenzoic acid- this pathway target for sulphonamides & trimethorprim
o In vitro tests- sulphonamides & trimethoprim synergistic but not when this combo used to treat bacterial infecs
• Trimethoprim
o Commonly used to treat UTI (broad spectrum for gram –ves that cause UTI)
o Oral
• Co-trimoxazole (trimethoprim-sulfamethoxazole)
o Mainly an antibacterial agent but also affective against Pneumocystis jirowecii (funagal cause of pneumonia in HIV patients & other immunosuppressed agents)
What are Quinolones & Fluoroquinolones?
• Inhibit 1 or more of 2 related bacterial enzymes
o DNA gyrase & topoisomerase IV
o Involved in DNA remodelling during DNA replication
• E.g. nalidixic acid, ciprofloxacin (used in UTIs), levofloxacin
• Quinolones only effective against bacteria
Quinolones can cause tendonitis and convulsions if given with steroids/NSAIDs
Anything that gives clues that say don’t take with milk- can cuase sticky mess in your stomach
What DNA Synthesis Inhibitor is used for Fungi ?
- 5 fluorocytosine (5FC) is a fungal DNA inhibitor, developed as a putative anti-cancer drug
- Selectively taken into fungal cells by a fungal enzyme (cytosine permease- fungus specific enzymes, so gets into fungal cells not human cells)
- Selective toxicity poor
- Not used very much
What is Rifampicin?
RNA Synthesis Inhibitor
• Rifampicin- RNA polymerase inhibitor- prevents mRNA synthesis
• Cornerstone of anti-tuberculous chemotherapy
Anything that gives clues that say don’t take with milk, or antacids- can cuase sticky mess in your stomach
What are Antibacterial Cell Membrane Agents?
o Colistin (gram –ves)- only effective against gram –ves
o Daptomycin (gram +ves)- only used against gram +ves
Cyclic lipopeptides (have a ring struc)
Destruc of outer memb/ cytoplasmic memb (lipophilic tail inserts in here)
What are Anti-fungal Cell Membrane Agents ?
o Azoles (e.g. clotrimazole (vaginal thrush), fluconazole)
o Terbinafine- inhibit ergosterol synthesis (a component of fungal cell membs but not human/ bacterial cells)
o Amphotericin B (& nystatin)- bind to ergosterol causing physical damage to fungal memb
What is Antimicrobial Combination Therapy? Why is it used?
Antimicrobial Combination Therapy
Reasons for combining antimicrobial agents;
• To provide adequaltely broad spectrum- single agent might not cover all required organisms;
o Polymicrobial infec (multiple n=bacteria or bacteria + fungi)
o Empiric treatment of sepsis
• To increase efficacy - synergistic combination may improve outcome
o β-lactam/aminoglycoside in streptococcal endocarditis
o Cell wall agent/ protein inhibitor in severe Group A streptococcal infec
• To reduce resistance- organism would need to develop resistance to multiple agents simultaneously
o Antituberculosis chemotherapy
What is Amphotericin B?
Inhibits Cell membrane integrity in fungi
What is Daptomycin?
Inhibits cytoplasmic membrane in bacteria
What is Colistin?
Inhibits outer membrane in bacteria
What are the Causes of Antibiotic Resistance?
- Mixture of sensitive & resistant bacterial strains exposed to antibiotics
- Sensitive strains- die out, resistant strains- become dominant colonising strains of that person
- Antibiotic-resistant strains more likely to cause subsequent endogenous infec (infec caused by patient’s colonising flora)
- Antibiotic-resistant strains may be transferred to other people (potential recipients of resistance)
- Large bowel- repository for many organisms
- If treat patient with antibiotics then take sputum sample- will grow gram –ve bacteria resistant to antibiotic (no clinical relevance here- but shows once give antibiotics will effect normal bacteria)
What are innate Resistance Mechanisms ?
• Fundamental property of bacterium/ antibiotic condition
• Usually relates to permeability/ entry of antibiotic into cell
o Gram –ves resistant to; glycopeptides, daptomycin (cell wall active agents, large molecules, can’t penetrate gram –ve outer memb- not effective against gram –ve bacteria so innate resistance in all gram –ve bacteria to glycopeptides & daptomycin)
o Gram +ves resistant to; aztreonam (only affective against gram –ves), colistin (works on gram –ve outer memb, gram +ves don’t have outer memb so won’t work on them)
o Anaerobes resistant to; aminoglycosides (e.g. gentamycin) don’t work (as need oxygen dependant transport mechanisms to get aminoglycosides into cells but anaerobes don’t have this mechanism)
o Streptococci resistant to; aminoglycosides but treated with gentimycin & penicillin together (as penicillin allows gentimycin to work)
What are acquired Resistance Mechanisms ?
• Organism acquires gene that encodes antibiotic resistance mechanism
o New mutation in organism (gives organism survival adv in presence of an antibiotic)
o Horizontal transfer of genetic material to another source
• Usually an antibiotic-modifying enzyme (destroys antibiotic) or target alteration (causes gene for antibiotic target to be altered in some way)
What are types of acquired changes in bacteria?
- Absent target
• Antibacterial agents/fungi
• Antiviral agents/ bacteria
• If treat fungal with anti-bacterial agent, fungal cell walls made of diff substance so antibiotic won’t work, or e.g. antibiotic won’t work in viral infec - Decreased permeability (common cause of innate resistance)
• Vancomycin: Gram –ve bacilli (gram –ves have an outer memb impermeable to vancomycin)
• Gentamicin: anaerobic organisms (aminoglycosides uptake needs O2 dependant active transport mechanism not present in anaerobes)
• Most antibacterial needs to get INSIDE cell e.g. bacterium coverings may not allow antibiotic to get inside - Target modification
• Antibiotic can no longer react with agent
• Flucloxacillin: MRSA
o Altered penicillin-binding protein (PBP2- encoded by MecA gene) doesn’t bind β-lactams
• Vancomycin resistant enterococci: VRE
o Acquired a gene which altered peptide sequence in gram +ve peptidoglycan (D-ala D-ala D-ala D-lac)
o Reduces vancomycin binding 100 fold so it doesn’t work
• Trimethoprim: gram –ve bacilli
o Mutations in dhr (dihydrofolate reductase gene) - Enzymatic degradation
• Drug acquired gene which codes enzyme (β lactamases)- causes enzyme to be hydrolysed (common resistance mechanism for β lactems) e.g. saph aureus rarely sensitive to penicillin now (has penicillinase)
• Penicilins & cephalosporins: β-lactamases
o Staphylococcal penicillinase (inactive penicillin/ amoxicillin)
o Extended-spectrum β-lactamases (ESBL); act against larger spectrum of β lactems, inactivate penicillins & cephalosporins, common resistance mechanism against gram –ve bacteria
o Carbapenemases (enzyme- degrade carbopenems)
• Gentamicin: aminoglycoside modifying enzymes modify it
• Chloramphenicol: degraded by chloramphenicol acetyltransferase (CAT) - Drug efflux
• Multiple antibiotics, specially in gram –ve organisms
• Antifungal triazoles & Candidia spp
• Transfers drug if gets into organism straight back out of organism
What are is vertical/horizontal spread of resistance?
• Many resistance mechanisms encoded by single genes e.g. antibiotic modifying enzymes, altered antibiotic targets
• Resistance genes encoded in plasmids; circular DNA sequences transmitted within & between species mainly by conjugation
• Bacterial genome- plasmins replicate around bacterial genome; can transfer gene to bacterial genome (free transfer between genome & plasmid), can cause resistance in cell plasmid is in, can transfer to other cells
• Horizontal transfer;
o Enabled by transposons & integrons
o Integrons- DNA sequences designed to be transferred from plasmid to plasmid and/or from plasmid to chromosome
o Often contain ‘cassettes’ with multiple resistance genes
o Usually intra-species
• Vertical transfer;
o Bacterial cells divide transferring chromosomal or plasmid-borne resistance genes to daughter cells
o Once bacteria has resistance gene- undergoes replication by binary fission so all replicated cells have it (is propagated through the generations)
What is pan-resistant gram –ve bacilli?
resistant to everything
What are the Practical Consequences of Antibiotic Resistance?
Bacterial infecs become resistant to antibiotics traditionally used to treat them e.g.
• Meticillin-resistant Staphylococcus aureus (MRSA) resistant to flucocycilin ?
• Vancomycin/glycopeptide-resistant enterococci (VRE/GRE)
• Extended-spectrum β-lactamase-producing Enterobacteriaceae (ESBL)
• Carbapenemase-producing Enterobacteriaceae (CPE)
• Multi-drug resistant tuburculosis (MDR-TB)
• Extremely-drug resistant tuberculosis (XDR-TB)
• Others;
o Enterobacteriaceae resistant to amoxicillin, ciprofloxacin, gentamicin, carbapenems etc.
o Pseudomonas resistant to ceftazidime, carbapenems etc.
How do you Monitor Antimicrobial Resistance?
Sensitivity testing;
• Culture micro-organism in presence of antimicrobial agent
• Determine whether minimum inhibitory conc (MIC) ia above a predetermined ‘breakpoint level’
o High enough to kill organism
o Sustained at site of infec for long enough using practicable dosing regimens
• If grows at a level of antibiotic that is available in body- is resistant
• If doesn’t- is sensitive
Dis-sensitivity testing;
1. Add organism
2. Put antibiotic impregnated filter discs- antibiotics diffuse out from disc outwards so highest in middle & lowest in outer edges
3. Incubate
4. Read & interpret results- zone of inhibition (if too small- doesn’t kill microoganism/ need such a vast conc of antibiotics to kill microorganism can’t give in human)
• In pic- microorganism resistant to 3, 1 not enough sensitivity, 2 antibiotics with enough zone on inhibition
Liquid media- microtitre plate susceptibility testing;
• Liquid culture
• Has wells
• Add antibiotics to plate e.g. have 8 diff antibiotics in plate, then do doubling dilutions with pipette so conc of antibiotic is reduced as move to R of plate (when get to e.g. n.o. 12 conc of antibiotic very low)
• Then add organism (add same amount to each well incubate it, get growth in some wells)
• Further to highest conc of antibiotic it grows- more resistant it is
• Green arrows- breakpoint MIC, compare black arrows with this (lowest where see antibiotic growth)
What is the Epidemiology of Antibiotic Resistance?
- Resistance rates vary markedly throughout the world e.g. in Europe resistance highest in the South
- Worldwide resistance ‘hotspots’ e.g. carbapenem resistance in Indian subcontinent
- Diff organisations collect, collate and publish comparative resistance data; European Antimicrobial Resistance Surveillance Network (EARS-Net), Surveillance atlas of infectious disease
- Worldwide epidemiological data- informs local antibiotic & infection prevention & control policies e.g. if person recently been to area that has high carbopenem resistance- screened to see if picked up resistance
What are Viruses?
• Consist of;
o Nucleic acid (DNA or RNA)
o Protein (coat- structural, enzymes- non-structural)
• They are obligate intracellular pathogens
• Virus genomes e.g. enterovirus genome less complex compared to DNA herpes virus genome
What are Acute Viruses?
Influenza
Measles
Mumps
Hep A
What are Chronic Viruses?
Chronic (generally DNA viruses)
• Latent (with/ without recurrences, stay with us lifelong- can still be passed on in secretions); herpes simplex, cytomegalovirus
• Persistent; HIV, HTLV, Hep B, Hep C
• (HIV & HTLV- RNA viruses but act as DNA viruses as convert RNA to DNA
What are examples of Virus Syndrome Rashes?
Non-vesicular rashes (red lumpy rash); • Measles • Rubella • Parvovirus • Adenovirus • HHV6 Vesicular rashes (start as macular popular rash then become fluid filled); • Chickenpox (HHV3) • Herpes simples (HHV1/2) • Enterovirus
What are examples of Virus Syndrome Respiratory Infections?
- Influenza A/B
- Respiratory Syncytial Virus (in young children causing bronchiolitis- in nov-Dec time)
- Parainfluenza virus
- Human Metapneumovirus
- Rhinovirus
- Coronavirus (including SARS)
What are examples of Virus Syndrome Gasteroenteritis ?
- Rotavirus
- Norovirus (often food associated & in hospitals)
- Astrovirus
- Sapovirus
- Adenovirus (group F)
What are examples of Virus SyndromeNeurological Disease ?
Encephalitis/ meningitis; • HSV (reactivation) • Enteroviruses e.g. caused by herpes simplex • Rabies • Japanese encephalitis virus • Nipah Virus
What are examples of Virus Syndrome Blood-Borne Viruses ?
- Hepatitis viruses; HBV, HCV
- Retroviruses; HIV 1,2 & HTLV 1,2
- (Can be spread via blood, mum to child, injecs)
When do you use Antivirals
Acute infections in general population;
• Primary HSV and Herpes simplex encephalitis
• Chickenpox in adolescents and adults
• Shingles in eye (reactivation of varicella zoster virus)
• Extremes of life e.g. neonate or Elderly when T-cell response starts to wane (shingles, influenza)
Chronic infections (signif morbidity & mortality);
• HIV, HBV, HCV
Infections in immunocompromised;
• Post transplant
• Individuals receiving immunosupressive therapies
What are the steps in Virus Replication?
- Virus attachment to cell (via receptor)
- Cell Entry
- Virus Uncoating
- Early proteins produced – viral enzymes
- Replication
- Then switches to make late transcription/translation – viral structural proteins
- Virus assembly
- Virus release and maturation
• Many viruses don’t affect cell, disease can be caused by immune resposne e.g. hep B in liver, liver will continue function- only if have immune response to hep B will see disease
• Not always the case e.g. with chicken pox- is virus causing the disease
- Viruses take over much of host intracellular machinery
- All viruses encode unique proteins many of which are vital for virus replication and infectivity
- These unique proteins are targets for molecular inhibition (anti-viral activity)
Which Polymerases can be inhibited in viruses?
Viruses contain polymerases that convert;
• DNA to DNA; eukaryotes, DNA viruses
• DNA to RNA; eukaryotes, DNA viruses
• RNA to RNA; RNA viruses
• RNA to DNA (reverse transcriptase- DNA then can integrate); retroviruses (HIV), hep B virus
What is Azidothymidine?
AZT (Azidothymidine)
• Developed in 1965 as anti-cancer drug
• In 1985 found to inhibit HIV replication (1st HIV drug)
• Nucleoside Reverse Transcriptase (NRTI)- affects reverse transcriptase in polymerising HIV
• Inhibits HIV replication (initially for cancer drug but too toxic dose required, can use for HIV as can use lower dose)
What are HIV NRTI?
Pyrimidine analogues • Thymidine analogues- Zidovudine • Cytosine analogues- Lamivudine Purine analogues (Adenosine and Guanosine) • Abacavir • Tenofovir
How do you treat Hep B?
• Contains reverse transcriptase enzyme (diff struc to HIV but function in similar way)
• Some NRTIs also active vs HBV
o Lamividine
o Tenofovir
• If duel infection then use these drugs in regime (as Lamividine &; tenofovir treat HIV & hep B)
What are Herpesvirus Polymerase Inhibitors?
• Aciclovir
o HSV (herpes simplx) and VZV (vaircella zoster)
o Aciclovir can work at low concs
• Ganciclovir
o CMV (cytomegalovirus), HHV6 (as well as HSV and VZV)
o Broad spec activity, need to use at higher levels
What is a HCV RNA Polymerase Nucleotide Inhibitor ?
- Hep C treatments
- Used to use interferons- part of our innate immune system (molecules that target viral infecs- triggers enzymic reacs in cell to destroy virus)
- Can use endogenous interferon to treat viruses
- Have now direct acting anti-virals- sofosbuvir; is monophosphate, is safe & effective
- Strain 3 of Hep c not sensitive to sofosbuvir but is to interferon
What are
• Viruses contain their own to control their own replication- diff from ones in our own cell so drugs that inhibit these shouldn’t be toxic to our own cells • HIV o Atazanavir o Darunavir o Ritonavir (now used to boost levels of other PIs) • HCV o Paritaprevir o Grazoprevir
What is Aspartate Protease of HIV?
• Complex molecule of inhibitor- bind to cell to inhibit it’s replication
What are Integrase Inhibitors?
• Integrase Inhibitors (unique enzyme to retroviruses)- make molecules to inhibit this enzyme, not very toxic as we don’t have these enzymes in human genome
o Raltegravir
o Dolutegravir
What is Enfuviritide?
• Entry inhibitor (as virus needs to bind to & enter cell) o Enfuviritide (T20, given by IM injection as is a polypeptide) –fusion inhibitor (molecule in HIV which causes fusion)
What is Maraviro?
• Entry inhibitor (as virus needs to bind to & enter cell)
o Maraviroc - Chemokine receptor antagonsit binds to receps so virus can’t (Co-receptor & CCR-5 receps- predominatly used by HV- needs to bind to this recep)
What is Highly Active Antiretroviral Therapy ?
Highly Active Antiretroviral Therapy (HAART)
• For HIV treatment need combo of 3 drugs- as if use 1 drug virus quickly replicates & gets ressitant to it but if use 3 drugs HIV can’t become resistntn to all 3 on time
• Attack at least 2 sites of viral replication e.g. 2 drugs attacks 1 part & ; 3rd drug attacks another part
• HAART;
o 2 NRTIs + NNRTI
o 2 NRTIs + boosted PI
• Started when CD4 falls
• Aim to switch off virus replication
• Taken life long, suppression >10yrs achieved
• Now problems with toxicity
What are the steps in viral maturation that can be targeted?
- Fusion of HIV to host cell surface
- HIV RNA reverse transcriptase, integrase and other viral proteins enter the host cell
- Viral DNA is formed by reverse transcription
- Viral DNA is transported across the nucleus and integrates into host DNA
- New viral RNA is used as genomic RNA and to make viral proteins
- New viral RNA and proteins move to cell surface= new immature HIV formation
- Virus matures by protease releasing individual HIV proteins
What is HIV Mutation?
- HIV genome contains ~9,000 nucleotides (is an RNA gene)
- Every genome contains at least one mutation (replication has high error rate)
- A strain will become predominant if it has a selection advantage over fellow progeny
- e.g. M184V mutation results in resistance to Lamivudine, so in presence of Lamivudine the rare pop of strains with this mutation will soon predominate
- Can sequence- reverse transcriptase HIV; from nucleic acid we can infer protein so resistance mutation
What is HIV Cure by CCR5∆32/∆32 Stem Cell Transplantation?
• HIV needs interac with receps CCR5 or CXCR4 to enter CD4+
• Successful CD4+Tcells reconstitution at systemic level &; in gut mucosal immune system after CCR5∆32/∆32 stem cell transplantation & patient remains without an y sign of HIV infec
• During immune reconstitution process- replacement of long-lived host tissue cells with donor-derived cells indicating size of viral reservoir has been reduced over time
• Hep C- RNA virus, if can supress virus, immune system will clear it (has no latent form)- so can be cured
• Mutation in CCR5 (some of us have mutation)
• CCR5- is a co-recep needs to be binded to by HIV for it to invade cell
• CCR5 delta 32 allele freq;
o Mutation geographically determined where you live (in North Europe)
o Many of these genes come about because certain genes give survival ADV
o People with this deletion LESS likely to be infected with HIV
What is the current HIV ‘cure’?
- HIV suppressed on antivirals
- Existing CD4 lymphocytes destroyed by conditioning
- Stem cells reconstituted with HLA-matched but delta 32 homozygous allogeneic donor
- Antiviral therapy stopped following transplantation
- Remained HIV negative (by PCR)
- HIV antibody titres have declined post bone marrow transplant (so true latency of HIV in T cells, even though affects lots of cells)
What is Aciclovir?
Antivirals
o For Rx of Herpes Simplex Virus (HSV) and Varicella Zoster Virus (VZV)
o Nucleoside analogue (phosphorylated by herpesvirus thymidine kinase)
What is Ganciclovir?
Antivirals for cytomegalovirus (CMV)
What is Oseltamivir and Zanamavir?
Antivirals
(neuraminidase inhibitors)- block coating of virus
o Influenza
What is Ribavirin?
Antivirals
(broad spectrum of activity)- RSV, HCV and HEV
What are Interferons?
Antivirals
(can be naturally immune)- Hepatitis B virus cure
What does a Urine Dip Test show that may support diagnosis of infection?
• WC;
o Leucocyte esteratse- enzyme made by neutrophils
o May reflect pyuria associated with UTI
o WBCs anywhere in GU tract (including the vaginal vault) will produce LE.
o +ve in patients with chlamydia infecs, urethritis, TB, bladder tumors, viral infecs, nephrolithiasis, foreign bodies & corticosteroid use
• Nitrites;
o Nitrates excreted by kidney
o Some bacteria reduce urinary nitrates to nitrites
o +ve nitrite test usually means infec
o Need over 10,000 bacteria per ml to turn the dipstick +ve (specific but not a very sensitive test).
o A -ve nitrite test doesn’t rule out a UTI; infec with non-nitrate-reducing organisms causes a -ve nitrite test.
o Nitrate- deficient diet may cause a falsely negative test in the presence of infection
o Improperly stored dipsticks are a common cause of a false-positive test for nitrites.
• RBCs;
o ‘RBC’= peroxidase activity of erythrocytes
o Myoglobin and hemoglobin also catalyze this reaction
o High doses of vitamin C inhibit this process (vit C can produce a false-negative occult blood in stool).
o A +ve dipstick for blood in absence of RBCs by microscopy = myoglobinuria or heamoglobinuria, not true hematuria
What are common UTI Pathogens?
• Bacterial
o Enterobacteriaciae; GI organisms, E coli also proteus sp, klebsiella sp
o Enterococci; GI organisms
o GI organisms; E.coli (gram –ve bacilli, usually found in gut), other gram –ves from gut, anaerobes also in gut but don’t generally cause UTI, enterococci (e.g. streptococci in gut) which can cause UTI,
o Staphylococci; skin organisms, S saprophyticus, (S aureus)
o (nb anaerobes, pseudomonas sp)
- Fungal- Candidia (catheter/ stents, diabetes)
- Viral- adenovirus (haemorrhagic cystitis)
